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Revision #: 03

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Title: 

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Sections in This Document

1. Introduction............................................................................................................................... 1

2. FDA Law and Filth .................................................................................................................... 5

3. Use of Microscopes for Examination ........................................................................................ 6

4. Collecting and Preserving Whole Insects and Arthropods......................................................21

5. Taxonomy...............................................................................................................................27

6. Identification Keys...................................................................................................................28

7. Digital Photography and Photomicrography ...........................................................................31

8. Dissection ...............................................................................................................................35

9. Insect Morphology ..................................................................................................................37

10. Fragment Recognition ............................................................................................................39

11. Mites .......................................................................................................................................41

12. Hair Identification....................................................................................................................44

13. Feather Identification ..............................................................................................................47

14. Excrement, Urine, Uric Acid (Morphological and Chemical)...................................................49

15. Sample Analysis .....................................................................................................................52

16. Reporting ................................................................................................................................59

17. Visual and Macroscopic Methods ...........................................................................................61

18. Microscopic Methods..............................................................................................................65

19. Sedimentation and other Specialized Techniques..................................................................68

20. Mold Detection........................................................................................................................71

21. Analysis of Factory Filth Samples and Filth Analytical Worksheets .......................................76

22. Specialized Microscopy Techniques, Optical Crystallography (a.k.a. Polarized Light 

Microscopy), and ID Spot Tests..............................................................................................82

23. Glossary/Definitions................................................................................................................83

24. Document History ...................................................................................................................84

25. Change History.......................................................................................................................85

1. Introduction

The Food, Drug, and Cosmetic Act protects the public from the presence of filth, putrid or 

decomposed material in food products, and those products that may have been exposed 

to insanitary conditions that may contaminate the product with filth or render it injurious to 

health. The terms filth, foreign material, or extraneous material are used 
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interchangeably. The courts define filth in a common-sense manner; filth does not have 

any specialized or technical definition. Filth is any type of matter that obviously does not 

belong in a food product. Representative examples of filth in food products include but 

are not limited to rodent excreta, insects, parasites, and extraneous materials such as 

metal and glass shards. 

Filth can enter a product through many forms and sources; and is often invisible to the 

consumer. Filth may be present in food naturally and unavoidably, or as the result of an 

intentional or unintentional controlled bad practice. The identification, confirmation, and 

quantitation of the type of filth can help determine how the material was found in the 

product, if it was a natural and unavoidable event, an accidental event, a controllable 

event, an unintentional event and/or a deliberate intentional event. 

More importantly, analysts can assess the real or potential health risk involved with these 

adulterants. Given today’s food production operations and storage facilities, when the 

presence of a health hazard or vector potential is confirmed, the findings may place 

added emphasis on certain problems and the surrounding circumstances. The objectives 

of this chapter are to introduce the visual/macroscopic and microscopic techniques, 

practices, and procedures used to identify and confirm the presence of adulterants in 

various commodities and provide practice in reporting such findings in a clear and 

concise manner. 

Given the nature of the work and the broad definition of filth, analysts with a strong 

background in the biological/agricultural sciences with special emphasis in entomology, 

pest control, botany, agricultural and food production have an advantage in this course 

work. However, FDA filth analysts come from diverse disciplines. This chapter is not 

intended to be all inclusive but will provide FDA analysts the basic six to nine-month 

orientation program for beginner filth analyses.

1.1. General Laboratory Trainings

The trainees will receive Basic Orientation Training through ORA web-based 

modules, discussions, exercises, and videos; the trainees are expected to 

complete the ORA Analyst Bingo card within six months of employment. 

Before beginning to work in a district laboratory, the trainee should be given a 

thorough safety orientation that includes familiarization with the laboratory's safety 

features and regulations, and the local laboratory's Standard Operating 

Procedures (SOP’s) and Quality Assurance Procedures. Emphasis is placed 

towards the policies and procedures inherent in the filth analysis program. 

Additional information can be found in MAN-000045 ORA Lab Manual Vol. III 

Section 1 - Environmental Health and Safety (III-01). 
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1.2. Reference Books and Materials

It is recommended that the trainer should provide the following instructional 

materials for the trainee’s general use:

A. Gorham, J. R. (Ed.). (1991). Ecology and management of food-industry pests

[Formerly released as FDA Technical Bulletin No. 4]. Gaithersburg, MD: 

AOAC International. 

B. Vail, D. J. (n.d.). Micro-analytical biology workbook for food sanitation control 

analysts (out of print, Vol. 1). Atlanta, GA: FDA, Southeast Regional 

Laboratory.

C. AOAC official methods of analysis (current Edition). Gaithersburg, MD: AOAC 

International.

D. U.S. Food & Drug Administration, Center for Food Safety and Applied 

Nutrition. (1998). Macroanalytical Procedures Manual (FDA Technical Bulletin 

No. 5). [Originally released in print by FDA in 1984 by Olsen, A. R. (Ed.), 

Knight, S. A. (Tech. Ed.), Ziobro, G. C., Ph.D. (Assoc. Ed.)] 

E. Harris and Reynolds (Eds.) Microscopic-analytical methods in food and drug 

control (out of print, FDA Technical Bulletin No. 1). 

F. Gorham, J. R. (Ed.) (1981). Principles of food analysis for filth, decomposition, 

and foreign matter (FDA Technical Bulletin No. 1, 2nd Ed.). Gaithersburg, MD: 

AOAC International. 

G. Gorham, J. R. (Ed.). (1978). Training manual for analytical entomology in the 

food industry (out of print, FDA Technical Bulletin No. 2, HHS Publication No. 

(FDA) 77-2086). 

H. Gentry, J. W., Harris, K., Gentry, Jr., J. W. (1991). Microanalytical entomology 

for food sanitation control (Vols. I and II, revised reprint of O.D. Kurtz and K.L. 

Harris AOAC publication by the same name). Gaithersburg, MD: AOAC 

International.

I. Olsen, A. R., Sidebottom, T. H. and Knight, S. A. (1996). Fundamentals of 

Microanalytical Entomology, a practical guide to detecting and identifying filth 

in foods. Boca Raton, FL: CRC Press. 

J. Hui, Y. H., Bruinsma, B. L., Gorham, J. R., Nip, W., Tong, P. S., Ventresca, P. 

(2002). Food plant sanitation. New York: Marcel Dekker, Inc.

K. Bennett, G. W., (2010). Truman’s Scientific Guide to Pest Control Operations 

(7 ed.) Questex Media Group LLC & Purd.

L. Arnold Mallis; Stoy A Hedges and Dan Moreland, Mallis Handbook of Pest 

Control, 10th Ed., GIE Media, Inc.
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1.3. Recommended Sample Collections/Products for Training

The trainee receives training on a variety of methods. It is recommended that 

these 22 food samples are used for training assignments: 

A. Wheat or other whole grain 

B. Green coffee beans or cocoa beans 

C. Flour 

D. Whole or crude spice: capsicum pods 

E. Whole or crude spice: peppercorns 

F. Whole figs or dates 

G. Shell nuts (Canned/Mixed) 

H. Blueberries, raspberries, or cherries (Fresh/Frozen) 

I. Whole tamarind pods 

J. Fig paste or other fruit paste 

K. Chocolate 

L. High bran content bakery goods 

M. Dried ground spice 

N. Peanut butter 

O. Canned tomato juice 

P. Canned sliced button mushroom or dried mushroom 

Q. Whole fruit or vegetable (Canned or Fresh) 

R. Cinnamon or Cassia sticks 

S. Tomato concentrate product (puree, sauce or paste) 

T. Berry or citrus juice product 

U. Rodent, insect, floor sweepings, exhibits 

V. Rodent contaminated food and packaging

1.4. Investigations Training

The trainer arranges for the trainee to accompany a district investigator on the 

following types of inspections:

A. Establishment Inspection (EI) of a food processing plant and a food 

warehouse.

B. Field examination of import foods. 
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2. FDA Law and Filth

2.1. This exercise describes the legal basis for the agency’s regulatory activity in foods, 

drugs, and cosmetics contaminated (adulterated) with filth.

2.2. To analyze a filth sample, the analyst is to be intimately familiar with the 

requirements and prohibitions of the Food, Drug, and Cosmetic (FD&C) Act. The 

filth analyst demonstrates compliance or non-compliance with sections of the Act. 

Filth Analyst work primarily with evidence collected to show non-compliance with 

Sections 402(a)(3), 402(a)(4), 501(a)(1), 501(a)(2)(A), 601(b), 601(c), 801(a)(1), 

and 801(a)(3) of the FD&C Act. 

It is very important to have a clear understanding of the difference between 

sections 402(a)(3) and 402(a)(4). Section 402(a)(3) deals with a condition of the 

food that is objectionable in itself, whereas section 402(a)(4) deals with an 

objectionable practice. 

Sections 501(a)(1), 501(a)(2)(A) deal with adulterated drugs and devices 601(b), 

601(c), deal with adulterated cosmetics, and 801(a)(1) and 801(a)(3) deals with 

import and exports for the same types of offenses as described above. 

A careful reading and discussion of the assigned literature is essential towards a 

more complete understanding of these and other sections of the FD&C Act.

2.3. Assignment

Complete: Introduction to the FD & C Act, web-based training module.

Read and discuss starred (*) items with the trainer from the following sections of 

the FD&C Act:

A. 201(f)*, (g), and (i) Definitions

B. 301(a)*, (b)*, (c)*, (g), and (k)* Prohibited Acts

C. 402(a)(3) * and (a)(4)* Adulteration (Also, review 21 U.S.C. 342(a)(3), (a)(4), 

and (b)(2))

D. 501(a)(1) and (a)(2)(A)

E. 601(b) and (c)

F. 702(b)* Reserve Sample

G. 704(d)* Letter

H. 801(a)(1) and (a)(3)

2.4. Read and discuss with the trainer:

A. Gorham, J. R. (Ed.) (1981). Principles of food analysis for filth, decomposition,

and foreign matter (FDA Technical Bulletin No. 1, 2nd ed., pp. 3-9). 

Gaithersburg, MD: AOAC International.

B. Gentry, J. W., Harris, K., Gentry, Jr., J. W. (1991). Microanalytical entomology 
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for food sanitation control (Vols. I and II, pp. 3-11). Gaithersburg, MD: AOAC 

International.

C. Olsen, A.R., Gecan, J.S., Ziobro, G.C., Bryce, J.R. (2001). Regulatory Action 

Criteria for Filth and Other Extraneous Materials V. Strategy for Evaluating 

Hazardous and Nonhazardous Filth. Regulatory Toxicology and 

Pharmacology, 33, 363-392.

D. Olsen, A. R., Sidebottom, T. H., Knight, S. A. (1996). Fundamentals of 

Microanalytical Entomology, a practical guide to detecting and identifying filth 

in foods (pp. 1-9). Boca Raton, FL: CRC Press. 

E. U.S. Food & Drug Administration, Office of Regulatory Affairs, Office of 

Enforcement. Filth from Insects, Rodents, and Other Pests in Foods. 

Compliance policy guides, Sec. 555.600 (CPG 7120.).

F. U.S. Food & Drug Administration, Office of Regulatory Affairs, Office of 

Enforcement. Tree Nuts - Adulteration Involving Rejects (Insect Infestation, 

Moldy, Rancid, Otherwise Decomposed, Blanks, and Shriveled). Compliance 

policy guides, Sec. 570.425 (CPG 7112.05). 

G. U.S. Food & Drug Administration, Office of Regulatory Affairs, Office of 

Enforcement. Foods, Adulteration Involving Hard or Sharp Foreign Objects.

Compliance policy guides, Sec. 555.425. 

H. U.S. Food & Drug Administration, Office of Regulatory Affairs, Office of 

Enforcement. Foods, Rail Car Sanitation – Adulteration. Compliance policy 

guides, Sec. 545.300 (CPG 7117.08). 

I. U. S. Food and Drug Administration, Center for Food Safety and Applied 

Nutrition (May 1998 revision). The food defect action levels (read the 

Introduction). 

J. U. S. Food & Drug Administration, Office of Regulatory Affairs, Office of 

Enforcement. Apricots - Canned - Adulteration with Insects. Compliance policy 

guides, Sec. 550.150 (CPG 7110.02). 

3. Use of Microscopes for Examination

3.1. Objective

The trainee receives instruction towards the use and care of laboratory 

microscopes and their accessories and learns proper terminology and definitions 

of the various forms of microscopic examination. 

3.2. Discussion

A. Types of Examinations and Microscope Use
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1. Visual/Macroscopic Examination

This type of product examination generally depends on the direct sensory input 

of the analyst. Examination is typically conducted with the naked eye but also 

may include the use of a hand lens (generally in the range of 3-10X), 

magnifying ring lamps (3X), or a pair of jeweler’s loupes (3-10X). 

2. Widefield Stereomicroscope or Dissecting Microscope

Results from a macroscopic examination may not be conclusive because the 

adulterant/defect cannot be completely identified without the assistance of a 

stereo microscope. This instrument, the most frequently used in filth analysis, 

has a wide variety of applications. The main application is for examining gross 

filth and for reading (examining) extraction papers for microscopic filth. The 

most used magnifications are from 6-30X; higher magnifications (50-75X) are 

used to confirm the identity of small objects. The microscopes exhibit a large 

field of view and have large working distances (from the lens to the focal point) 

to move and manipulate objects under the lens.

3. Microscopic Examination 

Filth/adulterants in the product are usually not visible to the naked eye and 

therefore are examined microscopically. A widefield stereo is often used first to 

manipulate and separate the contaminated product and then a compound 

microscope is used to positively identify the material and to see fine 

microscopic detail, morphology, and sculpture. Compound microscopes 

optimize lighting techniques and are engineered to manipulate light to see 

details not observable in stereoscopic examination, thus they have a very 

limited field of view, and they have short working distances. The typical 

working magnifications are 100-400X but may be used at 1000X with proper 

technique and special oil that helps capture and retain the light.

B. Fundamental Microscopic Techniques and Procedures

1. Lighting and Ocular (eyepiece) Optimization Techniques

Whether using a widefield or compound microscope, eye fatigue is usually the 

most important limiting factor when working with microscopes for long time 

periods. To help reduce headaches and eyestrain, the analyst needs to learn 

the following:

Lighting: Adjustment begins with the light source, which should be daylight, 

blue, or white (not frosted). Light strength should be adjusted with the 

transformer or iris diaphragm, so that details can be seen clearly with a 

minimum of glare and a minimum of intensity or brightness. For a 

stereomicroscope, the light source should be positioned slightly to one side 

and at approximately a 70° angle from horizontal, or overhead, as with 
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fiberoptic ring lights. The light field should be centered so that both eyes 

receive the same intensity of light (i.e., balanced).

Oculars (eyepieces): are adjusted to the individual analyst's eyes and facial 

features- while the eyes are in their most relaxed state. This procedure should 

be done before each magnification. Note: Leave both eyes open while 

adjusting the oculars. Wearing eyeglasses is a matter of individual preference. 

People with astigmatism may find wearing their eyeglasses will reduce eye 

fatigue. If eyeglasses are to be worn, high eyepoint eyepieces may be ordered 

and should be used, and the ocular adjustments should be made with the 

eyeglasses on.

2. Procedure for adjusting oculars: 

Adjust the interpupillary distance of the oculars so that the oculars are 

centered on the respective pupils. 

Determine which ocular is independently focusable.

Place a specimen on the stage of the instrument, focus to the clearest image, 

and center it in the field of view. 

Hold a black or white index card between the focusable ocular and the eye, or 

simply remove the eyepiece, blocking vision on that side. Do not close the eye 

or squint; the eye remains in a relaxed eye position.

Using the main focusing adjustment knob, clearly focus the image of the 

specimen for the eye that is not blocked. 

Remove the index card from the first position and use it to block the vision of 

the other eye (or remove the opposite eyepiece.) Using the focusing ring 

mechanism of the focusable ocular (not the main adjustment knob), clearly 

focus the image of the specimen.

Remove the card and using both eyes, view the specimen. The image should 

now be clearly in focus for both eyes in their most relaxed state

3. Resolution (compound microscopes)

For compound microscopes, the trainee should learn how to achieve optimal 

or Köehler illumination. The Köehler Principle focuses the field iris in the same 

plane as the specimen, thereby obtaining maximum resolution. Step-by-step 

instructions for achieving Köehler illumination are found in "Training Manual for 

Analytical Entomology in the Food Industry," Chapter 2 Part III D; FDA 

Technical Bulletin No. 2 and/or in the microscope manuals. The trainer 

demonstrates how to accomplish Koehler illumination and the trainee repeats 

the work on their unit.

4. General Microscope Maintenance
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The analyst is responsible for simple maintenance procedures when using the 

instrument e.g., tightening loose focusing mechanisms, properly cleaning the 

lenses, and changing the light bulbs. These and other maintenance 

operations are described in the instruction manuals accompanying each 

instrument and should be addressed in the laboratory’s Standard Operating 

Procedures. Maintenance records should be kept in the instrument logbook, 

and more complicated maintenance is directed to the assigned microscope 

monitor’s attention and should not be attempted without prior approval.

C. Special Types of Microscopy and Accessories

1. Phase-Contrast Microscope

The phase-contrast microscope is a compound microscope that has special 

sub-stage accessories and objective lenses designed to produce optical 

contrast between the specimen and the mounting medium. Using phasecontrast optics, the analyst can observe many details that are obscure or 

indiscernible under a conventional compound microscope. This type of 

microscope is widely used for examining mites and small, somewhat 

transparent insects, like maggots, and in glass identification.

Success with the phase-contrast microscope begins with and is highly 

dependent on the refractive indices of the specimen and the mounting 

medium. To get good contrast, the mounting medium should have the 

greatest possible difference in refractive index from the specimen. The 

difference between the refractive indices creates variations in light intensity. 

To the observer, it appears that a halo of intense light is surrounding the 

very dark edges and surface structures of the specimen, starkly silhouetting 

otherwise vague details.

The instrument is adjusted in the same manner as a conventional 

compound microscope. Once optimal illumination is achieved, the phasecontrast optics are aligned in the following manner:

Each phase objective lens has a metal ring of a defined size imbedded 

between two of the lens elements. The ring size is indicated by a number 

(usually "Ph 1," "Ph 2," or "Ph 3") printed on the lens casing. 

Similarly, the condenser has annular rings glued to a rotating clear plate 

that rotates into set position They are marked "1," "2," "3," and "clear (or J)" 

This latter position is the “normal” position for transmitted light microscopy, 

or the “starting" position for phase microscopy.

In phase microscopy, the analyst properly selects the matching objective 

and condenser ring numbers, positioning the ring of the condenser in the 

light path, so that it will just encircle the ring in the objective. That is, the 
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analyst pairs the objective ring to the condenser ring. 

Initially, the adjustment of the instrument proceeds in the same manner as 

conventional transmitted light microscopy, using the “J” condenser position. 

Establish Köehler illumination first, and then focus on a mounted slide 

object (like a mite’s hairy leg). Finally, select the low power objective and 

begin aligning the phase-contrast optics, i.e., each objective with its 

corresponding condenser ring. Phase-contrast microscopes vary 

significantly by manufacturer and the proper alignment techniques are 

unique to each (some use focusing eyepieces, while others use swing-in 

focusing lenses). Therefore, consult the manual for the specified 

microscope, or have the trainer demonstrate how to critically align the 

rings.

2. Polarized Light Microscopy (PLM)

A polarizing light microscope is a compound microscope fitted with 

polarizing prisms, called "Nicols," below and above a rotating circular 

stage. When two Nicols are placed in the optical train, the first acts as a 

polarizer and the second as an analyzer. The vibration direction of the 

plane-polarized light produced by the polarizer is conventionally the northsouth direction. If the analyzer, which can be rotated in most instruments, is 

set in the same relative position as the polarizer (parallel Nicols), then 

through light is transmitted, producing a light field of view. But, if the 

analyzer is rotated through 90 degrees so that its plane of vibration is at 

right angles to the polarizer (crossed Nicols), no light will pass, except that 

refracted into the analyzer’s plane, producing a dark field of view.

Optically active substances show interference colors when placed between 

crossed Nicols. Observations of optical activity can be useful to the analyst 

for identifying to some extent such diverse things as glass fragments, 

plastics, synthetic fibers, crystals, starches, and mites. This segment of 

training gives the trainee the basic principles of PLM techniques. 

Additional descriptions of PLM techniques will be found in Advance 

techniques Chapter 4.6.2 Optical Crystallography. 

3. Comparison Microscopy (Forensic microscopy)

Comparison Microscopy involves “bridging” the optics of two microscopes, 

so that they may be viewed independently, side by side, or overlapped for 

direct comparison purposes. In this manner, filth analysts can compare 

known specimens, with unknown specimens, and confirm similarities or 

identify differences. 

4. Lightfield/Darkfield Stereomicroscopes

Lightfield/Darkfield stereomicroscopes are conventional stereomicroscopes 
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mounted on a special light base, the base of which produces either a white 

background (fully illuminated field of view) or a dark background (with 

incident light coming in at an angle). The technique is very useful in 

increasing contrast (similar in a way to phase contrast) and in viewing light 

subjects against a dark background. Examples include using darkfield 

illumination when trying to count mites, versus counting white mites or 

maggots against a white background. 

5. Scanning Electron Microscopes (SEM)

Scanning electron microscopy (SEM) is used to visualize samples at higher 

magnifications (10X - 500,000X) and can provide morphological information 

about a sample. SEMs have particular applications in particle analysis and 

detailed micro-structural analysis. Several publications describe the use of 

electron microscopes in the examination of mites and stored product beetle 

mandibles, antennae, animal hair and related structures. 

Tabletop/Benchtop SEMs can be configured to easily image an object in 

situ or without many modifications. This may include using a low vacuum 

or atmospheric mode to allow for the imaging of biological samples. An 

Energy Dispersive X-ray Spectrometer (EDS) can be added to an SEM and 

is used to obtain qualitative elemental information about the sample. In the 

area of particle analysis, SEM/EDS can help to identify particles using not 

only their structure and morphological form, but also their elemental 

composition. 

