United States Department of Agriculture

Agricultural Marketing Service

Federal Grain Inspection Service

Directive 9180.47 April 23, 2021

Distribution: FGIS, OSP, Industry Originating Office: FMD PPMAB

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Washington, DC 20250-9410, or call (202) 720-5964 (voice and TDD). USDA is an equal employment opportunity 

employer.

EXAMINATION OF GRAIN FOR THE PRESENCE OF TCK SPORES

Contents

EXAMINATION OF GRAIN FOR THE PRESENCE OF TCK SPORES ��������������������������������1

1. PURPOSE �������������������������������������������������������������������������������������������������������������������������

2. REPLACEMENT HIGHLIGHTS �����������������������������������������������������������������������������������������

3. POLICY �������������������������������������������������������������������������������������������������������������������������

4. BACKGROUND �����������������������������������������������������������������������������������������������������������������

5. REQUEST FOR SERVICES �����������������������������������������������������������������������������������������

6. RESPONSIBILITIES ���������������������������������������������������������������������������������������������������������

7. GENERAL SAMPLING AND ANALYTICAL PROCEDURES ����������������������������������������

8. REVIEW INSPECTIONS �������������������������������������������������������������������������������������������������

9. CERTIFICATION ���������������������������������������������������������������������������������������������������������

10. FEES ���������������������������������������������������������������������������������������������������������������������������������

11. AUTHORIZING FGIS PERSONNEL ���������������������������������������������������������������������������������

12. PROFICIENCY VERIFICATION �����������������������������������������������������������������������������������������

13. QUESTIONS �����������������������������������������������������������������������������������������������������������������

14. ATTACHMENT �����������������������������������������������������������������������������������������������������������������
FGIS Directive 9180.47 April 23, 2021

Page 2

1. PURPOSE

This directive transmits sample handling, testing, and certification procedures for grain 

examined for the presence of Tilletia controversa Kühn (TCK) smut spores.

2. REPLACEMENT HIGHLIGHTS

This directive replaces Program Directive 9180.47, Examination of Grain for the 

Presence of TCK Smut Spores, dated July 24, 2012. It updates the background; 

responsibilities; sample size; testing procedures; and certification information. 

Additionally, it