D. Microscope Accessories

1. Eyepiece Micrometer (or graticule) 

The eyepiece micrometer consists of a clear disc with a graduated scale or 

pattern printed on it, which is inserted in the eyepiece of the microscope for 

use in measuring specimens. To obtain meaningful measurements, the 

graticule is calibrated for each magnification, by using a stage micrometer 

with a graduated scale of known increments (usually 0.1 mm for 

stereomicroscopes or 0.01 mm for compound microscopes). 

a) Calibration Procedure for micrometer

Place the stage micrometer on the microscope stage and focus on the 

stage micrometer scale. (The eyepiece graticule should always be in focus; 

if not, the disc is probably improperly inserted or out of adjustment.) Move 

the stage micrometer so that the zero end of its scale coincides with the 

zero end of the eyepiece scale and the two scales are superimposed on 

each other over their entire lengths. Reading from the eyepiece scale, find 

the farthest division from zero that coincides with a division on the stage 

micrometer.
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b) Record the following information:

• Magnification

• The number of eyepiece scale divisions between the zero coincident 

and the farthest eyepiece scale coincident. This value is designated 

EMD (eyepiece micrometer divisions).

• The number of stage micrometer scale divisions between the zero 

coincident and the far stage scale coincident. This value is 

designated SMD (stage micrometer divisions).

• The millimeter value of one division of the stage near scale. This 

value is usually found printed on the micrometer. This value is called 

mm/SMD (millimeters per stage micrometer division).

c) Calculate the millimeters per eyepiece micrometer division (mm/EMD)

mm/EMD = (mm/SMD) X (SMD)/(EMD) 

The mm/EMD is the number of millimeters per eyepiece micrometer 

division for that specific magnification. This value is used to convert 

eyepiece micrometer divisions to millimeters by multiplication. (Note:

These calculations need to be determined for each set of eyepiece and 

objective combinations.)

Example: At 10X magnification, 12 EMD coincide with 18 SMD on a 

stage micrometer in which 1 SMD = 0.1mm 

(0.1mm/SMD) X (18 SMD)/(12 EMD) = 0.15 mm/EMD

If an object is observed to be 5 EMD long at 10X magnification, then 

(0.15 mm/EMD) X (5 EMD) = 0.75 mm

The object's length is calculated as 0.75 mm, the last digit being only an 

approximation. The significance of calculated numbers should be 

carefully considered for the mathematical rules concerning significant 

figures. Refer to MAN-000048 ORA Lab Manual Vol. III Section 4 - 

Basic Statistics and Data Presentation (III-04)

2. Mechanical Stage Micrometer

This “accessory” consists of two graduated scales that are engraved or 

inscribed on the mechanical stage. One scale is on the moving portion and 

the other scale is directly parallel to the first on the stationary portion of the 

stage. As the length of the specimen's image is moved through a fixed 

point in the field of view, the number of divisions on the stationary stage 

scale can be counted by observing the starting and finishing positions of 

the sliding scale's end point. By substituting this number for the EMD in the 

equation found in the previous section, the trainee can calculate a 

conversion factor using a stage micrometer with a graduated scale of 
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known increments. Quite often, mechanical stage micrometers will have a 

provision for interpolating the final digit of a reading, which adds to their 

accuracy.

Note: The eyepiece micrometer has more versatility than the mechanical 

stage micrometer; the eyepiece micrometer can be positioned over the 

image at exactly the angle desired for determining longest and shortest 

dimensions.

3. Camera Lucida (Abbé type)

The Camera lucida (Abbé type) consists of a set of prisms that can 

superimpose the image of a specimen onto a piece of paper lying on the 

bench top beside the microscope. With practice, while viewing the 

specimen through the scope, the trainee can see the paper and pencil at 

the same time as the specimen, thus producing accurate outlines or 

detailed drawings of the specimen under observation. (Hint: Illuminate the 

drawing paper with a strong light.) 

This technique is particularly useful when photography does not show the 

desired details (poor depth of field or cluttered information), that line 

drawings can produce. 

3.3. Assignment

A. Read the following:

1. Gorham, J. R. (Ed.) (1981). Principles of food analysis for filth, 

decomposition, and foreign matter (FDA Technical Bulletin No. 1, 2nd ed. 

pp. 219-228). Gaithersburg, MD: AOAC International.

2. Gorham, J. R. (Ed.). (1978). Training manual for analytical entomology in 

the food industry (out of print, FDA Technical Bulletin No. 2, chap. 2).

3. Möllring, F. K. Microscopy from the very beginning (Brochure G41-100, p. 

58). U.S. and Germany: Zeiss, Inc. Or, Möllring, F. K. (1973). Beginning 

with the microscope. New York: Sterling Publishing. Both out of print, but 

useful if one can obtain a copy.

4. The microscope manufacturer’s manuals for the scopes the analyst will be 

using. 

B. Demonstrate the optimal set-up and illumination with specimens provided by 

the trainer using the following:
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1. Widefield stereoscope

2. Compound microscope

3. Phase-contrast microscope

C. Prepare a table of measurements for an eyepiece micrometer on a compound 

or stereomicroscope in the laboratory.

3.4. Preparing Microscopic Slide Mounts 

A. Objective

The trainer provides basic instructions for properly preparing permanent and semipermanent microscopic slide mounts of hairs, insect fragments, minute whole 

insects, or other "filth" elements. Slide mounts may be prepared for use in 

reference collections or as teaching aids or, regarding regulatory samples, as 

evidence in a court of law.

This training is applicable to all analysts involved in the identification of filth 

elements found in foods. General entomological knowledge and/or training are 

not a prerequisite for this section.

B. Discussion

No amount of microscope alignment, focusing, or other manipulation can undo the 

damage done by the improper mounting of a specimen on a microscope slide. 

The mounted filth specimen prepared for regulatory work is an item of evidence, 

ultimately subject to the scrutiny of a court of law. Fine detail observations may 

provide significant clues and to the identity of the object. Additionally, the need for 

quality mounted materials for use in a reference collection is essential and is 

tantamount to good museum practice. As such, the analyst strives to become 

proficient at producing professional-quality slide mounts.

C. Equipment and Reagents

1. Dissection microscope

2. Dissection needles

3. Fine (needle) point forceps

4. Microscope slides

5. Microscope slide cover slips (square and/or round, 1-11⁄2 thickness)

6. Adhesive labels

7. Hot plate

8. Alcohol lamp (if the laboratory allows open flames)

9. Slide warming plate (typically at 45-50oc)

10.Water bath

11.Glycerin
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12.Gelatin

13.Phenol

14.Gum arabic (crystalline form)

15.Chloral hydrate

16.Distilled water

17.2% solution of aqueous acid fuchsin or lignin pink

18.Commercially purchased "permanent" microscope mounting media (i.e. 

Permount, euparal, and/or canada balsam)

19.Ringing compounds- nail polish (clear preferred) or glyptal® (electrical 

insulating varnish)

20.Fume hood

21.Standard safety equipment (laboratory coat, eye protection, gloves)

D. Specimens for Observation

There are many textbooks and articles written on mounting specimens for 

observation under the scope. Many techniques date back to the early 

development of the microscope, as people worked with various formulas to 

accomplish the perfect slide mount for whatever material they were studying. Over 

time, people and disciplines developed preferences based on their needs, ease of 

use, or understanding of the media. Selecting the media of choice varies 

significantly based on the following factors:

• How easy is it to use?

• What is the refractive index and how well does it work with certain 

specimens of higher or lower index? 

• What preparation steps are needed?

• How long does it take to prepare the finished slide? 

• How long will the slides last - temporary, intermediate, or permanent, 

before they discolor, crack, or cloud over?

• What effect does the media have on the specimen or stain specimens?

• Does it need ringing?

• Is it expensive?

• Does it call for the use of noxious chemicals?

• How valuable is the specimen - what type, an authentic, forensic value 

as evidence, or quick, non-permanent observation?

Based on these questions this next section offers some discussion of the 

media FDA analysts have found to be of the most value and the easiest to use.
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4. Media

4.1. Wet Mount

A wet mount is a quick and temporary slide mounting technique. A drop of water 

or glycerin alcohol is placed on the microscope slide using a dropper or pipette. 

The specimen is then added to the drop and a cover slip is placed on top of the 

specimen. The edge of the cover slip is placed on the slide at a 45o angle. It is 

then gently lowered over the specimen reducing the chance of air bubbles. 

Tapping the cover slip gently can release trapped air bubbles. To insure the 

specimen is covered a sufficient amount of liquid needs to be on the slide. If too 

much liquid is on the slide the cover slip will float off the specimen. This can be 

corrected by placing a tissue or paper towel along one edge of the cover slip to 

draw up the excess fluid. If the slide mount is too dry, add a small amount of fluid 

on the side of the slide that is lacking fluid.

One advantage with a wet mount, a liquid stain can be added to the specimen, 

while the specimen is still under the cover glass. Along one edge of the cover slip 

place a drop of the stain to be used. At the opposite edge of the cover slip place a 

tissue or paper towel to absorb the water under the cover slip. The stain will get 

drawn under the cover slip and stain the specimen.

4.2. Glycerin Jelly (GJ) Media

The most used and preferred medium for mounting hairs and insect fragments is 

glycerin jelly (GJ). 

The formulation is 10 g gelatin, 70 ml glycerin, 60 ml H2O and 1 g phenol. The 

gelatin is poured on cold water to soak, and then heated over a water bath to 

completely dissolve the gelatin. The glycerin and phenol are mixed while hot. 

When cooled, the mixture has the consistency of semi-hardened gelatin, and it 

melts around 35-40oC. Glycerin jelly can also be ordered from some chemical 

supply houses. The phenol can be substituted with an antiseptic mouthwash like 

Listerine, using 1-2ml of the mouthwash., thus creating an alternative mounting 

media called Kaiser’s Glycerin Jelly.

To mount a specimen, a small piece of glycerin jelly is placed on a slide and 

warmed to the point where it becomes fluid. As an alternative, the analyst can use 

pre-melted material, from a glass rod dropping bottle, held on a slide warming 

plate. With practice, the analyst will have more control of the media droplet size 

using pre-melted media. The specimen is placed, then pushed into the media with 

a needle probe into the bottom/center of the liquid medium, then oriented to the 

desired position, and covered with a coverslip. Warming the slide again is 

sometimes needed for the jelly to engulf the specimen and fill the space under the 

coverslip. If trapped air is present near the specimen, the air can be removed by 

gently heating it over an alcohol lamp or low temperature hot plate.
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Caution: This may cause the specimen to migrate to the edge of the coverslip. If 

this happens, the analyst may need to remount the specimens and make another 

preparation. It is important for the analyst to practice mounting specimens in order 

to get a "feel" for the peculiarities of glycerin jelly.

To mount hairs in glycerin jelly, the analyst uses a little extra heat to drive out the 

air in the center (medullary) portion of the hair. The characteristics of the hair 

cannot be observed until the air inside the hair has been replaced with glycerin 

jelly. One common method of removing air is to heat the mounted specimen 

carefully over an alcohol flame or hot plate until the glycerin jelly under the 

coverslip begins driving the air out of the hair (Note: just below the media’s boiling 

point), then cool and observe the specimen at high magnification to see if the air is 

gone. Continue heating and observing until sufficient air is driven out to make 

definite identification. Again, practice is needed, as too much heat will curl and 

distort the hair, warp the coverslip, and denature or discolor the medium. See 

also Reference: LIB 2243, “Improved Procedure for Liquid Replacement of 

Entrapped Air in Mammalian Hairs.” 

Note: Some analysts make a slide by piling the specimen and coverslip on top of 

a solid piece of glycerin jelly and then warming the slide so that the glycerin jelly 

engulfs the specimen. Two problems may occur using this method. One, the 

specimen migrates with the melting media towards the edge; secondly, large air 

bubbles can be formed and trapped under the coverslip. These are considered 

permanent mounts when ringed (ringing is discussed below, Section C). They 

should be held flat, even when rung. They are stable for at least 5 years and 

longer if rung, and generally do not discolor or cloud over time. Specimens do not 

need special drying (water removal) processes as in other resin or Canada 

balsam mounts, however, mounting from dishes wet with glycerin-alcohol (50/50) 

or 70% alcohol does reduce some of the trapped air problems. 

4.3. Hoyer's Solution

Hoyer's solution is a mounting medium that has been used by entomologists for 

decades and is now gaining popularity in some areas of food analysis. It is found

commercially, but has many formulation variations, all principally gum-chloral 

hydrate derivatives. In addition to its excellent optical properties (~1.47 refractive 

index), Hoyer’s solution renders muscular and visceral tissues transparent 

(clearing effect), allowing the analyst to observe cuticular structures on the intact 

specimen without interference. This medium is used primarily for mites and small 

insects, but it can also be used for insect fragments. It is not used for hairs, except 

as a temporary mount, as it will disintegrate a hair over time. When rung the slides 

will last several years, but eventually moisture will enter, and the slides will cloud 

over. 

Hoyer's solution consists of 50 ml distilled H2O, 30 g gum arabic, 200 g chloral 
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hydrate, and 20 g glycerin. The gum arabic should be in crystalline form since the 

powdered form is difficult to wet. Ingredients are mixed in the given sequence; 

allowing time for one ingredient to be completely dissolved before adding the next. 

The final product is filtered through bolting cloth or glass wool. This medium has 

numerous modifications with names such as Berlese's fluid and de Faure's Fluid. 

(Safety note: Care is to be taken with chloral hydrate compounds; breathing the 

fumes and exposure to the chemical are not recommended. Use only in a hood. 

Additionally, chloral hydrate is a schedule IV-controlled substance so has 

additional requirements for its use and acquisition.)

Specimens may be mounted in Hoyer's solution directly from aqueous solutions or 

may be mounted live. This medium has good optical properties for phase-contrast 

microscopy. To obtain a longer-lasting slide, the slide mount is cured for 48 hours 

to one week at 45oC (113°F) and then held at room temperature for one week 

before sealing. Slides left undisturbed at relatively uniform room temperature will 

cure naturally in about three to four weeks. Temperatures above 45oC will harm 

the medium. Whenever using this medium, care is taken to properly vent fumes.

4.4. Canada Balsam, Permount, Euparal

For permanent slide mounts, Canada balsam is the most commonly recognized 

medium. Other commercially found mountants include Permount® and Euparal. 

They are desirable for museum quality work and for extremely long-term storage 

of authentic materials. All of these are natural or synthetic resin based mountants. 

The major drawback to these materials is timeliness and almost all typically call 

for tedious and often difficult specialized water removal drying techniques to 

prevent clouding. The drying techniques employ a series of gradient alcohols, to 

xylene, to mixed xylene/mountant solutions, prior to mounting in the diluted resin. 

Euparal, an alcohol based mountant, is an exception to the full xylene-based

systems, but it still calls for gradient alcohol fixing stages. For purposes of this 

training, these mountants demand advanced techniques beyond the scope of this 

section, however students should be aware of their usefulness and need for long 

term storage. Note: Use xylene and toluene in vented areas only. Histological 

hoods are recommended if these compounds are used with any frequency.

5. Ringing (Preserving Slides) 

Preservation of slide mounts is accomplished through a technique referred to as 

ringing. Glycerin jelly and Hoyer's solution are primarily media for nonpermanent 

slide mounts. However, with careful preparation and maintenance, they can be 

made to last many years. Once the medium has set (hardened), the coverslip 

edges should be sealed to prevent moisture exchange and to hold the coverslip in 

place. The most common sealants are nail polish or Glyptal, (a flexible sealant for 

electrical connections). The sealant is painted on with a small brush. Round cover 

slips slides are typically centrally mounted on a rotating table (like a Petri dish 
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turntable). The table is spun, and while holding the brush steady overhead, the 

sealant is applied around the edge as the coverslip rotates underneath.

5.1. Clearing (Removal of Interfering Material)

Clearing is a process that clears, removes or dissolves excess proteinaceous, gut 

or optically interfering materials from specimens. Clearing renders a specimen 

more optically usable for mounting, as a cleared specimen often shows more 

detail and surface characteristics. 

Some common clearing agents are dilute (10%) potassium hydroxide, dilute 

(10%) sodium hydroxide, (50-85%) lactic acid (the strength depending on 

sclerotization; stronger sclerotization needs stronger acidic concentration), and 

lacto-phenol, which consists of (85%) lactic acid, phenol and water in the ration 

Specimens cleared in any of these solutions can be rinsed in water and mounted 

directly in Hoyer’s solution or other water-based media.

Since this is a destructive process, do not let the specimen stay too long in the 

solution. Consideration should be given to neutralizing the hydrolyzed specimens, 

to prevent undesirable continued hydrolysis compatibility problems with 

subsequent mounting media.

This technique is not to be confused with bleaching, which removes excess 

pigmentation and coloration. However, some bleaching action may still occur in 

cleared specimens. 

5.2. Staining Specimens

After clearing, most small arthropods are more clearly observed with differential 

staining. Two percent solutions of aqueous acid fuchsin or aqueous lignin pink 

have been used successfully on aphids, mites and Collembola. Lactophenol 

cotton blue solution can be used to stain yeast and mold.

5.3. Specimen Orientation on Slides

Specimens being mounted for examination under a compound microscope should 

be mounted in the middle, oriented with the head or front end directed towards the 

bottom of the slide, and centered under the coverslip. Normally, mount one 

specimen per slide. However, there will be situations when it is acceptable to 

mount multiple specimens on one slide. Examples include similar items mounted 

next to each other to show similarities or differences, sex differentiation in the 

same species, or to demonstrate different orientations of the same species. When 

mounting multiple specimens, keep in mind labeling space and the added 

difficulties of orienting multiple objects without them moving from the desirable 

positions.
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5.4. Labeling Slides

Promptly identify every slide. Label the right side (lot label) with the sample 

number, sub number, date, analyst's name, or initials. If space allows include the 

product, country of origin, lot, or location. Label the left side with the specimen 

identification (to the correct taxonomy level) and include any additional information 

that is useful, e.g., sex, stage, size, and fragment. The identifier’s name or initials 

and date, if different from the preparer, should be on this label. Either side may 

include the mounting medium or ringing material, to facilitate later remounting.

5.5. Storage of Slides

Slide mounts should be stored flat. The mounting media may retain a small 

amount of fluidity if excessive media is used, or if it is not properly cured or rung. 

Over time, gravity may cause the coverslip or the specimen to migrate downward 

if held vertically. Protect the slide from crushing or accidental inversion. 

5.6. Exercise

A. Prepare the following:

1. Glycerin jelly

2. Hoyer's solution 

B. Prepare a wet mount:

1. Make a slide mount of some mold

2. Stain the mold with lactophenol cotton blue solution

Properly mount and label specimens of:

a) Any whole insect which measures less than 3mm

b) Insect fragment(s) elytra, a pair of mandibles, antenna, and legs

c) A mouse hair

d) A mite

5.7. References and Supporting Documents

A. Gorham, J. R. (Ed.). (1978). Training manual for analytical entomology in the 

food industry (out of print, FDA Technical Bulletin No. 2).

B. Martin, J. E. H. (1977). Collecting, Preparing and Preserving Insects, Mites 

and Spiders (The Insects and Arachnids of Canada Series, Part 1). Ottawa: 

Agriculture Canada.

C. Uribe, M., Vail, D. J. (1980). Improved procedure for liquid replacement of 

entrapped air in mammalian hairs. FDA Laboratory information bulletin, No. 

2243.

D. Bullington, S. W. How to mount maggots on microscope slides. Forensic 

Entomology website: http://www.forensic-ent.com/.
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E. Schauff, M. E. Collecting and preserving insects and mites: techniques and 

tools. Washington, D.C.: Systematic Entomology Laboratory, USDA, National 

Museum of Natural History. 

F. Horobin, R. W., Kiernan, J. A. (Eds.). (2002). Conn's biological stains: a 

handbook of dyes, stains and fluorochromes for use in biology and medicine

(1st ed.). Abingdon, UK: Bios Scientific Publishing, Ltd.

6. Collecting and Preserving Whole Insects and Arthropods

This section will provide basic instruction for collection and preservation techniques for 

whole insect and arthropod specimens found in regulatory samples and those specimens 

collected for the laboratory's authentic reference collection. A filth analyst does not 

routinely collect insects in the traditional sense (for example with a butterfly-net), and we 

rarely preserve them as a pinned specimen. Although most of the laboratory’s work will 

deal with insect fragments, whole specimens are first discussed. Analysts do find whole 

insects, (and in some cases, whole live insects) in regulatory samples. These insects 

are evidence that are properly preserved for courtroom presentation. If the specimens 

are collected for use as authentics, as reference materials and/or direct comparison with 

unknown specimens, only the best museum preservation techniques are applied. 

6.1. Recommended Equipment

A. Vials (1/2-dram, 1 dram, and/or 2-dram sizes, glass with screw-on cap with 

polyseal® cone insert recommended)

B. Sieves (U.S. Standard #8, #20, #40 and pan; a "collar" is also recommended)

C. Berlese Funnel and/or Tullgren Funnel

D. Aspirator (recommend "exhalation" style or "inhalation" style with in-line filter)

E. Artist's camel hairbrush (recommend #2 or #3)

F. Jeweler's or needle point forceps

G. Flexible steel forceps

H. 5X Magnifying lamp

I. White plasticized butcher paper

J. Various sized white or stainless-steel pans

K. Recommended Reagents

L. 95% Ethanol/Isopropanol

M. Glacial acetic acid

N. Kerosene

O. Dioxane ("Triton X-100" or "Tween 80" may be substituted)

P. Formalin (40% formaldehyde)

Q. 10% KOH, 10% NaOH, 50-85% lactic acid, or 2:1:1 lacto-phenol

R. Distilled Water
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S. Ethyl acetate

Note: There are commercial killing fluids, clearing agents, and preservatives. If 

these commercial products are used, the above list of reagents could be reduced.

6.2. Collecting Insects

Insects found in regulatory samples by macroscopic examination or microscopic 

examination are collected and preserved as evidence. In some cases, these 

insects may be alive. The method used to extract insects from a product will vary 

based on the composition of the product. Although a method may be dictated, 

there are some "traditional" entomological methods that may be of value.

6.3. Sieves

Insects may be removed from some products by placing the product in nested 

sieves (generally, a #8 is placed over a #20 or finer.) If the product being sieved 

needs to be contained, a collar or lid is added to the uppermost sieve, and a pan 

to the lower. Generally, the product is placed in the uppermost sieve and shaken, 

causing insects and other foreign material to fall onto the lower sieve and into the 

pan. Conversely, if the filth analysis is looking for larva in flour, the larva is 

retained in the uppermost sieve, and the flour passes through to the pan below.

6.4. Berlese/Tullgren Funnel

The Berlese Funnel, originally developed by Antonio Berlese to remove live mites 

from leaf litter, is an option for removing live insects and mites from reasonably 

dry leafy food materials (i.e., taro or palm leaf). The device is a large funnel with a 

coarse (U.S. STD #2, #4, or #8) woven metal screen inserted above the neck to 

hold back the product yet allow insects to pass through into a jar of preserving 

fluid. Insects and mites will be driven down by a heat source (generally a 40–60-

watt light bulb) in a lid that covers the mouth of the funnel. The Tullgren Funnel, a 

modification using a series of baffles rather than a screen, has been used to 

remove insects and mites from dry powders or granules that are too small for a 

typical Berlese. A workable Berlese apparatus is described in LIB 883 and some 

useful modifications in LIB 1816. See also J. Assoc. Off. Anal. Chem., 55(1), 51-

56, 1972.

6.5. Aspiration

An aspirator may be used to vacuum up small insects and/or mites. One type of 

aspirator involves inhalation or a vacuum pump to draw the insects into the 

container. Another type uses exhalation or an air pump to collect the specimen. It 

is recommended if an inhalation type aspirator without a vacuum pump is used, 

insure that there is an in-line filter between the user and the collection chamber, 

where the substrate, insects and/or mites are harmful (from microbes, spores, or 

chemicals). A simple aspirator is described in LIB 1637 and in J. Assoc. Off. Anal. 
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Chem., 55(1), 51-56, 1972.

6.6. Killing Insects

A. Freezing

When dealing with live insects or where insects are in dry products, freezing is 

the easiest and safest technique, provided the analyst allows time for proper 

penetration of the cold temperatures to the center of the product or exhibit. 

This can be checked with a thermometer. During the freezing process, insects 

are driven away from the cold temperatures and into the center of the product 

or exhibit. Care is taken to maintain the specimens in a frozen condition, until 

they can be examined. This is needed because allowing them to come back to 

room temperature for extended periods of time (e.g. a day), will cause 

discoloration and damage to a specimen. Freezing does not stop enzymatic 

and gut microbes from continuing their actions inside the warmed specimen. 

Once frozen, the specimens can be manipulated and picked out of the exhibits 

for preservation as identified below.

B. Killing Fluids

Immersing a larva in hot (near boiling) water is the best killing fluid for larvae in 

a laboratory situation. This treatment stops enzymatic and gut microbe action 

and distends the larva. The larva is then removed and put into cold water, or 

directly into 70% alcohol. Killing fluids such as KAAD (a mixture of kerosene (1 

part), ethanol (7-10 parts), glacial acetic acid (2 parts), and dioxane (1 part)) 

may be used to kill and "fix" a larva to avoid discoloration or distortion. The 

killed larvae will need to be removed from the KAAD solution and transferred 

to a preservative within 24 hours. 

Safety Note: Dioxane may become unstable if stored more than 12 months. 

Use of "Triton X-100" or "Tween" is recommended instead.

C. Fumigants

Generally used for adult insects, a fumigant is a substance that generates a 

poisonous gas. 

Fumigants of choice in a filth laboratory are quick, easy to use and lethal to 

insects and mites, but relatively safe for the analyst. 

Gas fumigants like CO2, can be used to temporarily stun insects to slow them 

down for photography. 

Liquid fumigants are more common, (e.g., ethyl acetate); a small amount of the 

liquid can be placed on an absorbent pad and placed in an airtight container or 

killing jar. Note: Cotton balls are NOT recommended as the absorbent pad, 

as insects may become entangled in the fibers.
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Solid form fumigants (e.g., paradichlorobenzene) may be used; the solid form 

is held inside a screened chamber built into the lid of the killing jar. (Note: 

Paradichlorobenzene is a slow acting fumigant.) 

In both cases, the insect is placed into the airtight container with fumigant and 

asphyxiated.

Safety Note: Numerous fumigants are used, but all generally have higher 

human health risks. Regardless of the fumigant used, the analyst works in a 

well-ventilated area or fume hood; care should be used to avoid breathing the 

fumes. 

Caution: Ethyl acetate and other fumigants may have solvent actions that may 

dissolve the container, lid, or seals. Always check compatibility first, before 

using solvents. 

6.7. Preserving Insects

A. Preservatives

Most specimens are stored in poly-cone capped glass vials of 70% ethanol, 

others (soft bodied maggots) in a 1:1 of 70% ethanol and glycerin, some 

(insect eggs) in pure glycerin, and still others (pigmented soft bodied) in 

commercially prepared solutions such as Pampel's or Kahle's. Mites are 

generally stored in 70% ethanol, lactic acid, or in AGA (87 parts 70% ethanol, 

8 parts glacial acetic acid and 5 parts glycerin). 

The analyst is reminded that some preservation agents, like formaldehyde, are 

also fixatives. They may actually cause damage to the specimens through 

subtle color loss or by tissue shrinkage from the denaturing of the proteins. 

Analysts are encouraged to ask the senior analyst questions as the need 

arises, or consult the references cited below or in the Reference Appendix. 

B. Pinning and Air Drying

Pinning, spreading, and air drying are techniques commonly used for whole 

adult insects that are generally placed in museum boxes. Smaller insects (too 

small to be properly pinned) can be placed (glued) onto triangular shaped 

paper points, the points of which are then mounted on pins. This technique is 

commonly used for authentic specimens, where handling is minimized and 

easy access to dry specimens is needed. Normally, given the size of most 

stored product insects and their brittle nature when pinned, we do not preserve 

regulatory sample specimens by pinning them, as the specimens are too 

fragile to stand much handling, especially where handling is out of the 

analyst’s control. However, some flies and thin cuticle specimens are better 

preserved on pins and careful packaging measures would be needed to 

assure that the pinned regulatory specimens are not damaged from dropping, 
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crushing, or shaking of the finished exhibits.

See Schauff’s document for details on pinning specimens for the best results.

6.8. Labeling Preserved Specimens

A. Every specimen (or group of like specimens) collected are to be labeled 

immediately. The value of a specimen is seriously diminished if the specimen 

is left unlabeled or incompletely labeled. 

B. Depending upon the type of preservation method used, labels should be on 

acid-free 28-60# index-weight paper using indelible ink or laser printing. Inkjet 

printing is not usable for wet vialed specimens. The labels can consist of one 

or more (broken up into sample collector’s or lot label(s) and identification label 

as grouped below) labels and they are placed in the vial or dish or on the pin 

with the specimen(s). The complete label should include the following 

information: 

Group 1

• Sample number (Lot)

• Sub number

• Date collected (or date extracted by analyst)

• Collector's or Analyst's name or initials 

• Type of preservation fluid (if applicable)

Group 2

• Product

• Country of Origin

Group 3

• Identification of specimen

• Who identified specimen and date

6.9. Supporting Documents

A. AOAC official methods of analysis (current Ed.). Gaithersburg, MD: AOAC 

International.

B. U.S. Food & Drug Administration, Center for Food Safety and Applied 

Nutrition. (1998). Macroanalytical Procedures Manual (FDA Technical Bulletin 

No. 5). [Originally released in print by FDA in 1984 by Olsen, A. R. (Ed.), 

Knight, S. A. (Tech. Ed.), Ziobro, G. C., Ph.D. (Assoc. Ed.)] 

C. Brickey, P.M., Gecan, J.S., Thrasher, J.J., Eisenberg, W.V. (1968). Notes on 

Microanalytical Techniques in the Analysis of Foods for Extraneous Materials. 

Journal of the Association of Official Analytical Chemists, 51, 872-876.

D. Brickey, P. M., Thrasher, J. J., Vazquez, A. W. and Gecan, J. S. (1972) Notes 
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on Microanalytical Techniques in Analysis of Foods for Extraneous Materials, 

Part (2). Journal of the Association of Official Analytical Chemists, 55(1): 51-

56.

E. Gorham, J. R. (Ed.). (1978). Training manual for analytical entomology in the 

food industry (out of print, FDA Technical Bulletin No. 2, HHS Publication No. 

(FDA) 77-2086). 

F. Olsen, A. R. (1973) Apparatus and Technique: Insect Filth Exhibits. FDA 

Laboratory Information Bulletin, No. 1637.

G. Stehr, F. S. (1987). Immature insects (Vol. I). Dubuque, IA: Kendall/Hunt 

Publishing Co.

H. Schauff, M. E. Collecting and preserving insects and mites: techniques and 

tools. Washington, D.C.: Systematic Entomology Laboratory, USDA, National 

Museum of Natural History. 

I. Vazquez, A. W. Examination of bulk samples of food products for infestations 

with living insect and mites using the Berlese funnel technique. FDA 

Laboratory Information Bulletin, No. 883. 

J. DeCamp, P. W. Modifications of the Berlese funnel for filth work. FDA 

Laboratory Information Bulletin, No.1816. 

K. Biological Survey of Canada. (2001). Brief: Label data standards for terrestrial 

arthropods. http://www.biology.ualberta.ca/bsc/briefs/brlabelstandards.htm. 

6.10.Glossary

Collar - An old sieve with the weave removed; extends the height of a sieve.

Fix - Chemical process that prevents or minimizes pigment discoloration, loss 

and/or tissue distortion, to preserve in place.

Overs/Throughs - After a sieving operation, anything retained on top of the screen 

is referred to as overs, those that pass through, as throughs. 

6.11. Exercises

A. Prepare the following solutions:

1. 60% ethanol

2. Glycerin-Alcohol (1:1)

3. A.G.A.

4. 10% KOH

5. KAAD

B. Kill and preserve the following, including a label:

1. adult stored product beetle (for regulatory sample, for authentic use)

2. larval stored product insect (for regulatory sample)
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3. a mite 

4. a cockroach

5. a spider

7. Taxonomy

7.1. Objective

This exercise will provide background in taxonomic principles and zoological 

nomenclature.

7.2. Discussion

Taxonomy is the science of giving names to organisms in order to classify them. 

The system used to name organisms was designed by the 18th century botanist, 

Linnaeus. It consists of a basic name for each kind of organism and a hierarchy of 

categories for grouping similar kinds of organisms together. Identifying an 

organism, then, is simply finding the proper name to call it.

The basic name or scientific name of an insect or any other biological entity 

consists of two Latin or Greek based words. This two-name system is often 

referred to as binomial nomenclature system. Each name combination is unique; 

there is no duplication of names under the system of Linnaeus. Scientific names 

also have a strict format. To check the validity of a scientific name and see the 

phylogenetic relationship, see the Integrated Taxonomic Identification Service at: 

http://www.itis.gov. 

The format is as follows:

a) The scientific name is always underlined or italicized.

b) The first letter of the first word of the scientific name (the genus) is 

capitalized.

c) The second word (the species) is not capitalized.

d) Immediately following the scientific name is the name of the scientist 

who originally named the species.

The basic unit of the system is the species. Next is genus, a group of species that 

is closely related phylogenetically (by ancestry). The system continues building 

larger and larger categories, each indicating a more remote phylogenetic 

relationship. The general progression of categories is given below with examples 

of each category.

a) Genus: Apis = honeybees

b) Family: Apidae = bees in general

c) Order: Hymenoptera = bees, wasps, and ants
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d) Class: Insecta = all insects

e) Phylum: Arthropoda = insects, crustaceans, spiders, etc.

f) Kingdom: Animalia = animals

Apis mellifera (Linnaeus) is the scientific name of the insect commonly called the 

"honeybee." To be sure however, that this is the correct name for the insect in 

question, a test is applied. For insects, and most other organisms, the test 

compares a specimen to either a validated (or authenticated) specimen whose 

identity is assured, or to compare the specimen to a written description of the 

validated specimen. In FDA work, both methods are used to confirm the identity of 

an insect.

In entomology, "validated" specimens are called "type" specimens. Type 

specimens are specimens that have been designated as examples of a particular 

species by the scientist who originally named the species (the author). In FDA, 

"authentic" specimens are similar to type specimens in that they have been 

verified by experts to be good examples of a particular species of insect.

Technical descriptions are written, detailed descriptions of a type specimen. Since 

most type specimens are housed in large museum collections and not provided

for casual examination, published technical descriptions are needed and useful. 

Although we may not have the original author’s description, most district 

laboratories will have literature containing technical descriptions of common foodinfesting insects, which we use to make identifications.

7.3. Assignment

A. Examine the laboratory collection of authentic whole insect specimens.

B. Determine the books or journals that contain technical descriptions of storedproduct (food-infesting) insects.

7.4. Questions

A. Give a literature reference for a technical description of the coffee bean weevil, 

Araecerus fasiculatus (DeGeer).

B. What is the full scientific name, including author, of the confused flour beetle? 

C. Based on the scientific names of the insects in "a" and "b," would one expect 

them to be like each other or dissimilar?

8. Identification Keys

8.1. Objective

The trainee will learn how to use identification keys.
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8.2. Discussion

A key is a guide to the identification of some specimen. Although there are various 

kinds of keys, for each kind of key attempt to arrange the characteristics of a 

particular group of specimens into an orderly format with the intent of guiding the 

analyst through a series of observations until every species but one is eliminated. 

Keys are not infallible, final, or all-inclusive. Keys provide a tentative answer; the 

final identification of a specimen depends on direct comparisons with technical 

descriptions and authentic specimens. The value of a key tells the analyst which 

technical descriptions and authentic specimens to look at first and helps narrow 

down the search in a structured fashion. Keys can be grouped into two broad 

categories: single access keys and multiaccess keys.

A. Single access keys are keys with a fixed number of sequenced identification 

steps, each with multiple alternatives. By making a single choice you 

determine the direction of your next step. The advantages to these types of 

keys is that they use many reliable characteristics which can be used in 

specific groupings. Some of the disadvantages to these types of keys: you 

can get ‘lost’ in the terminology if you are not familiar with it, if a characteristic 

is missing it may become difficult to identify your specimen, and finally the key 

is only as good as it is designed for the specimens or species in question. 

Common single access keys are dichotomous keys and pictorial keys.

B. Dichotomous keys: the most common entomological identification keys. They 

can be of two types: Diagnostic (artificial) or Synoptic (natural). Diagnostic

keys are convenient and reliable, they can incorporate geographic distribution 

or even habitat preferences, and contain artificial groupings. Synoptic keys 

follow taxonomic classifications, often incorporate difficult characteristics to 

observe, and difficult terminology. The basic composition of both keys are a 

series of pairs of mutually exclusive statements about the specimen being 

identified, called couplets. By choosing the statement that best satisfies the 

specimen being observed, the analyst is directed to another couplet. This 

process continues until a couplet is reached that indicates a name (typically to 

the species level). That species name is the most likely identification of the 

specimen. However, FDA demands the analyst confirm this name 

identification with authenticated material, or at least to a literature description.

There are variations on the basic dichotomous key format. Sometimes a key 

will include a triplet (three mutually exclusive statements) or even a quadruplet. 

In these cases, the analyst still chooses only one statement. As a kindness to 

identifiers who occasionally backtrack, lengthy keys will often provide 

parenthetical reference to the previous couplet immediately following each 

couplet number. 
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C. Pictorial keys: although dichotomous keys are often illustrated, pictorial keys 

rely on illustrations to guide the analyst towards an identification. Using a 

series of illustrations with terse legends, the pictorial key guides the analyst by 

directional arrows, much like an agency personnel table of organization.

D. Multiaccess keys give the user the freedom to pick multiple characteristics 

which are convenient to evaluate. These keys can be printed in a tabular or 

matrix type format. They can even be designed as computer generated 

interactive keys. The advantages of these types of keys: they can be used on 

damaged specimens, not limited to a few characteristics, easily updated when 

new specimens are added to the key and can have computer aided 

identifications with hyperlinks to authoritative text and images. The 

disadvantages to these types of keys are that they can become cumbersome 

in selecting the multiple characteristics and takes practice in knowing how to 

select the various characteristics. Examples of multiaccess keys are tabular 

keys and interactive keys.

E. Tabular keys are useful for distinguishing members of a small group of similar 

appearing specimens. A table compares the distinguishing characteristics of 

each specimen. Frequently, a single characteristic may be duplicated, or 

characteristics may be overlapping, but each species will have a unique total 

set of characteristics that will distinguish it from others.

F. Interactive key with the aid of a computer, the analyst will select a series of 

characteristics, then the computer gives the best choice (s) based on the 

characteristics which were selected.

Hint: Experience shows that the analyst doesn’t always get the answer expected 

when using keys, especially if the keys are complicated, or some of the 

characteristics are ambiguous. To help remedy mistakes, and keep from going 

back to the beginning, on a separate piece of paper, try keeping a running list of 

choices, i.e., 1, 3, 4, 17, 18, 23, 30 etc., and circle the couplets that are major 

breaking or grouping points in the key. Also, put a question mark (?) above the 

ambiguous or questionable couplet choices. This will allow one to retrace steps 

and review decisions. This will save a lot of time, especially in unfamiliar territory. 

Other techniques which can be helpful to confirm the specimen is keyed out 

correctly: send the specimen through a second key, compare the specimen to the 

authoritative literature and authentic specimens or have a second analyst send 

the specimen through a second key. 

8.3. Assignment

A. Examine one or more examples of each of the four types of keys: 

dichotomous, pictorial, tabular and interactive. 
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B. The trainer presents a set of objects. Construct a simple dichotomous key.

C. The trainer provides an unknown specimen(s). (Beginner Level). Identify the 

specimen(s), using each of the four types of keys.

9. Digital Photography and Photomicrography

9.1. Objective

This procedure applies to all analysts using digital cameras or scanners for photo 

documentation of evidence/sample casework. This procedure is written for filmless photography with images stored on magnetic or optical (CD) media.

9.2. Definitions and Acronyms

Aperture - Circular hole in the camera that controls the amount of light reaching 

the sensor or film emulsion.

Blooming - The bleeding of signal charge from extremely bright pixels resulting in 

over-saturated pixels. “Blooming” in digital photography compares with overexposure in film photography.

BMP - Bitmap (.BMP file extension): this is a standard image file format for 

Windows®.

GIF - Graphic Image Format (.GIF file extension): gif format is what is termed as a 

"lossless" compression format. This format is referred to as a "paletted" image or 

a 256-color image. It is limited to 256 colors.

JPEG - Joint Photographic Experts Group or jpeg (.JPG file extension) this format 

is a lossy compression format. The higher the compression ratio the more the 

pixelization or "blockiness" occurs.

Cropping - The act of cutting out a portion of a digital image for blow-up/display as 

a separate image. 

Photo documentation - The process of recording images representative or 

demonstrative of a sample or object.

PNG - Portable Network Graphics (.PNG file extension): this file format is an 

alternative to the GIF (Graphic Image Format) format.

TIFF - Tagged-Image File Format (.TIF file extension): a lossless bitmap image 

format supported by virtually all paint, image-editing, and page-layout applications.

9.3. Discussion

A. The replacement of film emulsions with digital sensors for imaging brings many 

conveniences to laboratory photo documentation. Digital imaging provides an 

instant review of composition for quality and facilitates archival through modern 

digital storage techniques.
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B. Due to variations in equipment from laboratory to laboratory, the trainee learns 

to use equipment with the guidance of an analyst familiar with its use. As with 

conventional film photography, it is important to pay attention to the 

composition, lighting, and overall contrast of a scene when photographing 

samples. A well-composed picture has the following qualities:

1. The subject is positioned and contrasted to attract the viewer's 

attention. 

2. Important details are highlighted. 

3. The subject is magnified so it is not lost as an inconspicuous speck in 

the photographic field. 

4. Sample identification is included in the scene, if possible

5. When possible, a scale of known distance intervals is included in all 

images. If not possible, all photographic conditions, including 

magnification and lenses used, are recorded and a calibrated bar 

marker inserted in the photomicrograph. 

C. For digital photographs or scans, the following equipment (if used) should be 

documented on the worksheet or a separate attachment:

1. Brand and Model of the Digital/Video camera 

2. Image Capturing device

3. Scanner type 

4. Printer Brand and Model

5. Microscope(s) 

6. Illuminator(s) 

7. Lens Brand and Model

8. Focal Length and Aperture Site

9. Exposure time (if applicable)

10.Filters (if applicable)

11.Image Storage (location, file names, etc.)

D. Digital cameras often have numerous settings that can be set to automatic or 

manual modes of operation. Such features will usually call for an SOP, 

tailored to the equipment, that outlines which settings should be left as 

automatic and which should be set manually to maximize quality, repeatability, 

and to facilitate in archival.
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E. Original images are not to be modified. When an image is modified 

(contrast/color adjustments, sharpening, or cropping for instance) a copy of the 

original file as obtained from the camera is kept. The new, modified file should 

be named by appending the word “modified” to the original file name. It is 

vitally important to document what adjustments were made to the original file in 

creating the modified one. A print of the original image alongside or below the 

modified image may not be needed if the analyst has safely stored the original 

file.

F. Both the modified and unmodified files should be written to media for 

submission with the analyst report. Each print of an image should be identified 

with the image name, description, and variables recorded at the time of 

capture, as previously described.

9.4. Considerations

A. Printer

Only paper recognized by the printer manufacturer as fade resistant and fade 

resistant ink (cartridges) are used. Printer paper stack should be covered when 

not in use. Printer heads cleaning is conducted as needed. (See instructions with 

printer manual). 

B. Camera

Mount the camera on a tripod and set the exposure manually by adjusting the 

aperture to maximize depth of field. Set the image quality to maximum and turn 

off (or manually set) as many automatic features as possible to increase 

repeatability/consistency. In-camera sharpening, white balance, and use of a 

flash (as well as flash intensity) should be fixed. Choose a lossless file format if 

possible; an option to produce a TIFF file is often provided and is desirable seeing 

as how the TIFF format uses a lossless compression scheme (or no compression 

at all).

C. Scanner 

Output Resolution should be set to a minimum 150DPI*. Sharpening level should 

be set to low or none (some scanners automatically sharpen a scan). Output 

Dimensions: the file format chosen should use lossless compression, or none, as 

described in step (a).

* The concept of DPI (dots per inch) is often confused with camera, or scanner, 

resolution. The two terms are not interchangeable. Resolution as it pertains to 

digital imaging refers to the number of pixels captured by the sensor while DPI is a 

display (i.e., print) characteristic which indicates how many of those pixels are 

displayed per inch. Thus, the DPI of an image may be adjusted to no end without 

affecting the amount of data (pixels) in an image. The human eye is incapable of 
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perceiving more than 340 pixels per inch (approximate) at a viewing distance of 

10 inches. In practice, a print with a DPI of 150 pixels (dots) per inch or greater 

will appear acceptable. As an example, the analyst is to use the maximum 

resolution of a Nikon Coolpix 4500 digital camera to determine what the DPI of an 

uncropped image should be set to for printing on an 8.5x11inch sheet of Photo 

Quality Inkjet Paper:

The resolution of an uncropped Coolpix 4500 image is 1704 pixels wide x 2272 

pixels high (using a portrait aspect ratio). Due to printer margins, we could 

assume the maximum printable height of the image on the paper will be 10 

inches. The equation describing the relationship between resolution and DPI in 

the vertical dimension is thus 2272/n = 10. Solving for n, we see that 

approximately 228 DPI is needed. If we carry this same DPI over to the width, we 

see that the image will be 1704/228 = 7.47 inches wide. Thus, the solution is to 

adjust the DPI of the image to 228 before printing. This is greater than 150DPI so, 

for most subjects, this print should appear acceptable. This 150DPI rule of thumb 

is common in the graphics profession.

Adjusting the DPI as in this example does not change the amount of data in the 

image since no pixels are being added or removed. By default, some image 

editing applications will inappropriately “resample” the image (i.e., interpolate to 

increase or reduce the number of pixels) when given the command to change the 

DPI. Ensure that this is not occurring by checking the number of pixels in both 

dimensions before and after the DPI adjustment.

9.5. Acquiring and Saving Images

A. A scale (ruler) should be positioned in the field of view when a camera or 

scanner is used to capture an image. A proportional scale may then be used 

in the worksheet to provide distance and size information.

B. Avoid “blooming” an image by checking the exposure prior to capture. For 

many scanners this entails running a test scan so as to allow an automatic 

calibration. The reflectance properties of some surfaces may need manual 

exposure adjustments. For digital cameras, ideal exposures are often a result 

of experience or trial-and-error. The LCD preview screen on the rear of most 

digital cameras can provide some indication of the effectiveness of an 

exposure, but such displays are un-calibrated and are highly dependent on 

ambient lighting or brightness/contrast controls. As a result, apart from 

providing a check for sharpness they may be far less useful than at first 

imagined. One exception is the display of tonal or color information in the form 

of histograms by some cameras. Such histograms can provide detailed 

exposure information when interpreted correctly.

C. Images are saved on removable media. Image storage on the local hard disk 

should only be temporary; there is no need to retain copies of images on a 
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local computer or server once they have been written to the media to be 

submitted with the worksheet (multiple analysts doing so would quickly 

overwhelm the storage capacity of the server or perhaps even a central 

computer designated for managing digital photography). A preferred medium 

for storing images is the CD-R disk. Such disks can be “closed” after being 

written to and thus offer an unalterable means of storage with the added 

benefit of tolerance to environmental conditions that would otherwise destroy 

data on common magnetically based media.

9.6. Printing

Images are to be printed on photographic quality paper using the printer’s highest 

quality and resolution. Often the best paper for a given situation will be branded 

by the printer manufacturer itself. Specialty papers should be covered when not in 

use. Printer head cleaning is conducted as needed with periodic checks to 

counter nozzle clogging.

A photograph does not replace written results and descriptions. Normally FDA 

does not require the photographic documentation of negative results. In short, 

use discretion. A photograph can be particularly useful, for example, when it can 

demonstrate the lack of an item that should be present in a product.

9.7. Assignment

Read a suitable introductory text; Low, A. (1991). Introductory computer vision 

and image processing. New York: McGraw-Hill Book Co. Ltd.

10. Dissection

This stage of the training program is the dissection of insect specimens to learn insect 

morphology and recognize insect fragments. Practice is needed for the development of 

dissection skills, and the following guidance will enable the trainee to begin properly.

10.1. Dissection Equipment

A. Dissecting microscope. Dissecting widefield microscopes are the best choice.

B. Probes. Standard dissecting probes, either straight or bent tip, are purchasable

from most biological supply companies and are acceptable for general use. 

Microprobes are probes made by inserting a minuten or #2-3 insect pin into the 

end of a thin wood dowel, such as a cotton swab stick. A spatula-type 

specimen lifter may be fashioned by flattening the tip of a microprobe, and a 

micro-scapula can be fashioned from a flattened # 2 pin which is then 

sharpened on a wet stone or fine grinding wheel. 

C. Forceps. In addition to tweezers, a pair of fine-tipped jeweler's forceps 

(Dumont #3 or 5, or equivalent) are needed for handling small objects, and a 

pair of coverslip forceps. The tips of the jeweler's forceps should be protected 
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against damage when not in use by a sleeve of small diameter rubber or 

plastic tubing. Always keep a spare pair on hand.

D. Additional equipment. Insect pins, a fine (#11) scalpel, and a pair of fine tip 

surgical or iris scissors will be useful. Small hotplate (for boiling solutions), an 

alcohol lamp (if open flames are allowed), and a slide warming plate (50° C.). 

Disposable petri dishes of various sizes (100X10, 100X15, and tight-fitting lid 

65X10) are needed and lined (S&S # 8 ruled filter paper) and unlined filter 

papers are needed. A No. 2 or No. 3 artist’s camel hair paint brush is an 

excellent tool for picking up insects without damaging them.

E. Glassware. A wax bottom dissecting dish is useful as an arena (an alcohol 

insoluble pinning board) for the dissections. To make one, melt paraffin/bees 

wax in the bottom of a small glass petri dish to the depth of approximately onehalf of the height of the dish, and cool. After a period of use, the paraffin/bees 

wax surface may become rough and full of holes. Simply remelt the 

paraffin/bees wax and cool again to obtain a smooth surface.

F. Other useful items. Syracuse watch glasses (2 - 5/8" diameter), Coors 

porcelain casseroles (size 00) or 10-25 ml beakers with glass watch glass 

covers for operations that call for heating specimens in liquid, and various 

shallow watch glasses for use as lids. 

Note: Clean white beach sand can be wetted in a petri dish and used as a 

formable orientation dish for wet specimens (to orient specimens for different 

views under the stereomicroscope).

10.2. Dissection Techniques

Most dissections are performed in liquid (usually alcohol) to prevent the specimen 

from drying and to better control the movement of small pieces of the specimen. 

Dry dissection can be performed in shallow petri dishes with filter papers wetted 

with 50/50 glycerin alcohol; however, the specimens should be softened by gentle 

boiling in pure water prior to the dissection.

Dissecting a specimen essentially dismantles it. Pulling off or teasing apart is the 

easiest technique and works well for most large appendages such as antennae, 

legs, elytra, wings, or even smaller exposed appendages such as labrum and 

labia (top and bottom mouth parts). The insect's body is firmly held at a point near 

the appendage while the appendage itself is pulled off or severed at its base using 

forceps and the microprobes. Leg and antennal segments may be separated in 

the same manner.

Main body plates or structures (sclerites) can be broken free by judicial application 

of pressure from a probe or forceps. Ventral abdominal plates may crack however, 

so it is advisable to cut them free along the sides and then separate them. Cutting 

the plates free, especially if only a few straight cuts are needed gives more control 
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than breaking the plates.

Mouthparts and other small structures like genitalia can usually be teased free 

using probes. Adhering excess muscle and visceral tissue should also be teased 

away. Tissue that cannot be teased free can be macerated with a caustic solution 

(5-10% potassium hydroxide) but the specimen is thoroughly rinsed in distilled 

water before mounting. Gentle heat will speed this maceration process.

Slide mounting media are described in Section 4. If a specimen is very thick, it will 

cause the coverslip to rest unevenly. This can be remedied by propping up the 

coverslip with bits of glass (broken coverslip fragments), nylon fishing line, or 

other material strategically placed in the medium before laying on the coverslip.

Finished slides are labeled, ringed, and then stored flat, as the specimen is not 

fixed to the slide surface. Remember to mount only one specimen/fragment/hair 

per slide.

11.Insect Morphology

11.1. Objective

The trainee should review and become familiar with the "Micro-Analytical Biology 

Workbook for Food Sanitation Control Analysts," Volume I (1981) by Don J. Vail, 

Jr., FDA Atlanta Regional Office, Atlanta, GA. or materials provided by the trainer. 

An excellent, additional approach to learning insect morphology is described in the 

exercise below.

11.2. Discussion

To learn basic insect morphology, the analyst needs to recognize the various 

parts, which make up a particular insect. The analyst will need to use their

dissection techniques learned in section 10 to aid in this process. 

11.3. Assignment – Insect morphology

This exercise is geared towards learning insect morphology and serves as a 

useful tool for later analyses. The trainee sets up a series of petri dishes, each 

petri dish representing a particular insect or series of insect fragments from a 

group of insects. For example, the analyst will label a dish for Tribolium 

confusum, and another larger dish for stored product beetle mandibles, with adults 

on one side and larva on the other.

The trainee will perform the following two step process:

A. Step One:

The trainee performs a series of dissections on various stored product beetles. 

These are referred to as Known Species Plates. Using the airtight lid small 

petri dishes with the bottom lined with glycerin/alcohol wetted filter papers, the 
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trainee labels the lid in indelible ink with the species name and source 

information or lot number (one species per plate.) The trainee places 3-4 intact 

whole adult insects of that species on the wetted filter paper and begins their 

dissections, one insect at a time. The dissection serves as a learning tool for 

morphological terminology and how the insect comes apart. This information 

will prove useful towards understanding what the fragments look like when 

separated from the body. (Note: this exercise will also provide some insight 

into what may happen in a milling, flour making operation when the insect is 

crushed or broken up). Complete the dissections of a group of 2-4 identical 

specimens; some species may show some size variation or will call for several 

dissections to get intact fragments representing all body exoskeleton parts. For 

example, some beetles (like the rice weevil) have closed coxal cavities which 

prevent the analyst from getting either a complete prothoracic fragment or 

complete coxae without sacrificing one fragment for the other. When this 

segment is completed, then dissect the mature larva on the same plate, 

keeping the fragments segregated.

Upon completion, these plates can be used throughout one’s career for 

additional dissections or for reference.

B. Step Two:

Step Two consists of a series of Fragment Plates, where the trainee uses the 

100 X 10 petri dishes and labels the lids describing the morphological 

fragments, e.g., all stored product beetle mandibles on one plate. Once Step 

One dissections are completed, the trainee will code in India ink each line of a 

S&S #8 ruled filter paper with numbers or the actual species name of the 

insects dissected. When dry the labeled filter papers are placed in the petri 

dishes and wetted with glycerin/alcohol. Then species by species, the trainee 

places the corresponding fragment on the species line with a representative 

number of fragments present to show size variations, or larval/adult conditions 

if possible. 

When the analyst completes the plates, the plates serve as a rapid 

identification tool for unknown species, but clearly identifiable morphological 

fragments. The analyst can pick the fragment up on the end of a probe and

move the fragment next to the known fragments on the plate, comparing size 

and gross shape or character. Narrowing down the final identification without 

having to do a slide-by-slide analysis reduces analytical time. 

Note: The trainer identifies the common stored product insects the trainee 

should work with in preparing the initial plates. The trainee can and should set 

the plates up for a lifetime of learning; the plate collections will take 

considerable time to develop and may never be completely done throughout 

the employee’s career.
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The object of this section is simply to set up the system in which the trainee 

can systematically learn insects. It is difficult and tedious work and has a very 

steep learning curve relying heavily on memorization skills and the ability to 

perform minute dissections under the stereomicroscope. The initial training 

time should be limited to one or two weeks to learn the approach alone, with 

additional time granted as needed. The time for these exercises needs to be 

granted to the employee as continuing education and quality assurance 

throughout their career. For initial training, the trainees should not attempt to 

memorize the fragment to species as Section 12 covers this aspect. However, 

if needed, and if the trainee already possesses a strong entomological 

background, these two sections can be combined and taught as one section. If 

done effectively, the trainer/trainee may also spend some time learning more 

about the specific insect’s biology. Descriptive literature accompanies each 

dissection. 

11.4. Assignment – Dissecting insect genitalia

This exercise is geared specifically towards learning how to dissect genitalia from 

an insect. The genitalia of adult Coleoptera and Lepidoptera are important 

characteristics used in their identification. It takes lots of practice to tease out 

genitalia and mount them on a microscope slide for identification. 

A. The trainer supplies similar beetles to the trainee. The trainee will dissect out 

the genitalia from the beetles. Then mount the genitalia on a microscope slide 

and observe the differences in the genitalia. Some examples to use: 

Oryzaephilus mercator and O. surinamensis; Stegobium paniceum and 

Lasioderma serricorne; Cryptolestes sp. and Ahasverus advena

B. The trainer supplies the trainee with at least two different species of moths. 

The trainee will dissect out the genitalia from the moths. Then mount the 

genitalia on a microscope slide and observe the differences in the genitalia. 

Examples of some moths to use: Plodia interpunctella, Sitotroga cerealella, 

and Ephestia figulilella. 

12.Fragment Recognition

12.1. Objective

The purpose of this exercise is to learn how to distinguish microscopic fragments 

of insects, and how to segregate and identify them from other plant tissues on the 

plate.

12.2. Discussion

To accurately distinguish microscopic fragments, the analyst needs a thorough 

knowledge of insect morphology, and access to a reference collection of 

authenticated fragments. This knowledge is a prerequisite (see AOAC 970.66) 
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before completion of any filth analysis.

The previous insect morphology exercises were the starting point for learning and 

accumulating reference material. The learning process is never truly finished; the 

analyst should never pass up a new dissection opportunity to accumulate further 

knowledge and additional reference material.

There are certain qualities peculiar to cuticular fragments of insects that serve as 

proof of insect origin. Even though a suspect fragment may have a genuine insect 

appearance, it cannot be reported as an insect fragment unless there is proof of 

insect origin. As outlined in AOAC 970.66, the following diagnostic characteristics 

are the proofs by which a positive identification of insect fragments can be 

accomplished.

A. Diagnostic Characteristics of Insect Fragments

1. Shape. The shape of a fragment is diagnostic if it is recognizable as an 

entire or particular portion of, an appendage, body segment, or specialized 

structure of the insect body.

2. Setae. The presence of one or more non-cellular setae with associated 

setal pit (papilla) is diagnostic. Should the setae have become separated 

from the fragment, the presence of setal pits is sufficient for identification. 

3. Sculpture. Surface pattern (sculpture) that is typical of a particular part of 

an insect is diagnostic, quite often to the family or genus level.

4. Sutures. Fragments that consist of portions of interlocking plates (sclerites) 

are, by the complex form of the joining interface (suture), proof of insect 

origin.

Certain qualities of insect cuticle may alert the analyst to look closely for one of 

the above diagnostic characters. Even though these secondary qualities are not 

unique to insect fragments, they are useful to the analyst in that their absence 

casts doubt on the insect origin of a fragment.

B. Secondary Characteristics of Insect Fragments 

1. Texture. This is thinness combined with flexibility or toughness.

2. Luster. Insect cuticle often has a distinctive sheen that the trainee soon 

comes to recognize.

3. Lack of cellularity. This negative aspect separates insect fragments from 

many types of plant material. Some types of sculpture may give a 

superficial impression of cellularity, but close examination finds that plant 

cells exhibit much individual variability of size and configuration while insect 

cuticular sculpture tends toward repetitious uniformity of cell-typed units. 

Comparison with authentic reference material is the final, irrefutable proof of 
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insect origin and should be employed as often as possible. Identification of 

fragments to family and genus is routinely possible; occasionally species 

determinations are accomplished when reference materials (authentics or 

literature) are provided. 

12.3. Assignment 

A. Systematically arrange the fragment reference material from section 11.3 so

that it is most useful to the individual analyst preparing the material, OR, if the 

trainer prefers, integrate the reference material into the laboratory reference 

collection.

B. Using the laboratory library, compile a personal bibliography of literature 

concerned with insect fragment recognition, especially from AOAC and FDA 

publications.

C. Review and discuss with the trainer what has been learned thus far on 

fragment identification. Additionally, discuss the difference between whole, 

whole/equivalent (w/e), and large body parts of insects. Maggot mouth hooks: 

fragment or (w/e)?

D. Practice identifying unknown specimens supplied by the trainer until the 

analyst is familiar with the literature and confident in their own ability to identify

insect fragments.

E. Using any literature resources and reference material, identify at least 10 

unknown fragment specimens supplied by the trainer. Identifications are to 

the lowest taxonomic level supportable by literature resources and reference 

materials found in the laboratory.

13.Mites

13.1. Objectives

The purpose of this exercise is to learn the basic morphology of mites.

13.2. Discussion

A. Mite Features

Mites are chiefly recognizable by their small size (usually 0.5mm or less), general 

lack of body segmentation, and four pairs of legs. In order to understand mite 

taxonomy, analysts learn basic mite morphology, which differs considerably from 

insect morphology.

Body regions are defined in relation to the positions of the legs and mouth. 

Anteriorly, the gnathosoma bears the mouth and oral appendages. Following this 

is the propodosoma, whose area is defined by the first and second pairs of legs. 

Collectively, these two regions, gnathosoma and propodosoma, comprise the 
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proterosoma. The metapodosoma bears the third and fourth pairs of legs. The 

remainder of the body behind the last pair of legs is called the opisthosoma, and 

collectively the last two regions are called the hysterosoma. The term idiosoma

refers to the entire body exclusive of the gnathosoma.

Appendages of mites are of three basic kinds, each of which may exhibit varying 

degrees of modification. The chelicerae are the front-most pair of oral 

appendages. They are basically pincer-type appendages, although in some 

groups they may be highly modified for specialized feeding while in other groups 

they may be greatly reduced. In addition to chelicerae, the gnathosoma may bear 

a pair of leg-like segmented appendages called pedipalps. Although generally 

very small, the pedipalps sometimes have the proportion of true legs, which can 

be distinguished by position, segmentation, and lack of claw-type structures or 

pretarsi. Legs are usually eight in number for adult mites, although certain 

immature stages may have only six legs. Like other arachnids, the mite has a sixsegmented leg consisting of a proximal coxa, trochanter, femur, genu, tibia, and 

distal tarsus. The latter exhibits no secondary segmentation as found in insect 

tarsi. The tip of the tarsus bears a pretarsus that is often fleshy or membranous 

and may bear one or more claw-type structures. Mite pretarsi exhibit literally 

hundreds of variations among the different mite groups. 

Mite setae are basically similar in general appearance to insect setae. The base of 

a mite seta is slightly swollen, and a papilla is usually evident. Due to a central 

cytoplasmic core, mite setae exhibit optical activity between crossed Nicols on a 

polarizing microscope. As with insect setae, mite setae may be variously modified.

Solenidia are hair-like structures found on mite legs that differ from setae as there 

is no basal swelling, no optical activity, and very little, if any, modification of the 

basic hair-like form.

Other mite features include a postero-ventral anus, genital structures whose 

position varies among species, leg and anal suckers, various sclerotized body 

areas called shields, and simplified respiratory structures roughly analogous to 

tracheae (peritremes) and spiracles (stigmata). These structures may each be 

modified or absent in any given group of mites.

B. Preparing Mite Microscope Slide Mounts

Mites are mounted on a microscope slide and observed under a compound 

microscope for identification. Prior clearing (See Section 4.2.3 Preparing 

Microscope Slide Mounts) may be needed. Because of its desirable optical 

qualities, the mounting medium of choice is Hoyer's solution or one of its variants. 

For general work, the specimen is mounted venter up with the gnathosoma 

pointed towards the bottom (south) edge of the slide. (This is so the compound 

microscope image will appear with the gnathosoma at top). The specimen is 

centered, pushed to the bottom of the drop of medium, and the legs spread as 
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much as possible, before placing the coverslip. The weight of the coverslip may 

produce further leg spreading, but a coverslip that is too heavy will burst the 

bodies of delicate specimens. The smallest sized, lightest weight coverslip found

should be used.

Small amounts of heat may be applied to the mount to help spread the legs and 

aid penetration of the body by the mounting medium. Hoyer's-type solutions are

not to be boiled as this affects the storage life and may release harmful fumes.

(Safety note: Work with proper ventilation to avoid breathing fumes.)

C. Effects on Human Health 

The effects on human health of mites in foods have not been completely 

documented, but some deleterious attributes of mites are becoming evident.

1. Mites can cause considerable physical damage to stored foods.

2. Mites can impart a distinctive disagreeable, sweetish musty odor to foods 

they infest.

3. Mites can produce a sugary, white, encrusted coating on dried fruits.

4. Some kinds of mites can induce allergic reactions, including asthma-type 

symptoms, in sensitive individuals.

5. Mites can transport spores of molds that will grow on and spoil food 

products.

6. Certain mites are potential intermediate hosts for parasitic organisms that 

infect mammals.

13.3. Assignment

A. Read pp. 63-82 of: Gorham, J. R. (Ed.) (1981). Principles of food analysis for 

filth, decomposition, and foreign matter (FDA Technical Bulletin No. 1, 2nd 

ed.). Gaithersburg, MD: AOAC International.

B. Review mite slides and discuss with the trainer.

C. Compile a list of references concerning mites found in the laboratory. Check 

intra-agency documents such as the Laboratory Information Bulletins and FDA 

By-lines.

D. Under the direction of an experienced trainer, practice mounting mite 

specimens until good quality whole mounts for microscopic examination can 

be produced.

E. With the specimens mounted in "4," practice using the phase-contrast 

microscope.
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14.Hair Identification

14.1. Objective

The trainee will gain experience in the identification of mammalian hairs, 

especially rodent hairs.

14.2. Glossary

Commensal - one who eats at the same table with others; an organism, not truly 

parasitic, that lives in, with or on another.

14.3. Discussion

The regulatory analyst can identify hairs or hair fragments from murine rodents or 

commensal rodents. The commensal relationship is between certain murine 

rodents and man, and not some other commensal relationship they might have 

with other animals.

Murine rodents are so termed because they are placed taxonomically in the family 

Muridae. Rodents are all those animals placed taxonomically in the order 

Rodentia. Examples of rodents familiar to us include squirrels, ground squirrels, 

chipmunks, various field mice, cotton rats, muskrats, beavers, and porcupines. 

These are placed taxonomically in families other than Muridae but within the 

larger taxonomic unit, the Rodentia. This very general description serves us 

simply by pointing out that the term "rodent hair," used in general by 

microanalysts, is simply too general to use for describing the hair of commensal 

rodents. The hairs of most concern to the microanalyst are those from the 

commensal rodents, but not necessarily limited to these. The commensal rodents 

of most concern are the Norway rat (Rattus norvegicus), the roof rat (Rattus 

rattus), and the house mouse (Mus musculus). These animals are not native to 

North America but were introduced by commerce. Because of their close 

relationship to man and documented evidence of their role in disease 

transmission, they are considered probable health hazards, and evidence of 

contamination by these animals is weighed heavily by regulatory and health 

officials. Other commensal rodents of concern is the lesser bandicoot rat 

(Bandicota bengalensis). This rat is found in the area surrounding India, Pakistan, 

Burma and some of the islands in the area. The Polynesian rat (Rattus exulans) is 

widespread throughout the Pacific islands and Southeast Asia.

Another important commensal animal is the Asian musk shrew (Suncus murinus). 

An insectivore, found in Southern Asia and eastern Africa, this animal belongs in 

the shrew family Soricidae. The hairs of this animal are found on a regular basis 

and they have a distinctive appearance. The Asian musk shrew has been known 

to be a reservoir for Salmonella and plague.

Other animals, many of them native rodents, often establish a temporary 
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commensal relationship with man. These include various squirrels, muskrats, etc. 

Contamination by these animals, domestic animals, pets, and human hair are also 

be considered and recognized by the analyst.

It is not assumed that an analyst can learn to identify hairs by reading about their 

various characteristics. This ability can only be acquired by careful study of 

authentic specimens. Suspect material should always be compared to authentic 

specimens.

The basic structure of most hairs consists of an external layer of scales underlaid 

by a cortex of generally amorphous tissue. In the center of the hair is the central 

core, called the medulla. Striated hairs have discontinuous medullae that give 

these hairs their characteristic banded appearance. Striated hairs cause the most 

concern since the mammals that pose the greatest threat to world food supplies, 

the commercial rodents, all have striated hairs. The primary task of the trainee is 

to learn to identify hairs of the commensal rodents. This knowledge can then be 

applied to learning the identification of other types of hairs.

Hairs are examined under the compound microscope for identification. Most 

striated hairs contain considerable amounts of air trapped in the medulla. This air 

is removed by heating to prevent interference with microscopic observation by 

diffracting light away from the objective lens. As heating procedures vary widely, 

the trainee chooses a personal technique under the guidance of the trainer. The 

simplest techniques involve heating the hair in the mounting medium, glycerin 

jelly, so that the air is replaced entirely by medium (See section 3.4 Preparing 

Microscope Slide Mounts).

A. General Microscopic Characteristics of Rodent Hairs

1. Prominent scales. Under the compound microscope the edges of rodent 

hairs have a serrated appearance due to the projecting tips of the external 

scales.

2. Clear cortex. The usual color with unfiltered light is bright hyaline green 

with virtually no dark spots. The cortex is also typically very thin.

3. Discontinuous medulla. This type of medulla is thought to be composed of 

cell remnants embedded in a solid matrix. Each cell or segment contains 

numerous pigment granules packed tightly in one end leaving the other end 

clear. An intervening clear air space separates each cell from the next. This 

contrasting alternation of dark pigment and clear areas in the medulla gives 

the hair its striated appearance. 

B. Guard Hair Characteristics

Guard hairs are the long, coarse hairs of the rodent pelt. Microscopically, the 

medulla is seen to consist of several rows of segments or cells, each with 
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pigmented and clear areas as well as separating air spaces.

C. Fur Hair Characteristics

The most striking feature of rodent fur hair is the zig-zag configuration of the 

hair itself. This is evident even at low magnification and is a result of bending 

of the hair at the internodes. A single row of medullary cells is typical of rodent 

fur hairs. These hairs are thinner than guard hairs.

1. Internodes. Fur hairs exhibit this rapid constriction of the hair diameter at 

one or more points along the length of the hair. The area where an 

internode occurs shows proportionate size reduction of the medulla.

2. Air spaces. The most singular characteristic of rodent fur hair medullae is 

the shape of the air space between each cell. This is typically the shape of 

a capital "I" with the stem of the "I" at right angles to the hair's length.

3. Cortical pegs. This small extension of cortical material into the medulla 

appears as a single indentation on each side of the medullary cell, usually 

in the pigmented area.

A hair possessing the above characteristics is probably a rat or mouse hair 

and should be identified by comparison with authentic specimens. The analyst 

compares the sizes and configurations of all structures, including scales, 

cortex, air spaces, medullary cells, pigment granules, and internodes.

Mammalian hairs can be deceptive look-alikes. Shrew hairs are virtually 

identical to some mouse hairs except for the tips, which are more elongated, 

and the scale pattern, which is asymmetrical, the scales on one side projecting 

more prominently than those on the other and the internodes tend to be 

narrower. Squirrel and rabbit hairs are like rat or mouse hairs in general, but 

differences in the shapes of the air spaces and medullary cells can be used to 

differentiate them.

Hairs of mammals are sufficiently different between families and genera to 

permit identification to these levels in most cases. The analyst pursues this 

expertise through the study of authentic specimens with guidance from 

experienced analysts and from the literature.

Note: As noted with insects and fragment identification, only time and 

experience, or specialized study will improve the analyst’s proficiency in this 

area of expertise. The initial objective is to distinguish commensal rodents from 

non-commensal, but analysts are encouraged to continually study these 

materials throughout their careers through continuing education and QA 

programs. 
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14.4. Assignment

A. Read pp. 125-170 of: Gorham, J. R. (Ed.) (1981). Principles of food analysis 

for filth, decomposition, and foreign matter (FDA Technical Bulletin No. 1, 2nd 

ed.). Gaithersburg, MD: AOAC International.

B. Read pp. 157-216: Olsen, A. R., Sidebottom, T. H. and Knight, S. A. (1996). 

Fundamentals of Microanalytical Entomology, a practical guide to detecting 

and identifying filth in foods. Boca Raton, FL: CRC Press. 

C. Prepare acceptable slide mounts of authentic hairs supplied by trainer.

D. Practice identifying rodent hairs until one feels confident in their own ability.

E. Examine the following types of hairs and discuss how each can be 

distinguished from rat or mouse hairs:

1. rabbit

2. shrew

3. bat

4. dog

5. cat

6. human

15.Feather Identification

15.1. Objective

The trainee will gain experience in bird feather identification, especially 

recognizing the difference between feather, feather barbs and barbules. The 

trainee will also learn how to recognize the difference between feather fragments, 

hairs, and gill fragments. They will also learn how to identify the feather barbules 

to commonly encountered bird Orders.

15.2. Glossary

Feather-the primary covering on a bird and aids in flight.

Feather Barb-the individual units which grow from the feather shaft or rachis.

Feather Barbule-are like mini barbs growing from the shaft of the barb.

Node: the swollen structure spaced at regular intervals along the feather barbule. 

The shape of the node can vary in shape from having spines, prongs, round

points or even flared.
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Rachis-the central shaft of the feather. 

15.3. Discussion

The regulatory analyst can identify feather, feather barbs and feather barbules 

found in samples and with experience can identify those fragments down to order. 

There are numerous commensal birds including English sparrow (Passer 

domesticus), feral pigeons (Columba livia domestica) and crow (Corvus sp.). 

Food can become contaminated by birds from direct contact of the bird, feather 

material or their excreta. Feather barbules being the most common bird 

contaminate found in samples. Feather barbules often can be confused with 

animal hairs like bat and gill tissue from shrimp and lobster. Birds are common 

carriers for diseases like histoplasmosis, salmonella, and E. coli, just to name a 

few.

The birds most found contaminating samples can be broken down into 4 main 

orders with general characteristics to separate their barbules: 

1. pigeons/doves (Columbiformes): The nodes are flared on the proximal 

barbules. The flaring is so distinctive that they resemble small flowers and 

are often called crocus shaped. 

2. chickens/turkeys (Galliformes): The proximal nodes often are slightly 

expanded with small spines. Typically, the distal nodes will be encircled 

with rings. These rings can sometimes slip at the node juncture and slide 

along the barbule. 

3. ducks/geese (Anseriformes): The node can have an expanded triangular 

shape or more prong shaped. The nodes typically are isolated toward the 

distal end of barbule or proximally on the barb

4. songbirds/passerines (Passeriformes) The Passeriformes is the largest 

group encountered in samples. the nodes are variable but typically are 

very slightly expanded with flared or rounded transparent like projections. 

Nodes normally are not prolonged or spined. 

15.4. Assignment

A. Read pp.15-61: Dove, C. J., Koch, S. L. (2010) Microscopy of Feathers: A 

Practical Guide for Forensic Feather Identification. Journal of the American 

Society of Trace Evidence Examiners Vol. 1 (1),

B. Prepare acceptable slide mounts of authentic feather barbs and barbules 

supplied by the trainer.

C. Prepare acceptable slide mounts of feather barbules supplied by the trainer 

and compare with mammalian hairs especially, bat. Also compare the feather 

mounts to gill material from either shrimp or lobster. Discuss with the trainer on 

how to recognize the differences. 
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D. Practice identifying barbs and barbules until one feels confident in their own 

ability.

E. Examine feather barbs and barbules from each of the four bird orders until one 

feels confident in their own ability to recognize the four orders.

1. Columbiformes

2. Galliformes

3. Anseriformes

4. Passeriformes

16.Excrement, Urine, Uric Acid (Morphological and Chemical) 

16.1. Objective

The trainee will become familiar with the major types of animals (including insect) 

excrement that may be found in foods, and the potential health hazards 

presented, such as Hantavirus, coccidiosis, and related diseases, carried by 

vermin pests.

16.2. Discussion

Excrement is a term that may be applied to feces as well as other excretory 

products such as urine and various glandular substances, including sweat.

A. Feces

Feces is the word that is commonly used for the material ejected from the 

intestine through the anus. Fecal pellets are feces ejected in discrete units, as in 

the case of rodents and many insects. Feces consist mainly of undigested food 

remnants. Alternate terms are "dung" or "manure."

Fecal pellets are identifiable by visual examination under a widefield microscope 

with comparison to authentic material. The salient characteristics of fecal pellets 

are size, shape, color, and, in the case of rodents, surface coating and embedded 

hairs.

Rodent fecal pellets are elongated with pointed or tapered ends. The color ranges 

from tan to dark brown to black under dry conditions and is also dependent upon 

what the animals were feeding. Interesting color variations may occur in rodents 

that have fed at bait stations, with blood inclusions observed. One of these color 

variants can be greenish blue, depending upon the coloration of the bait. 

Immature rodents undergoing weaning may, for a short period, produce pellets of 

a light brown color. Color variations of these sorts are not routinely encountered 

alone, but are mixed with other, more typical, pellets. Size range is 5-20 mm for 

rats and mice, with mouse fecal pellets rarely exceeding 10 mm. When 

moistened, rodent fecal pellets exhibit a surface coating of grayish-white mucous. 

Mice do not need free water to survive, therefore typically exhibit dry pellets with 
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heavy mucous coatings, while rats need a source of free water to survive and 

have thinner mucous coating and moister pellets. To confirm the mucous coating, 

a small drop of water is placed on the surface, usually softening the pellet and

producing a mucous like characteristic. In addition, as the animals are constantly 

preening themselves, embedded hairs may often be seen protruding from a 

rodent fecal pellet, or they may be disclosed by crushing the pellet. These hairs 

offer vital information and are essential in determining the kind of rodent involved, 

especially commensal rodents (rats or mice, as opposed to muskrats, squirrels, 

etc.).

Bat fecal pellets have a similar size range to mouse excreta pellets. They are 

slightly spindle-shaped with a mucous coating. The pellets will contain bat hairs 

and normally contain insect fragments.

Fecal pellets of deer, sheep, goats, or rabbits are rounded, without an intact 

mucous surface coating. They are usually less dense than rodent fecal pellets.

Insect fecal pellets are generally small, although some grasshopper and 

cockroach pellets may approach the size of rodent pellets. Pellets of the 

orthopterans and larval lepidopterans are characteristically barrel-shaped, having 

truncated ends and longitudinal ribs. Coleopterans and some other insects pass 

small, elongated, irregularly shaped pellets. Insect fecal pellets are often the same 

color as the food substrate. This is especially true of stored-product beetles; these 

pellets do not have a mucous surface coating. Sowbugs and pillbugs (Crustacean: 

Isopoda) often found in damp areas in manufacturing plants. They normally feed 

on detritus including excreta pellets. The fecal pellets produced by Isopods are 

rectangular in shape and relatively thin and flat. They can contain animal hairs, 

due to their behavior of feeding on excreta pellets. 

Due to the dietary habits and digestive processes of cockroaches, their fecal 

pellets may resemble mouse pellets in color. Generally, of smaller size (1/8 inch 

or less), cockroach pellets exhibit longitudinal ridges and often have a somewhat 

six-sided appearance caused by pressure from the internal rectal glands prior to 

expulsion through the anus. Cockroach pellets do not have a mucous coating. 

Since cockroaches habitually eat their own cast skins, the presence of these 

fragments in a pellet is an additional clue to the pellet's origin.

Caution: The contents or components of a particular pellet (mammalian or insect) 

should not be the sole basis for the pellet's identification but rather one of many 

observations, the sum total of which constitutes the basis for identification. For 

example, rodents living in the same environment as cockroaches may feed on 

dead cockroaches, resulting in rodent fecal pellets that contain cockroach 

fragments. Conversely, cockroaches may feed on rodent pellets, with the result 

that a rodent hair may occasionally be found in a cockroach fecal pellet. Bats are 

insectivores and their pellets consist almost strictly of insect fragments and exhibit 
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no mucous coating. Therefore, the analyst carefully weighs all of the 

characteristics observed (size, shape, color, surface coating, and embedded 

components) in order to identify the source of the fecal pellet.

B. Bird Excrement

This term is applicable to bird droppings, which consist of a mixture of glandular 

excretions and feces. Bird excrement exhibits a texture varying from liquid to 

semi-solid. Drops of bird excrement usually take the familiar form of a chalky white 

amorphous material mixed with darker food and watery residues. Morphologically 

suspect material is chemically tested for uric acid to confirm the identification as 

bird excrement. Important: only test the white amorphous material from the 

excreta pellet for uric acid.

C. Reptile Excreta

The excreta pellets of the common house gecko (Hemidactylus sp.) range in color 

from a light to dark brown, often with a whitish circular mass at one end. This 

whitish mass easily breaks off from the pellet and will test positive for uric acid. 

The excreta pellets are normally tapered at both ends and have a similar size 

range to rat and mouse excreta pellets. The surface of the excreta pellets is 

relatively smooth to wrinkled without a mucous coating. The matrix of the pellets 

is densely packed with insect fragments. Besides the common house gecko, the 

day gecko (Phelsuma sp.) can be found in dwellings as well. The excreta pellets 

of the house gecko have been known to contain various bacteria including E. coli, 

Salmonella, Staphylococcus and Shigella, while the day gecko can contain at 

least Salmonella.

D. Urine

Urine is a term describing the fluid excretions of a mammalian kidney. This term 

also has applications, for birds and reptiles, which are not usually encountered in 

FDA work.

Defilement of food with rodent urine is usually detected initially by observing urinestained packaging. These stains exhibit a typical greenish fluorescence under 

long-wave ultraviolet light. Rodent urine stains often exhibit "streaking" 

configurations caused by the rodent urinating while running or by dragging its tail 

through a wet urine spot. Many times, even with dry stains, a urine odor is evident. 

Suspect stains are confirmed chemically under many circumstances, as defined 

by agency policy.

Safety Note: Handle these materials as biohazards; practice universal 

precautions. Discuss with the trainer safety practices needed for the handling of 

these materials. Rodents and other animals serve as potent carriers for numerous 

diseases. Diseases such as Hantavirus, Histoplasmosis, some tapeworm, and 

related organisms are the principle concerns. Exhibits should be prepared for 
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safe presentation in the courtroom, while retaining recognizable characteristics.

16.3. Assignment

A. Read pp. 201-216 of: Gorham, J. R. (Ed.) (1981). Principles of food analysis 

for filth, decomposition, and foreign matter (FDA Technical Bulletin No. 1, 2nd 

ed.). Gaithersburg, MD: AOAC International.

B. Examine authentic specimens of rodent and insect fecal pellets, rodent urine 

stains, and bird excrement. Review and discuss with trainer.

C. Under the direction of the trainer, learn to perform the various AOAC methods 

for the xanthydrol chemical confirmation of mammalian urine, identify fecal 

materials and find the citations for confirmation of fecal material, and perform 

the chemical identification of bird excrement for Uric acid following AOAC 

method 970.13. 

17.Sample Analysis

17.1. Preliminary Guidance

Before beginning each analysis, the analyst should ask their supervisor if any 

administrative guidance has been issued concerning the product about to be 

analyzed. Analysts should be aware and familiar with the guidance or program for 

which the product was collected under, shown on the Collection Report as the 

“PAC/PAF” number. The analyst may also want to study and gain insight into the 

history of the product or the firm, pest problems, processing, and production as it 

relates to the agency’s regulatory policy and the philosophical/economic impact on 

the regulated industry. 

Sources of information range from the Sample Collection Report (C/R, FACTS, 

CMS, and OASIS), the supervisor and senior analysts, the sample collector, the 

EIRs in the firm’s jacket, the FD484, and photographs taken by the Investigator. 

The analyst can request additional information not commonly found with the 

sample. 

While reading the documentation provided with the sample, the analyst asks the 

following questions: (who, what, when, where)

• What does the analyst need to determine (problem area) for this 

sample? (i.e., insect, rodent, mold, and/or particulates- like glass or 

metal adulteration). 

• What is the scale of the analysis? (i.e., visual, macro and/or 

microscopic?)

• What methods, preferably official, are found for this kind of analysis?

• What equipment and reagents will be needed, and how much of each is
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needed?

• What steps/timeline will be followed before, during, and after the 

analysis?

• Is there enough material/sample/time to do the analysis? E.g., can the 

analysis be modified such that one can accommodate the situation?

• Does this analysis require a 702(b) portion and is there one provided?

• Will help be needed, and when will it be needed? 

• What kind of problems/interferences can one expect to see, and what 

can be done to avoid them? E.g., does the ingredient label show any 

unexpected, unusual ingredients that the analyst will have to deal with 

by method modification?

• Is there any additional analysis needed, if so, what, when, where, and 

with whom does one coordinate? 

• What kind of results does one expect to see, how are the results going 

to be reported and what kind of format is to be used?

• Is the sample fit for use? (i.e., accountability, storage, seal, damage or 

integrity issues, lack of 702(b) portion).

If these questions cannot be answered, seek help and discuss the issues before 

proceeding with the analysis. Information from the investigations branch can be 

obtained through the supervisor; sample investigators may have valuable 

information not always present on the C/R, (e.g., photographs). 

The Observant Analyst: If the above questions can be answered, then, the 

analyst is ready to begin the analysis, with this added note of caution. Regardless 

of the stated or implied objectives of the C/R or the analysis chosen, the FDA 

analyst is always to be alert for unexpected developments. The purpose of 

regulatory sample analysis is to discover evidence of a violation of one of the 

various laws enforced by the agency. Many routine analyses have taken a sharp 

change of direction due to a chance observation by an alert analyst. Each analysis 

represents a new and potentially provocative situation that challenges the 

analyst's powers of observation and scientific curiosity. An alert, inquisitive 

approach to sample analysis is every bit as valuable to the agency as any 

scientific expertise the analyst may have.

17.2. Scale of the Analysis

Macroscopic and microscopic procedures for characterizing defects in foods tend 

to supplement each other, and together provide a comprehensive evaluation of 

defects in the product. It is important that the analyst realize the close association 

of the macroscopic and microscopic methods for use as a joint approach in 
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solving analytical problems

A. Macroscopic Methods of Analysis

To consumers, "macroscopic" analysis of a product refers to an evaluation of 

the substance using their unaided senses (primarily sight, smell, or taste). 

Every consumer in our society who exercises some judgment in the purchase 

of foods and other consumer goods, knowingly or unknowingly conducts some 

form of macroscopic examination to detect apparent or obvious defects. The 

examination may range from a cursory, perhaps unconscious visual check of 

the product to confirm that everything "looks right", to a much more detailed 

scrutiny for defects. Regulatory authorities, in fulfilling responsibilities for 

protecting the public health and safety, conduct systematic examinations to 

disclose not only apparent defects, but also hidden defects. Over the years, 

standardized methods of macroscopic examination have evolved for 

determining filth, decomposition, and foreign matter. These methods of 

analysis have evolved with the input of producers and consumers as well as 

regulatory authorities.

In general, "macroscopic" or macroanalytical methods for food examinations

primarily depend upon the direct sensory input of the analyst. For example, 

visual examinations are typically conducted with the naked eye. These exams 

are occasionally supplemented by low power magnification to confirm defects 

observed initially with the naked eye, or to describe the defects in greater 

detail. 

There are several major advantages to the use of macroanalytical procedures. 

They are inexpensive and call for little specialized equipment. They generally 

permit the analysis of a large quantity of product in a relatively short period of 

time, thus allowing the analyst to assess the overall condition of the lot quite 

rapidly. The analyst can quickly identify and isolate those portions of the lot 

which may contain defects and thus limit the amount of material which may 

need a more detailed, microscopic evaluation. 

Although macroscopic methods have many positive aspects, they may not be 

the method of choice for every analytical situation. In fact, the very features 

which add to their usefulness may also limit their application in some 

situations. Because macroscopic procedures deal with defects which are 

discernible to the unaided senses, they are not usable for defects hidden from 

the senses such as those defects too small to be visible to the eye, or those 

obscured through processing or other factors. In such cases, microscopic 

methods are essential for characterizing and evaluating the defects in the 

sample. 

B. Microscopic Methods of Analysis
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Microscopic methods of analysis involve the detailed examination of a very 

small portion of the sample; these procedures provide a different type of 

information than macroscopic methods. They are used to describe and 

quantify defects on a different scale than macroscopic methods, and to identify 

"hidden" defects that cannot be detected through a gross evaluation of the 

sample. However, microscopic methods also have limitations; they tend to be 

more time-consuming and more expensive, and they need more specialized 

equipment. Also, because they are limited to the analysis of a very small 

sample, the results are not always representative of the overall condition of the 

lot, thus representative sampling plays a more critical role in this type of 

analysis.

17.3. Method Selection for Filth Analysis

See MAN-000037 ORA Lab Manual Vol. II - Methods, Method Verification and 

Validation (ORA-LAB.5.4.5)

Filth methods commonly used have been published in the AOAC Official Methods 

of Analysis, Macroanalytical Procedures Manual (MPM) | FDA or Laboratory 

Information Bulletins (LIBs). These methods employed by FDA analysts have 

been proven to give reliable and consistent results. Many of these methods have 

been validated through collaborative study. If possible, the analyst should use 

validated or verified methods as written and without modifications. Any method 

modifications shall be documented on the analytical worksheet. 

A. Association of Official Analytical Chemists (AOAC)

Many of the analyses in the following sections will be found in the Official 

Methods of Analysis published by AOAC INTERNATIONAL (AOAC). The 

analyst should become familiar with the editorial conventions of this text. All 

editorial conventions are described in the front of the manual. Boldface 

reference numbers in the text of a method refer to safety precautions, 

apparatus, or other critical information. The entire method should be read, and 

each reference looked up before beginning the analysis. A star after the title of 

a method indicates that the method will be dropped from the next edition, as 

“Surplus”. “Surplus” methods can be used, but if one of these methods is used, 

the analyst should inform the AOAC Section Editor, so the surplus decision 

can be reconsidered. Journal references at the end of each method refer to the 

article that reported the results of the collaborative study for that method. 

These journal articles often contain useful additional information, such as 

problems encountered and expected recoveries. Formulae, such as (2+1), 

indicate volume ratios for mixing the reagents being discussed. 

The AOAC’s chapter, "Extraneous Materials: Isolation," initially discusses 

general techniques, apparatus, and reagents. It also cites how to record, and 

report results and gives counting/identification instructions. This information is 
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important and should be read by the trainee before the first attempt to use the 

manual; use the most current edition found.

B. Macroanalytical Procedures Manual (MPM) | FDA

Also known as the FDA Technical Bulletin Number 5, originally published by 

the AOAC, this publication is now out of print, but has been placed on the FDA 

intranet. The web version does include some minor changes (error corrections) 

that are not reflected in the printed versions. The web version also includes 

web citations to the Compliance Policy Guides for products, making it easier to 

find related guidance.

This one volume manual compiles and organizes the standardized methods of 

macroscopic analysis which are useful in determining defects in various types 

of foods. Although in a general sense, the term "macroscopic" is not as broad 

as the term "macroanalytical," for the purposes of this manual, the terms are 

used interchangeably. 

This manual compiles standardized macroanalytical procedures for identifying 

defects in food products. However, macroscopic procedures are frequently 

interrelated with and supplemented by microscopic ones, each providing the 

analyst with different types of information. For this reason, the Macroanalytical 

Procedures Manual will refer to microscopic procedures in some situations. 

These microscopic procedures may be grouped into three categories: 

1. Microscopic methods which have been published by the AOAC in Chapter 

16 ("Extraneous Materials") of the Official Methods of Analysis. Where 

needed, this manual simply refers the analyst to the applicable section of 

the AOAC volume for the correct method. 

2. Microscopic methods which have been published in the AOAC volumes, 

but which are adapted by the analyst for a particular situation. In these 

cases, special instructions are provided in this manual so that the analyst 

can modify the microscopic procedure as needed. Reference is made to 

the correct section of the AOAC. 

3. Microscopic procedures which have been developed and are in use, but 

have not been subjected to collaborative study and thus are not yet 

published by the AOAC. These procedures are included in full in this 

manual so that they are not lost to the analyst. 

Thus, when using this manual, the analyst may be instructed to combine both 

macroscopic and microscopic techniques. Examples of this can be seen in the 

method for determining decomposition in frozen strawberries, which utilizes 

macroscopic "pick-out" of defects (see MPM Chapter V, Section 9.N.(4)b.) 

supplemented by the microscopic mold count technique (MPM Chapter V, 
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Section 9. N.(4) c.). Information provided by the microscopic techniques will 

aid the analyst in interpreting and evaluating the macroscopic findings and in 

determining the overall quality of the food.

C. Laboratory Information Bulletins (LIBs) 

LIBs are developed within FDA and used as a tool for the rapid dissemination 

of laboratory methods and information. Some LIBs contain methods that have 

been tested on a limited basis and deemed to work but have not necessarily 

undergone the rigid inter-laboratory testing required for publication as an 

AOAC official method. Since LIBs do not report complete scientific work, the 

user must utilize appropriate validation procedures where applicable to ensure 

that the LIB method and techniques are reliable and accurate for the intended 

use. Filth specific LIB publications contain information related to 

extraction/isolation, insect identification, adulteration/substitution, equipment, 

and/or safety. 

D. Assignments

Read the following:

1. Chapter 16, Section 16.1.1 and 16.1.2 of the current edition of the AOAC 

Official Methods of Analysis and look at the general organization of the 

methods.

2. Preface and Introduction in the MPM and look at the general organization 

of the manual.

3. The lab’s Standard Operating Procedures that relate to the filth lab.

17.4. Sample Handling

Read and follow the local laboratory SOP’s dealing with cleanliness and quality 

control in operation. 

A. Techniques to Prevent Sample Contamination

The filth analyst exercises constant vigilance against the inadvertent introduction 

of any type of outside contamination into the sample(s) under the analyst's care. 

This vigilance extends to those who work in the laboratory if their work can 

contaminate the sample. 

1. Glassware and other equipment are kept scrupulously clean and stored in 

clean, enclosed areas when not in use. Equipment should be given a 

preliminary "extra" cleaning prior to analysis by rinsing with clean water or 

wiping with clean towels. Purity of reagents are assured by proper storage 

and analysis of reagent blanks. 
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2. Steps are taken to assure that the analyst's person or clothing do not 

contribute anything to the sample. A clean laboratory coat is essential. The 

analyst develops personal techniques of handling samples and equipment 

that avoid bodily contact with the sample or sample contact surfaces. 

Spoons, scoops, tongs, and rubber gloves are useful for this purpose. Hair 

(both human and pet) and lint are the primary contaminants to be guarded 

against. Precautions such as these combined with personal hygiene will 

preclude any possibility of analyst-related sample contamination.

3. During analysis, the sample is protected from airborne contamination as 

much as possible without interfering in the analytical procedure. Containers 

such as trap flasks, beakers, and percolators that are to be left standing for 

a period of time should be covered. Petri dishes are employed in the 

conventional manner, with the larger diameter part used as the top or lid for 

storage of filth extraction papers. Bench tops and other surfaces should be 

cleaned to avoid dust buildup, and reagents should always be stored in 

capped or stoppered vessels.

4. Guard against cross-contamination between two samples or two sub-units 

of the same sample. The same piece of equipment is never used twice 

without thorough washing. Unused portions of reagent are never returned 

to the original container but disposed of in a proper manner. Spatulas, 

tweezers, and other implements are cleaned before they are used to 

manipulate samples or reagents. Any reagent whose purity is in doubt is 

disposed. Aerators and wash bottle nozzles should never come in contact 

with any sample or piece of equipment. If this happens, a thorough 

cleaning is needed. These and other common-sense rules are followed to 

protect the integrity of the results of analyses.

B. Use of Quantitative Transfer

The concept of quantitative transfer applies to each sample portion from the 

moment the sample container is opened until the final results are reported. 

This demands that every vessel, implement, and operation be considered a 

possible cause of sample loss.

Each time an analytical portion is transferred from one vessel to another, every 

effort is taken to assure that no amount of material, however small, is 

inadvertently left behind in the old vessel. While some small loss appears 

inevitable in every transfer operation, the analyst can take steps to keep this 

loss to a minimum.

1. For methods using wet chemistry, the wash bottle is the analyst's most 

versatile tool for accomplishing quantitative transfer. Many methods specify 

wash bottle liquids for optimal recovery of filth, and care is taken to use the 

correct wash to avoid interference with the extraction process.
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2. The analyst also avoids losing small amounts of sample through dripping, 

splashing, or leakage. Once a loss has occurred, there can be no 

replacement or compensation, thus the results of the analysis may be 

compromised. 

3. Chipped or cracked vessels and overzealous heating (bumping) and sloppy 

or overpressure sieving operations should be avoided, as these have the 

greatest potential for sample loss. Sieves are also inspected for rips or 

deformities that could result in losses.

C. Apparatus Selection

For each analysis, the analyst chooses the proper apparatus. Often the 

analytical method will call for explicitly described apparatus. Regardless, the 

analyst is to be aware of a few general restrictions on apparatus.

1. Avoid sample contact with plastic vessels and implements. Hairs and 

insect fragments will stubbornly adhere by static force or surface texture to 

plastic surfaces resulting in reduced filth recovery.

2. Do not use chipped, scarred, or broken equipment. Irregular surfaces can 

snag material, causing problems.

3. Do not use glass equipment when analyzing a product for glass 

contamination.

4. Use plain weave (not twill weave) sieves. After each use, sieves should be 

backwashed and inspected for tears or deformities. Fine mesh sieves 

should never be touched by spoons, scrapers, etc. The most drastic 

cleaning allowable for fine mesh sieves is soaking in mild cleaning 

solutions such as bleach, pancreatin or soap. "Clogged" sieves should be 

discarded if they cannot be cleaned, or the fabric should be replaced.

18.Reporting

A complete, accurate report of the analyst's work (not just results) is essential. Agency 

and court decisions may be based, in part, on the analyst's written report of the analysis 

and reported results. The results should be reproducible, and the analyst's report 

contains an account of every operation performed and by whom, every result, and 

evidence of sample continuity. During the next phase of training, the trainee will be 

evaluated not only on performance of analytical methods, but on reporting of analyses as 

well. Reports should be clean, well-organized, and neat.

18.1. Measurements

A complete description of the filth found during an analysis always includes the 

sizes, in metric units, of the filth elements. This important factual evidence should 

never be omitted. The analyst should use the correct number of significant figures 
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indicated by the method in the measurement of filth elements, reagent amounts, 

net contents, etc. This will help avoid overstatement or understatement, rounding 

errors, and will increase precision.

See MAN-000048 ORA Lab Manual Vol. III Section 4 - Basic Statistics and Data 

Presentation (III-04)

18.2. When reporting filth elements, use guidance in AOAC 970.66. Do not report “Too 

Numerous to Count” on worksheets. Remember AOAC 970.66 B(i) guidance: “If 

amount of filth present makes exact count impractical, report either approximate or 

minimal figure rather than term “too numerous to count.” It is always better to 

report an estimated number (for example, >500, >1000, or other). It should be 

clear that the result was an estimate. Also, be sure to clarify which filth elements 

the estimate applies to. Example: “The number of insect fragments is an estimated 

count. This applies only to insect fragments, not hairs or other filth elements.”

18.3. When reporting arthropods other than insects or mites (examples include 

arachnids, other hexapods such as Collembola, Diplura, Protura, myriapods such 

as millipedes, centipedes, etc.), use an approach similar to that which is outlined in 

AOAC 970.66 for insects. Report number of whole arthropods or equivalents (i.e., 

separate heads or body portions with head attached). Distinguish whole 

arthropods and equivalents in subtotals. Give identity, stage of life cycle, and size 

(mm). State whether whole arthropods are dead or alive. For reporting arthropod 

fragments and other filth elements related to arthropods, use an approach similar 

to that which is outlined in AOAC 970.66 for insect fragments and other filth 

elements. If there are any questions, contact the MPM Council 

(MPMCouncil@fda.hhs.gov). 

18.4. Analytical Filth Worksheet 

A. Objective

To acquaint the trainee with the analytical worksheet (form FD-431) and other 

standard form worksheets, emphasizing proper presentation of analytical results.

B. Assignment

1. Read the AOAC Official Methods of Analysis, current edition, Chapter 16 

on "Light and Heavy Filth;" and see the reporting format described in 

Chapter 5 of the Macroanalytical Procedures Manual for various products 

(particularly the 10 sub dried peas and beans, sequential sampling plan for 

nut products, and others selected by the trainer).

2. Learn the procedures for completing filth analytical worksheets.

3. Examine recent filth food sample worksheets and review the laboratory’s 

various forms and formats used to report filth results. 
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19. Filth Laboratory Safety

Before working in the filth laboratory, the trainee should have laboratory safety training

that is specific to filth activities. Prior to starting an analysis, the analyst carefully 

considers the hazards that could be presented by the equipment and reagents in the 

analysis, and plan measures to avoid or minimize these hazards. The analyst should 

have read the SDS sheets and be aware of the location and use of the emergency 

showers, eyewash stations, first aid kits, safety hoods, personal protection equipment, 

and other safety or emergency equipment in the laboratory. Constant caution and care 

will assure a safe, accident-free working environment. 

During the next phases of training, the trainee should always discuss beforehand with 

the trainer the potential hazards involved in the proposed analysis and the precautions 

needed for a safe analysis.

See MAN-000045 ORA Lab Manual Vol. III Section 1 - Environmental Health and Safety 

(III-01)

20.Visual and Macroscopic Methods

20.1. Objective

The trainee learns and performs various common food analyses for gross 

contamination, and reports analytical results on the official forms, especially the 

analytical worksheet.

20.2. Discussion

Visual and Macroscopic methods examine relatively large amounts of product for 

contaminants easily detected with the unaided eye. A portable magnifying lens or 

magnifying lamp may be used, but the magnification range should not exceed 5X 

for general purposes. Lenses of higher power have restrictive fields of view and 

impractical, short focal working distances. If the analyst suspects the 

contaminants are likely to be much smaller than a poppyseed, or otherwise 

difficult to detect, then reliance should not be placed solely in a visual 

examination. A stereomicroscopic method should also be used to evaluate the 

contamination.

A. Sample Preparation

1. Sieves

Dry sifting is a common way of separating macroscopic contaminants from a 

large bulk of product. The material retained on a particular sieve is referred to 

as "overs" and the material passing through the sieve as "throughs." One 

versatile feature of analytical sieves is that they can be nested in order of 

increasing fineness so various sizes of particles can be separated out in one 

operation. Sieve mesh sizes are indicated by a standardized number scale, 
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with the larger numbers denoting successively finer meshes. In general, 

standard Number 8 mesh sieves are used to retain larger rodent fecal pellets, 

gross contaminants such as sweepings, or product units the size of a small 

pea or larger. Standard Number 20 mesh sieves will retain most adult insects, 

many larvae, and smaller rodent pellets. Sieves that are finer than Number 40 

mesh are usually too fine for macroscopic applications. Between operations, 

these sieves can be cleaned using an air current and a dry, clean towel, or 

they can be washed. Also note that for some products, like peppercorns, 

special sieves have been designed to sift and grade the product and to 

determine the “fines” in the finished product. 

2. Jones or Riffle Divider

This device can be used to thoroughly mix products such as wheat kernels or 

coffee beans. Passing the sample six times through a Jones divider produces 

a totally random distribution of product units. Similarly, it can be used as a 

sample-halving device; the device halves a sample on each pass through the 

divider. A portion of product can be isolated from the main bulk by passing 

through the Jones divider and discarding successive halves until the desired 

portion size is approximated.

3. Seedburo Grain Inspection (Picking) Tray and Cover

Commonly used in USDA Grain Inspection Services, the lower unit consists of 

a metal tray with linear groves in which the product rests. The top is also a 

tray, but with a compressible foam pad. Spread and examine the product on 

the bottom tray, then when finished, add the top tray, compress, then invert the 

two trays together, and remove the (now, top) grooved tray. This allows one to 

see the opposite side of the materials. It helps insure that all sides of the 

individual grains are examined without having to turn each piece individually.

4. 20” Wide Roll of White Butcher Paper

Paper on one side, plastic coated on the other—this inexpensive paper is both 

strong and large enough to examine almost any sample. Used plastic side 

down, it can be cut to whatever length needed, and when replaced, (i.e.,

between each subsample analysis), it insures subsample integrity and a new 

clean working surface. Samples can be shaken into a singular flat layer, and 

then the items can be manipulated as individuals, or grouped together. The 

paper can be folded, or closed, to accommodate easy transfers back to the 

original containers. Wet samples can also be examined by using the plasticcoated side. 

5. Pharmacist’s Tablet or Counting Tray

This small 6” X 8” tray has a flat surface with a round grove on one side of the 

tray. The grooved side has a matching cover that flips into place to cover the 
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grove. Tablets (or in our case, beans, peas, nuts) can be counted (in groups of 

five or ten) and slid into the groove until the desired number is reached, then 

the lid closed, extra product removed, and the counted units poured out for 

closer examination or weighing. It can be used for estimating product weight 

versus count, and for sequential sampling purposes. 

6. Examination Area

It is recommended that a 2’ deep by 4’ wide area be set aside that is clear of 

obstructions and superfluous equipment. It should be easy to clean, preferably 

with a white or wheat colored matte surface. The area is to be well lit, at 150–

200-foot candles, preferably with natural colored, shadow and glare free 

adjustable lamps. Ideally, the tabletop height can be adjusted to the analyst’s 

need and will be open underneath for knees, stool or chair work. 

7. Interpretive Line System

This is a series of photographic slides or photographs originally developed by 

USDA for their GIPSA grain inspection service. These materials are 

purchasable from Seedburo and provide exemplars of the types of damage 

that may be seen in various products including barley, beans, corn, lentils, 

peas, rye, safflower, sorghum, soybeans, sunflower, wheat and other products. 

It should be used in combination with FDA authentics, toxic and weed seed 

identifiers, and the FDA slide series.

8. X-ray machine

X-ray radiography can be used for rapid examination of grains, coffee beans, 

spices and legumes for internal insect damage. It can also be used for other 

types of contamination like metal and bone particles in processed foods. 

Modern bench top x-ray machines are rapid and relatively easy to use. They 

do not require radiographic film and developing, like their predecessors.

9. Other Tools

Various spoons, spatulas, knives, flour slick, small brushes, pans, trays, fine 

and coarse forceps, needles, and various sized petri dishes. 

20.3. Assignment

A. Perform the indicated visual and macroscopic analyses under the direction of 

the trainer. (Substitutions may be made based on product availability, 

preserving the type of analyses presented)

B. Report the results of each analysis using the correct forms and formats.

C. For each analysis, discuss with the trainer:

1. the method used and difficulties encountered
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2. results and their significance 

3. the quality of reporting

20.4. References

A. Whole cereal grains (Macroanalytical Procedures Manual (MPM), Chapter 

V.3.A.). The analyst is to pick out whole insects, webbing, fecal pellets, 

extraneous material, and kernels damaged by heat, mold, and/or pests. Good 

lighting and a light-colored, non-reflective background are needed. Insects 

cause damage to kernels either by surface feeding or by tunneling. Rodentgnawed kernels can be recognized by the scalloped edges, a result of the 

rodents' paired gnawing teeth. Use of a Jones divider may be needed.

B. Green coffee beans, dried beans, or lentils (MPM, Chapter V.1.A, V.11.G.). 

For bulk sampled product, a Jones divider is useful for mixing or compositing 

as well as for obtaining the analytical portion. The trainee looks for insects, 

insect damage, and mold. Mold is confirmed microscopically, by observing 

hyphae and/or fruiting bodies. If possible, the grading of coffee can be 

demonstrated by a trainer. (FDA By-lines 7:285-91, May, 1977.) Lygus bug 

damage to beans should be demonstrated.

C. Flour (MPM, Chapter V.2.B). The flour is sifted portion wise through a standard 

# 20 mesh sieve. Examine sieve "overs" for filth. The portion size is generally 

the entire contents of a consumer package. Sieve "throughs" may be saved for 

additional analysis in the next section.

D. Whole or crude spices (MPM, Chapter V.8.). Analysis of spices often entails 

breaking or cracking open a large amount of product in search of insects or 

signs of insect activity. Rodent defilement may also be encountered. A special 

sieve is used for peppercorns, over which the product is passed a specified 

number of times. The analyst should check beforehand whether the product, 

especially peppercorns, has been fumigated for bacterial contamination. 

Cinnamon or Cassia sticks are cracked for internal mold. The exterior and 

interior of Capsicum sp. pods need to be examined for insect damage and 

mold.

Safety Note: Precautions and clean-up for Salmonella-type organisms may be 

indicated if there was no fumigation. Mite infestations appear as “moving” dust 

or surface feeding on inner or protected surfaces. Feeding surfaces are 

sometimes discolored, with adhering, fluffy, granular material (cast skins) often 

encountered in surprisingly large quantities.

E. Whole figs or dates (MPM, Chapter V.9.F). This type of analysis may call for a 

statistically derived sequential examination plan that is faithfully executed for 

valid results. A sharp knife is needed to cut the fruit since the insect 

contamination usually occurs near the center or pit. An occasional small wasp 
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may be found in some types of figs. These insects are tolerated since they are 

essential to develop and pollinate the fruit and they enter into the fig to 

complete their life cycle.

F. Whole tamarind pods (MPM, Chapter V.9.F). The tamarind pods need to be 

broken open to look for the presence of insect contamination and mold. Whole 

insects like the tamarind weevil (Sitophilus linearis) and mites can be found in 

tamarind pods. Evidence of Lepidoptera larvae often can be seen by the 

presence of webbing material and excreta pellets.

G. Shell nuts (MPM, Chapter V.10.A). For this analysis, a sturdy hammer and 

pounding block are helpful. The various types of reject nuts are described in 

the method. Mites can sometimes enter pecans or walnuts through breaches 

between shell halves and can build up impressive populations inside the nut, 

causing considerable damage.

H. Cocoa beans (MPM, Chapter V.4.A.). Mold should be confirmed 

microscopically. The trainee should discuss with the trainer the best technique 

for mixing the sample, considering all factors including size of sample, size and 

number of subsamples, administrative guidance, etc.

I. Blueberries or cherries for maggots (AOAC Official Methods of Analysis, 

current edition, Chapter 16 on "Extraneous Materials: Isolation;" or MPM, 

Chapter V.9.C or D). Preliminary manipulation of the product is needed to free 

the maggots from the fruit tissue. Other insects may be encountered, as well 

as decomposed berries. If substantial decomposition is suspected, additional 

analysis may be indicated. Maggots are preserved in dilute (60-75%) ethanol.

21. Microscopic Methods

21.1. Objective

The trainee will learn how to perform the most common types of analyses for “light 

filth” in foods using various flotation techniques, examine the recovered material 

for “microscopic filth,” and report the analytical results on the worksheet.

21.2. Flotation Techniques

Flotation methods are designed to isolate microscopic filth by floating the filth 

upwards, typically in an oil/water-phased system. Insect fragments, mites, and 

hairs are lipophilic and like to be in the oil phase, thus they float to the surface with 

the oils, (hence the term “light filth”). Plant tissues and most related tissues are 

hydrophilic, and they prefer to stay in the water phase. Common gravity further 

helps this process, and larger particles sink. To accomplish the separation of filth 

from food, use a number of solution systems to insure that the majority of the 

product sinks, while the oils with trapped filth, floats. Often, the analytical portion 

undergoes a pretreatment, to enhance this effect. The analyte (filth) portion is 
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usually very small, both in a weight-to-weight relationship to the food (parts per 

million) and in size or scale. Typically, the recovered filth contaminants are 

examined under a widefield stereomicroscope. Once found, the filth items may 

have to be mounted for microscopic identification, thus the term “Microscopic 

examination”.

Most microscopic methods are found in Chapter 16 of the AOAC "Official Methods 

of Analysis," consisting of a compilation of validated methods. The trainee should 

be familiar with the initial information concerning reagents, apparatus, and 

techniques, and the safety precautions referenced parenthetically in the text of 

each method.

All microscopic methods begin with the analyst weighing out a prescribed amount 

of material to be analyzed. This step is critical because the guidelines for how 

much filth can be in a violative sample, is based on a set amount of product being 

analyzed. 

From this point on, the exact steps followed may vary significantly from product to 

product and are prescribed in the method; the method is to be followed precisely. 

Variations in methodology based on product

1. There may or may not be a pretreatment step to remove excess fats and 

oils found in some foods (e.g., spicy sauces). 

2. A digestion step is often used to hydrolyze or digest the product into small 

particles (e.g. - bread). Some foods, once digested, are wet sieved, that is 

washed with very hot water over a fine sieve, to flush away excess product 

and oil.

3. Some products may need a hydration, to swell, add water, or saturate the 

product (e.g., raisins)

4. Alcohol may be added to the water, to help saturate the product, penetrate 

the skins, and dissolve or solubilize excess fats or oils.

5. Detergents may be added to loosen the filth, to saturate the food, to 

emulsify or trap the oils.

6. Compounds such as salt or EDTA may be added to help products sink or 

make them heavier, or to make the water phase even heavier.

7. Light hydrocarbon products may be added, or the choice of flotation oil 

changed to help capture the lipophilic filth.

There are basically two physical systems to use in extracting light filth from foods, 

the Wildman trap flask, and the corning percolator. The Wildman trap flask is a 

closed system, that is, the volume and composition of the aqueous phase remains 

constant. The filth laden oil interface layers are captured in the neck of the flask. 
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Utilizing a rubber disk on a rod, the oil and interface layers are cleanly separated 

from the aqueous phase and removed (poured off) from the trapping system. The 

corning percolator is an open system which allows for repeated drain and refill 

cycles further isolating the oil filth interface layer. The final drain of the percolator 

results in a totally isolated oil phase with just a very small amount of the aqueous 

layer left.

The final step of most flotation methods is to transfer the oil and extracted filth to a 

ruled fast draining filter paper for microscopic examination. For this we use a 

Buchner funnel- fitted into a large side arm flask attached to a vacuum pump. A 

Buchner funnel is an open top funnel, with a porcelain screen about 1 cm below 

the opening. A 60-mesh brass or stainless-steel fine screen is cut to fit over the 

porcelain openings. This screen serves to evenly distribute the vacuum under the 

filter paper and to help distribute the product over the filter paper. The filter paper 

is wetted, and then centered over the opening, and the vacuum draws the paper 

down into the funnel forming a small cup-shaped filter. The trapped filth and oil is 

poured into the filter paper cup and the recovered tissues and filth are evenly 

distributed over the filter paper. Each extraction paper is then placed in a petri 

dish bottom plate, and glycerin alcohol, a (1 + 1) mixture of glycerin and 95% 

ethanol, is added to partially wet the material on the paper, but not to the point 

debris can float freely over the paper. The edges of the filter paper cup are laid 

down, and the lid is placed on the dish to keep out dust and minimize evaporation 

until the extraction paper can be examined. Each petri dish is immediately labeled 

with sample, sub number, date, and the analyst's initials. Extraction papers 

become part of the sample reserve and should be carefully preserved. 

Refrigeration or the addition of a few drops of formaldehyde are effective 

preservatives after the papers have been examined.

Note: The petri dish should not be inverted, with the filter paper in the larger 

diameter top, covered by the small diameter piece. This allows airborne 

contaminants to contact the filter paper and causes rapid evaporation of the 

glycerin alcohol. It may also cause loss of some filth by adherence to the parts of 

the dish that contact the filter paper.

21.3. Assignment

A. Read pp.173-180 in: Gorham, J. R. (Ed.) (1981). Principles of food analysis for 

filth, decomposition, and foreign matter (FDA Technical Bulletin No. 1, 2nd 

ed.). Gaithersburg, MD: AOAC International.

B. Perform the indicated analyses under the direction of the trainer.

C. Report the results of each analysis using the forms and formats.

D. For each analysis, discuss with the trainer:

1. the method used and difficulties encountered 
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2. results and their significance

3. the quality of reporting 

21.4. References

A. Flour (AOAC Official Methods of Analysis (OMA), Method 972.32 "Light Filth 

(Pre and Post Milling) in Flour (White)"). Acid hydrolysis is employed to digest 

the flour, and the filth is isolated by flotation in a percolator. Due to the 

uniformity of flour milling processes, pre-milling and post-milling contaminants 

can be roughly recognized by size. It is, therefore, important to record the size 

ranges of contaminants so that compliance personnel can accurately interpret 

the analytical results.

B. Fig or fruit paste (AOAC OMA, Method 964.23 for "Filth in Fig and Fruit 

Paste"). Filth flotation is accomplished using a Wildman trap flask. The trainee 

practices quantitative transfer from the flask to a beaker prior to attempting the 

analysis. A smooth motion is needed to pour the trapped material into the 

beaker while holding the trap flask rod out to exclude the aqueous phase of the 

liquid system. About 1/4"-1/2" of aqueous phase is trapped off along with the 

oil phase to ensure that no filth is left below the stopper. While holding the 

stopper in the "up" position, the flask neck is rinsed with the same aqueous 

solution and the rinses are poured into the beaker.

C. Chocolate (AOAC OMA, Method 965. 38 B (b) “Filth in chocolate”). This 

method uses a detergent to defat the product prior to the flotation. The 

extraction papers (filter papers containing the extracted filth) will give the 

trainee a challenging exercise in distinguishing between very similar-appearing 

insect and plant fragments and introduce bleaching techniques that may prove 

useful in other situations.

D. High bran content bakery goods (AOAC OMA, Method 972.36 for "Light Filth in 

High Bran Content Breads"). This method employs a defatting step to 

enhance the separation of filth from product. The defatting should be 

performed in a hood to avoid fumes.

22.Sedimentation and other Specialized Techniques 

22.1. Objective

The trainee will learn how to perform some common types of food analyses for 

various types of filth not easily recovered by flotation, and how to record the 

results. 

22.2. Discussion

Three very different problems that cannot be resolved by using any of the 

techniques described thus far will be demonstrated in this section:
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1. The first problem deals with “heavy” filth. As the name implies, these 

methods rely on a combination of gravity and density in a liquid system that 

allows the material to sink to the bottom of the container, while the lighter 

generally organic materials are floated off. 

2. The second problem deals with thin-skinned insects, like maggots, insect 

eggs, and mites. Because of their thin exoskeletons or shells, they are less 

oleophilic than the more mature stages of the animal, and they too prefer to 

sink- even in oil/water phased systems.

3. The third problem is how to count/detect maggots or mites if they are 

buried deep inside the plant tissues, and they are attached to tissue 

material?

A. Heavy Filth

In heavy filth methods, we are typically looking for heavy material 

contaminants such as sand, glass, metal fragments, and even feces. Basically, 

the analyst is going to make a liquid slurry of solid materials using different 

solutions, that, with stirring, act to both solubilize the material to loosen up the 

product and free the heavy materials, thus allowing the heavy materials to 

sink. In the past, carbon tetrachloride and chloroform were used, now solutions 

like chloroform alone or salts are used, which when added to water, solubilize 

and make the solutions much denser than water alone. The higher the density 

of the solution, the greater the chance that the light weight organic material will 

float and the heavy materials will sink. 

Keep in mind, that usually hairs and insect fragments are not recovered by 

these methods. The analyst often will use the heavy filth method as a prelude 

or preliminary step to a light filth extraction. These methods are a very good 

way to defat or remove excess oils from the food product. The analyst usually 

retains the poured off organic materials for his next analysis, which is typically 

light filth. Hairs and insect fragments come off in the organic material, and that 

material should be examined for these and other types of filth (especially fecal 

pellet fragments). 

Heavy sediments such as glass and metal shards, sand, minerals, bone, and 

other materials may adulterate some products. The analysis should be tailored 

to the specific kind of analytes suspected in the product. For example, if glass 

contamination is suspected, the analyst avoids using all glass apparatus such 

as glass beakers and stirring rods. The product's original container is always 

examined to determine if it could have contributed to the contamination.

Once these materials are recovered, they are described. Pictures help, but a 

physical description of the recovered material is annotated on the worksheet. 

The descriptions should be precise, accurate and brief. Although it is not 
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possible to cover all of the characteristics or terms used in material 

identification, for training purposes, one or more of the following characteristics 

may help describe these materials (this is not an all-inclusive list; use a 

thesaurus if needed, and modifiers when needed).

Material Descriptions: Size, shape, color, finish (matte/gloss), variegation, 

surface texture, surface coating, lamination, presence of a parental (original) 

face or markings, scoring, density, fracture, fraying, melting point, density, 

inclusion, assemblage, distance, interval, parallelism, obliqueness, angularity, 

position, curvature, softness, elasticity, voids, optical character, etc.

For example, glass has many characteristics, but it can be differentiated from 

sand grains or other crystalline look-alikes by two principle characteristics. 

Broken glass exhibits acute angular, conchoidal fractures (resembling the 

markings of a clamshell) and it shows no color (isotropism- no optical activity) 

when viewed between crossed Nicols on a polarizing microscope. When 

combined with size, shape, hardness, density, solubility, parental face 

characteristics (if present), and refractive index, there is little doubt left towards 

the identity of the material. The analyst must list the pertinent facts in the 

report and draw a conclusion based on those facts. Keep the report brief and 

to the point. 

B. Maggots and Insect Eggs

Another problem area deals with the isolation of thin-skinned insects, like 

maggots, insect eggs, and mites. As stated earlier, because of their thin 

exoskeletons or shells, they are less oleophilic than more mature stages of the 

animal, and they prefer to sink even in oil/water phase systems. In the olden 

days, people did not want to go anywhere near tomato canneries because the 

canneries would dump the waste tomato skins in huge piles outside the plant. 

This created ideal breeding grounds for flies and maggots. Some of these flies 

ended up inside the plant and the eggs and newly hatched maggots would be 

in the product. One of the first things filth analysts learn is if a mixture of oil and 

tomato tissue floats, then the eggs and the larva sink to the bottom of the flask. 

In the method below, Analyses Section D.2., the maggots and eggs are 

drained off, the oil and tomato tissue remain in the separatory funnel. This 

method is exactly opposite of the extraction procedure in “light filth” methods.

C. Maggots and Mites in Mushrooms

Maggots and mites present the filth analyst with a real challenge, especially 

when embedded deep in the product’s tissues. To extract them, the maggots 

and mites are mechanically freed; this is done in a blender. A challenge still 

exists when they are free; the maggots cannot float off, nor can they sink 

because of the extra tissues they are mixed with. This method demonstrates 

staining the maggots and mites, so they are much easier to see. It is a unique 
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method that works very well for all mushroom products and incorporates a 

bleaching technique that illustrates how easy it is to clear tissues, yet not affect 

the filth one is looking for. It also teaches the analyst to be careful in how long 

to blend the products and how many subs can be done in a timely manner. 

Analysts should practice with training samples before receiving samples for 

regulatory analyses. 

22.3. Assignment

A. Perform the indicated analyses under the direction of the trainer.

B. Report the results of each analysis using the correct formats.

C. For each analysis, discuss with the trainer:

1. the method used and difficulties encountered

2. results and their significance

3. the quality of reporting

22.4. References

A. Ground spices for heavy filth (AOAC Official Methods of Analysis (OMA), 

Method 978.21 for “Light Filth in Capsicums (Ground), sedimentation”). The 

liquids selected for these methods are dense enough to float the spice material 

but not heavy filth such as feces, rocks, sand, and dirt. Decanting the spice 

material leaves the heavy filth behind to be examined microscopically before 

the weight is determined. The cautions indicated in the text are to be observed. 

Substitute Chloroform for all Carbon tetrachloride citations. 

B. Tomato products for fly eggs and maggots (AOAC OMA, Method 955.46 “Filth 

in Tomato Products”). A large separatory funnel is used to separate product 

from maggots and fly eggs. The latter settle to the bottom and are drawn off 

through the stopcock. The analyst should become familiar with the appearance 

of fly eggs and maggots prior to attempting the analysis.

C. Mushrooms for maggots and mites (AOAC OMA, Method 967.24 "Filth in 

Mushrooms"). A dye is employed to make the filth highly visible (purple) 

against the background of bleached (white) product. The product is blended to

free maggots and mites that are embedded in the mushroom tissue or wedged 

tightly in the gills. The crystal violet dye is soluble in ethanol for clean-up 

purposes but try not to get it on the hands.

23.Mold Detection

23.1. Gross Mold Contamination

A. Objective
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The trainee will learn the general diagnostic characteristics of mold.

B. Discussion

Most people can recognize visible mold growth by its characteristic growth 

habit, colors, and musty odor, without the aid of magnification. Under a lowpower hand lens, however, typical mold consists of a mass of thread-like, 

branched filaments. The mass is called a mycelium and the individual 

filaments are called hyphae. Spore-bearing fruiting bodies, whose shape 

varies between species of mold, may also be found.

In most cases, however, macroscopic observations are insufficient for FDA 

purposes. Some non-mold plant diseases or other conditions may superficially 

resemble mold. Also, decomposition can sometimes mask the presence of 

mold. For these reasons, the analyst confirms the presence of mold 

microscopically. This can be done by preparing an aqueous slide mount of a 

small portion of the suspected mold, examining it for the presence of hyphae. 

Microscopically, mold hyphae can be distinguished by one or more of the 

following characteristics:

1. Parallel walls. Although individual hyphae may vary in size, the diameter of 

any single hypha is constant. Mold hyphae are basically tubular, with 

parallel walls. 

2. Septation. The presence of parallel cross walls (septa) in hyphae is a 

characteristic of many molds. They give the hyphae a segmented 

appearance. Some plant hairs may have cross walls but they are usually 

not parallel to each other.

3. Granulation. The protoplasm of mold may exhibit a distinct granular 

appearance. This is an especially useful characteristic among larger 

species that may not exhibit septation. 

4. Branching habit. When present, this is one of the most reliable 

characteristics. Mold hyphae typically have branches that are the same 

diameter as the main trunk. Typical mold branches extend out at right 

angles to the main trunk. 

5. Rounded ends. The natural tip of a hypha is normally rounded like a test 

tube bottom. Hyphae that are broken, however, typically break off squarely.

C. Assignment

1. Using moldy fruits or other foods, practice preparing aqueous slide mounts 

and distinguishing, under a compound microscope, the mold hyphae.

2. Examine at least two samples of different whole fruits, vegetables, or 

spices for macroscopic mold contamination, confirming the mold 
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microscopically. Methods for some of these products can be found in the 

"Macroanalytical Procedures Manual".

3. Report the results of each analysis on the correct forms, discussing each 

report with the trainer.

23.2. Howard Mold Count

Howard Mold Count procedures are empirical methods that are to be precisely 

followed in every detail for each type of product to obtain satisfactory results. 

Experience has shown that mold counting cannot be reliably learned without the 

help of an experienced instructor who can give the trainee personalized 

instruction.

A. Microscope

A Howard Mold Count is performed on a compound microscope with certain 

features. The first requirement is a lens system that has a standard 

microscope field diameter of 1.382 mm. The eyepiece has a micrometer disk 

ruled in squares, each side of which is equal to one-sixth the diameter of the 

ocular lens opening. A Howard Mold Count cannot be performed using a 

microscope that does not meet these requirements.

B. Howard Mold Count Chamber

This is a specially constructed slide and cover glass unit that is used. It is 

designed to contain 0.03 cc of material on a central platform with the cover 

glass in place. The platform is surrounded by a moat and flanked left and right, 

beyond the moat, by shoulders whose height is 0.1 mm taller than the 

platform. The combined exacting requirements of the microscope and Howard 

Mold Count chamber ensure that the analyst, at all times, views a precisely 

known amount of product. Each Howard Mold Count chamber has a scored 

calibration circle of 1.382 mm diameter, or has scored parallel calibration lines, 

1.382 mm apart, to use to check the standard microscope field diameter.

C. Sample Preparation 

The product is prepared exactly as stated in the method for that product. The 

sample is thoroughly mixed both before and after dilution. Immediately before 

each slide is prepared, the sample again is thoroughly mixed. This is important 

to assure uniform suspension of mold filaments.

D. Slide Preparation 

For each preparation, the Howard Mold Count chamber is completely clean 

and dry. A clean scalpel is dipped into the well-mixed sample and then 

touched against the platform of the Howard Mold Count chamber so that just 

enough sample is transferred to fill the platform when the cover glass is in 
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place. The drop of sample is evenly spread using the scalpel, and the special 

cover glass is lowered over the platform until it almost touches the product with 

the cover glass sides aligned with the shoulders. The cover glass is quickly 

pressed down, spreading the sample evenly over the platform and avoiding the 

trapping of air bubbles under the glass. A proper preparation has the platform 

entirely filled with the product, no air bubbles, and no spillage into the moat. If 

a proper preparation is not obtained, the entire chamber should be cleaned, 

dried, and another attempt made. 

E. Newton's Rings 

Newton's rings are a rainbow-type optical phenomenon produced between 

each shoulder and the cover glass when they are in contact without applied 

pressure. The rings are observed by holding the completed preparation at a 

slant so that light is reflected off the cover glass. The presence of Newton's 

rings assures that the depth of product on the platform is 0.1 mm. Their 

absence indicates that either the slide was not thoroughly cleaned and dried or 

that product solids thicker than 9.1 mm are holding the cover glass above the 

designated height. In either case, a new attempt is to be made with a clean, 

dry slide and cover glass.

F. Examination of the Slide

After the slide is placed under the microscope, it is brought into focus and the 

field examined. The fine adjustment is used to bring into view mold filaments 

that may be at different depths of the field. The method calls for a field be 

counted as positive when the aggregate lengths of not more than three 

filaments of mold exceed one-sixth the diameter of the field. One-sixth the 

diameter of the field is not enough to be counted as positive: the aggregate 

length exceed one-sixth the diameter of the field. The drop-in eyepiece 

micrometer disk, divided by etched lines so that 36 equal-sized squares are 

formed and any side of which measures one-sixth of the field of view, should 

be standard equipment for mold counting.

In the examination of a slide for mold, the field should be selected in a 

consistent manner. One method is to begin at what appears, through the 

microscope, to be the upper left portion of the counting area and go straight 

across, skipping every other field, then drop down approximately two fields 

and, reversing the direction, again cross the counting area, continuing this 

back and forth until 25 fields are examined. Fields to be counted should be 

selected at random. Under no circumstances should fields be selected for 

counting because they do or do not contain mold.

If it is readily observed that the field is positive, it should be so recorded and 

study of the next field begun. If not enough mold is observed at first glance, the 

field should be carefully examined and the fine focusing adjustment used to 
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bring different depths of the field into focus. In some instances, the mold can 

be seen better by changing the light intensity. A systematic search of every 

part of the field is needed before it can be concluded that a field is negative. 

When branched filaments or clumps of mold are found, the length of one 

filament is considered as the sum of all the branches or the sum of all the 

filaments in the clump. The fruiting heads, such as those of Alternaria, with any 

attached mycelia are counted as mold filaments. If the examination of a field 

reveals a piece of suspect material extending into the field from the edge, the 

material should be traced back so that its true identity can be established.

However, only mold found within the field should be considered in determining 

whether the field is positive or negative. Should the identity of any filament be 

in doubt, it may be studied at a magnification of approximately 200X, although 

the length is determined at 100X. Unless the suspected filament is 

unquestionably mold, the field should be counted as negative. Small air 

bubbles, which in aggregate do not exceed one-sixth the diameter of the field, 

may be disregarded. Occasionally, a field is largely obscured by a mass of 

opaque material or air bubbles. In this case, if a count cannot be determined, 

the analyst should move on to the next field.

G. Calculation of Results

The results are calculated from the findings on examination of 25 fields from 

each of two or more slide preparations. Because comminuted fruit and 

vegetable products are mixtures rather than solutions, mold filaments are not 

always uniformly distributed among the plant fibers and tissues in the 

individual droplets used for slide counts. Therefore, the count of several slides 

of the same sample may vary, even though the slides are prepared and 

examined with the greatest care. Studies of deviation in mold counts indicate 

that the results are grouped about the average in the same way that other 

mixtures follow the rules of random distribution. This may account for the 

occasional wide variations of results. 

A general rule is that two counts from the same sample should check within 

three positive fields; otherwise, two or more additional slides should be 

examined. For greater accuracy, more fields may be counted.

23.3. Assignment

A. Read pp. 191-200 of: Gorham, J. R. (Ed.) (1981). Principles of food analysis 

for filth, decomposition, and foreign matter (FDA Technical Bulletin No. 1, 2nd 

ed.). Gaithersburg, MD: AOAC International.

B. Perform a Howard Mold Count on at least two types of tomato products and 

one type of berry product under the guidance of an experienced trainer. Use 

the correct AOAC method for each product.
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23.4. Evaluation

With a sample provided by the trainer, perform a Howard Mold Count. The trainer 

performs an independent analysis to compare with the trainee's results for the 

evaluation of performance. The use of a compound microscope with a dualviewing body is recommended so that the trainer and the trainee can view the 

sample at the same time.

24. Analysis of Factory Filth Samples and Filth Analytical Worksheets

New Hire Analysts should have already reviewed the ORA U site on the 

preparation of Analysts Worksheets and should have discussed this with their 

trainers. Filth worksheets follow the same format and rules as all other worksheets 

follow, differing only in the final content and formatting. To some extent, they are 

easier to complete, yet more complicated in content. This section provides 

guidance and practice in describing what initially appears to be very complicated 

samples, but are relatively simple exhibits that need to be broken down into 

component parts. Also, submit photographs to help document findings and help 

others visualize in their minds what is being described. 

For most exhibits, a generic method statement, like the following, covers all the 

analytical methods:

Methods: Visual, Macro and Microscopic Examination, with pick out, for Gross 

Filth. For the Confirmation of Rodent and Bird Adulteration, See FDA Bylines #3, 

Nov. 1970, pp. 153-164, with method up-dates in the AOAC 17th edition, Chapter 

16, with Supplements. See also FDA Technical Bulletin #1 Chapters 9 and 13 and 

FDA Technical Bulletin #5 Macroanalytical Procedure Manual Chapter V Parts 2B 

and 3A(4)(B) and Chapter ∇II Part 4. See also Zimmerman, M.L. and S.L. 

Friedman, 2000, "Identification of Rodent Filth Exhibits", Journal of Food Science, 

Vol. 65 (8): 1391-1394, and “Insect Penetration through Packaging Material” 

(AOAC 16th 16.15.05 973.63). Exhibits placed in labeled petri dishes for 

examination. Insects examined as above, using visual and macroscopic exam. 

Where applicable, see Results below for method citations as they are used in the 

analysis.

For each subsample, the analyst identifies the subsample and the analysis called 

for (rodent, insect, or other filth, some, or all the above.). This is done by visual or 

stereoscopic examination and confirmation of the subsample's description versus 

the investigator's collection report. Begin by physically segregating the filth into 

general categories, being sure to capture loose elements first. The type(s) of 

analysis to be performed will be dependent on this information; do not limit the 

analyses to those requested on the C/R. Often, additional items of “filth” are 

found; include these items in the description. For example, the investigator has 

identified rodent adulteration, but the investigator may not have seen or identified 
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the mites or webbing from moth larvae also present. Also, keep in mind the 

differences between 402(a)(3) and (a)(4) evidence, and where needed 

highlighting (a)(3) evidence, as it could be a separate charge within the sample.

Consideration should be given to the preparation of the exhibits for use in the 

courtroom. These exhibits may be displayed or passed around; prepare these 

exhibits for optimum viewing yet present no hazard to those handling the exhibits.

Safety Caution: Generally, these are grossly contaminated samples, and the 

analyst should use caution and protect themselves in preparing the exhibits. 

Personal Protective Equipment, ventilation and preparation sites should be 

located and used to prevent aerosol release of potentially harmful agents or 

fumigants.

A. Whole insects 

In addition to identifying the insects, the analyst should record the following:

1. Counts or approximations- Count an area and estimate the number with 

multiplication factor - do not use “too numerous to count” (TNTC) 

2. Record if the insects were dead or alive.

3. Record stages of growth present.

4. Record evidence of fumigation or preservation by the inspector (This 

should be included on the C/R, but mistakes happen, and they should be 

noted.)

5. Pliability, moistness, presence, or absence of body fluids if it adds 

information regarding the relative age of the contamination.

6. Associated material (adulterated product, fecula or pellets, cast skins, etc.)

7. Size ranges.

B. Pellets and other excrement

1. Origin or source are identified, i.e., size and range, shape, presence of 

mucous coating and/or hairs. As applicable provide: 

• Description of odors (none, weak, strong, fecal, or urine-like) 

• Age is difficult to determine but can be estimated, note if pliable, 

moist, insect damaged, bleached or discolored, or if brittleness is 

present.

• Constituents make up 

2. If necessary, confirm source using chemical confirmatory tests. 

C. Urine stains and bag cuttings

1. For all layers, mark and identify the interior and exterior surfaces. Note 
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approximate size, shape, layers, and construction, e.g., “4-layer kraft paper 

bag cutting with inside 3rd layer plastic bag liner”.

2. Look for loosely adhering filth (hairs, mites, insects, etc.) stereoscopically. 

Pick them off, prepare and identify. Note their presence. 

3. Look for visible stains. Note size, shape, characteristics. 

4. Switch from visible light to Long Wave Ultraviolet black light, and in pencil, 

accurately outline the fluorescing stains. Note if the stains penetrate and 

how far. Note size, shape, characteristics.

5. Product beneath stains may also be contaminated, as evidenced by 

fluorescence, caking or lumping, or adherence to the packaging.

6. Finally, select the most characteristic stains and perform the chemical tests 

needed to confirm the presence of mammalian urine and its source, 

human, mouse, or other mammal.

D. Rodent/Insect Gnawing

For rodent gnawing, in addition to the gnawed hole itself, adhering hairs, 

pellets, or urine stains may be found at or near the gnawed site. Note and 

confirm these as above. Gnawed packaging or product should be examined 

macroscopically and characterized as rodent or insect gnawing. Rodent 

gnawing has a typical serrated appearance (i.e., paired crescent-shaped cuts, 

with double incisor tooth marks). Insect gnawing, exhibiting liner striations, 

may also exhibit webbing, pellets, cast skins, setae, etc. 

1. Record minimum-maximum diameter of each hole or gnawed area and 

location

2. Record direction of penetration of insect gnawed packaging (terracing) 

AOAC 17th ed. 973.63.

3. Record whether or not gnawing penetrated packaging completely

4. Record adhering product

E. Nesting Material

Rodents use their incisor teeth to pull apart and gather soft materials such as 

paper, cloth, cardboard, feathers, etc. Confirmation of nesting material 

includes:

1. Material appearance characterized as shredded, frayed, or ragged.

2. Presence of gnaw marks, urine, hairs, pellets, feathers, insects or 

parasites.

F. Dead Animals
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Occasionally dead rodents or related materials may be collected as exhibits. 

As with insects, these items need to be identified following traditional 

mammalian taxonomy procedures. They may also yield additional forensic 

information such as the presence of parasites (CDC washing and combing 

procedures,) and decay, desiccation, or putrefaction stages can be estimated. 

The references cited below can help in this work. 

G. Floor sweepings, trash collections

These exhibits may contain all the above, and more. The easiest procedure is 

to take a picture, then segregate out the important or significant items that may 

not be evident in the picture. A simple inventory of these items is typically 

sufficient, and unimportant items can be lumped together as “wastepaper, 

product, and/or debris” 

H. Product and packaging blanks

Product and packaging blanks (uncontaminated “control” portions) should have 

been included in the factory filth sample. These should be analyzed in the 

same manner as contaminated material. If no blanks are included in the 

sample, the analyst derives them from the uncontaminated portions of a 

subsample or by securing other credible blank materials.

I. 702(b) portions

These should be set aside and not analyzed; the reserve samples are required 

by law. When returning the sample to the sample custodian, consider creating 

or segregating the 702(b) portion. It should be in a clearly identified sealed 

package and the contents fully described on the C/R. This will save a 

considerable amount of time should the firm request the 702(b) portion. 

Note: 702 (b) portions are not required for exhibit samples that are not tied to 

or connected to a specific lot of product and are used to illustrate conditions at 

an establishment (e.g., rodent excreta pellets) or samples collected from 

equipment or surfaces to demonstrate the insanitary conditions of an 

establishment.

J. Reporting the Reserve sample

The reserve sample, including the 702 (b) portion (if present), is described 

completely on the analyst worksheet. Quotations of all identifying labels 

prepared by the analyst are included. The analyst preserves as much of the 

sample as possible in its original condition.

Fumigation or preservation applied by the analyst is noted. Finally, the seal (if 

needed) applied by the analyst is quoted and the disposition of the sealed 

sample is stated. Follow standard operating procedure when holding or 

returning the reserve sample, including the 702 (b) portion. Special storage 
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instructions should be pointed out on the worksheet and made clear to the 

sample custodian.

See MAN-000042 ORA Lab Manual Vol. II - Sample Management (ORALAB.5.8) Assignment

24.1. Exercise

A. Read Phillip DeCamps November 1970 article “A guide for the examination of 

rodent filth exhibits and related samples.” FDA By-lines No. 3, pp.153-164. 

B. Read Zimmerman, M.L. and Friedman, S. L. (2000). “Identification of rodent 

filth exhibits.” Journal of Food Science, Vol. 65(8): 1391-1394.

C. Under the guidance of an experienced analyst, analyze a factory filth sample. 

(Note: If the analyst has not received the training for chemical confirmation of 

rodent adulterants; the trainer will have to provide this training as needed.)

24.2. References and Additional Method Citations

A. Filth Exhibits: Urine

1. LWUV light, Urine Stains on Food & Containers (AOAC Official Methods of 

Analysis (OMA), current ed., Method 945.88).

2. Urease Test for Urea (AOAC OMA, current ed., Method 942.24).

3. Xanthydrol test for Urea (AOAC OMA, current ed., Method 959.14).

4. (AOAC OMA, current ed., Method 959.14, Xanthydrol test for Urea, 

modified to include 4 μg + urea response requirement from J. AOAC Intl. 

81(6): 1155-1161).

5. Magnesium Uranyl Acetate Test for Urea (AOAC OMA, 15th ed. (now 

surplused), Method 963.28).

6. Urease Bromothymol Blue Agar Test for Urea (AOAC OMA, current ed., 

Method 972.41).

7. TLC Method I (AOAC OMA, current ed., Method 980.28).

8. TLC Method II- with potential interference material (AOAC OMA, current 

ed., Method 973.64).

B. Filth Exhibits: Fecal Material

1. Alkaline Phosphatase Test for Mammalian Feces (AOAC OMA, current ed., 

Method 981.22).

2. Alkaline Phosphatase Detection Method for Mammalian Feces in Corn 

Meal (AOAC OMA, current ed., Method 986.28 - See 990.10).

3. Alkaline Phosphatase Detection Method for Mammalian Feces in Grain 
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(AOAC OMA, current ed., Method 990.10).

4. Alkaline Phosphatase Detection Method for Mammalian Feces in Ground 

Black Pepper (AOAC OMA, current ed., Method 993.27).

5. TLC Coprostanol for Mammalian Feces (AOAC OMA, current ed., Method 

988.17).

6. Microchemical test for Uric Acid (AOAC OMA, current ed., Method 962.20).

7. TLC Method for Uric Acid (AOAC OMA, current ed., Method 986.29).

8. Spectrophotometric Method for Uric Acid (in Flour) (AOAC OMA, current 

ed., Method 969.46).

C. References for bone and skull confirmation:

1. Popesko, P., Rajtova, V. and Horak, J., (1990). A color atlas of the 

anatomy of small laboratory animals (Vol. 1, Rabbit, Guinea Pig; Translated 

1992, ISBN 0 7234 1822 5). London: Wolfe Publishing, Ltd.

2. Popesko, P., Rajtova, V. and Horak, J., (1990). A color atlas of the 

anatomy of small laboratory animals (Vol. 2, Rat, Mouse, Hamster; 

Translated 1992, ISBN 0 7234 1823 3). London: Wolfe Publishing, Ltd.

3. Gottschang, J. L. (1981). A guide to the mammals of Ohio (ISBN 0-8142-

0242-X). Columbus, OH: Ohio State University Press.

4. Nowak, R. M. (Ed.). (1991). Walker’s mammals of the world (5th ed.). 

Baltimore: John Hopkins University Press. 

5. Hamilton, W. J., Jr., Whitaker, J. O., Jr. (1979). Mammals of the eastern 

United States (2nd ed., ISBN 0-8014-1254-4, LC 79-12920). New York: 

Cornell University Press. 

D. References for Forensic Entomology:

1. Smith, K. G. V. (1986). A manual of forensic entomology. London: British 

Museum (Natural History) and Ithaca, New York: Cornell University Press.

2. Hall, R. D. and Haskell, N. H., Wecht, C., (Ed. Forensic Sciences). (1995). 

Forensic entomology; applications in medicolegal investigations. New York: 

Mathew Bender.

3. Byrd, J. H. and Castner, J. L. (2001). Forensic entomology; the utility of 

arthropods in legal investigations. Boca Raton, FL: CRC Press 
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ORA Lab Manual Vol. IV Section 4 - Microanalytical and Filth Analysis (IV-04) Page 82 of 85

For the most current and official copy, check QMiS.

25. Specialized Microscopy Techniques, Optical Crystallography (a.k.a. Polarized 

Light Microscopy), and ID Spot Tests

25.1. Objectives

Microscope discussions in an earlier section (Section 3), were intended only to 

introduce the variety, principles, set-up, and maintenance of microscopes. It 

was not intended to teach the practical application of those tools. This chapter 

shows specialized microscopy methods and techniques, specifically those 

used in optical crystallography, as they apply to common problems often 

encountered in the filth laboratory.

25.2. Discussion

A thorough discussion of the principles of optical crystallography is beyond the 

scope of this manual, and in situations requiring advanced crystallographic 

techniques, e.g., measurement of more than one refractive index, measured 

structure, or precise optical characterization, these topics will be handled as a 

specialized course. An excellent discussion of this topic appears in the Food 

and Drug Administration Bulletin No. 1, Chapter X, reprinted in FDA By-Lines 

Vol. 6(1):20-53 (July 1975) and the instructor will identify the parts that should 

be read. It will give the trainee more insight into the science of crystallography 

and the need for advanced study. This chapter will be discussing problem

analytes and how they are analyzed in the lab, through polarized light 

microscopy or phase contrast microscopy, and the use of “spot testing” or 

micro-chemical tests to confirm visual findings. 

In the past, the main thrust of the science of optical crystallography or PLM 

has been the identification of crystalline drug substances. Even today, in the 

hands of a skilled analyst, PLM provides a rapid and accurate identification of 

many substances and avoids elaborate and costly chemical analyses. But the 

work can be tricky, after learning, practice frequently for reliable results. 

PLM has had a long history in FDA. The earlier work provided a reference 

catalog of known optical properties of crystalline substances. Most district 

laboratories have this index card catalog covering the thousands of 

substances (mostly drugs) studied. However, substances falling within the 

framework of filth analysis include glass, struvite, urea, dixanthylurea and the 

starches. Precise identification of these substances, and perhaps others, is 

most easily accomplished by optical crystallography, and/or with phase 

contrast microscopy; many are spot tests in and of themselves, or spot tests 

can be used as confirmation of observations.

25.3. Assignment 

A. Under the direction of an experienced analyst, study the principles, methods, 

and techniques of optical crystallography found in the Food and Drug 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Science

Document Number:

MAN-000057

Revision #: 03

Revised: 

08 Dec 2022

Title: 

ORA Lab Manual Vol. IV Section 4 - Microanalytical and Filth Analysis (IV-04) Page 83 of 85

For the most current and official copy, check QMiS.

Technical Bulletin No. 1, 2nd Ed. 1981. (Note: The trainee is to have the 

opportunity for first-hand observation of the optical phenomena described in 

that publication.)

B. Learn to apply the microscope and spot testing to the identification of the 

following substances:

• Glass

• struvite

• urea and dixanthylurea

• corn, wheat, potato, soy, and rice starch

25.4. Evaluation

A. Demonstrate an understanding of the following optical phenomena: Becke 

lines; birefringence; crystal habit; extinction; isotropism and anisotropism; 

Newton's rings; cross polarization; refractive index 

B. Using an unknown crystalline substance provided by the trainer, correctly 

determine the refractive index of an isotropic substance.

C. Identify the unknown substance given by the instructor (#2 above) making 

additional observations. 

26.Glossary/Definitions

Commensal - one who eats at the same table with others; an organism, not truly 

parasitic, that lives in, with or on another.

Collar - An old sieve with the weave removed; extends the height of a sieve.

Fix - Chemical process that prevents or minimizes pigment discoloration, loss 

and/or tissue distortion, to preserve in place.

Overs/Throughs - After a sieving operation, anything retained on top of the screen 

is referred to as overs, those that pass through, as throughs.

Aperture - Circular hole in the camera that controls the amount of light reaching 

the sensor or film emulsion.

Blooming - The bleeding of signal charge from extremely bright pixels resulting in 

over-saturated pixels. “Blooming” in digital photography compares with overexposure in film photography.

BMP - Bitmap (.BMP file extension): this is a standard image file format for 

Windows®.

GIF - Graphic Image Format (.GIF file extension): gif format is what is termed as a 
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Science

Document Number:

MAN-000057

Revision #: 03

Revised: 

08 Dec 2022

Title: 

ORA Lab Manual Vol. IV Section 4 - Microanalytical and Filth Analysis (IV-04) Page 84 of 85

For the most current and official copy, check QMiS.

"lossless" compression format. This format is referred to as a "paletted" image or 

a 256-color image. It is limited to 256 colors.

JPEG - Joint Photographic Experts Group or jpeg (.JPG file extension) this format 

is a lossy compression format. The higher the compression ratio the more the 

pixelization or "blockiness" occurs.

Cropping - The act of cutting out a portion of a digital image for blow-up/display as 

a separate image. 

Photo documentation - The process of recording images representative or 

demonstrative of a sample or object.

PNG - Portable Network Graphics (.PNG file extension): this file format is an 

alternative to the GIF (Graphic Image Format) format.

TIFF - Tagged-Image File Format (.TIF file extension): a lossless bitmap image 

format supported by virtually all paint, image-editing, and page-layout applications.

Feather - the primary covering on a bird and aids in flight.

Feather Barb - the individual units which grow from the feather shaft or rachis.

Feather Barbule - are like mini barbs growing from the shaft of the barb.

Node: the swollen structure spaced at regular intervals along the feather barbule. 

The shape of the node can vary in shape from having spines, prongs, round 

points or even flared.

Rachis - the central shaft of the feather. 

27.Document History

Revision

#

Status*

(D, I, R) Date Author Name and Title Approving Official Name and Title

1.2 R 09/01/2005 LMEB LMEB

1.3 R 06/06/2008 LMEB LMEB

1.4 R 02/02/2010 LMEB LMEB

1.5 R 02/14/2013 LMEB LMEB

02 R 06/15/2020 LMEB LMEB

03 R Refer to 

QMiS

Tyra Cooper, Lead Biologist 

NFFL; Heather Hawk, OFFLO 

Filth Program Manager

LMEB
FOOD AND DRUG ADMINISTRATION

OFFICE OF REGULATORY AFFAIRS

Office of Regulatory Science

Document Number:

MAN-000057

Revision #: 03

Revised: 

08 Dec 2022

Title: 

ORA Lab Manual Vol. IV Section 4 - Microanalytical and Filth Analysis (IV-04) Page 85 of 85

For the most current and official copy, check QMiS.

* - D: Draft, I: Initial, R: Revision

28.Change History

Revision 

# Change

1.2

Table of Contents – deleted Sections 4.3.5, 4.3.7, 4.39, and 4.6-4.9; changed 4.5 to 

Document History

Questions deleted from all sections.

1.3 4.2.3 D.- 4th Reference website updated

1.4

4.1.1 – bullet 4 web link updated

4.2.1 B. 3. – bullet 4-9 web links updated

4.2.4. C. 2 – web link updated

4.2.5.1 B. – revised second paragraph

4.4.1 C. 1. b – web link updated

Footer – web link updated

1.5 Header – Division of Field Science changed to Office of Regulatory Science

02

Updated references, types of samples to be used for training assignments, and 

regulatory references in FD&C Act and USC. Removed broken/outdated web links. 

Significant updates to some sections, including scanning electron microscope, wet 

mount media, introduction to identification keys, insect morphology assignment for 

dissection of insect genitalia, discussion and assignment related to hair identification, 

entire feather identification section, excreta and fecal pellets, X-ray radiography, 

Machinery Mold: Geotrichum, rodent nesting materials. Reformatted document and 

moved to new template.

03

Revised language in Reference Books and Materials

Removed section numbers in Sample Collections to align with IOM

Updated language in Investigations Training

Removed reference to local SEM SOP and operation manuals from Microscopic Examination 

and Microscope Accessories 

Clarified optical properties in Preparing Microscopic Slide Mounts

Link updated in Feather Identification

Added CMS, LIBs, reagent blanks, reporting of arthropods and estimated filth counts to 

Sample Analysis

Removed worksheet headers. Revised paragraphs for pellets and insect gnawing. Added 

details for rodent nesting material and 702(b) portion to Analysis of Factory Filth Samples and 

Filth Analytical Worksheets

Changes are highlighted in grey.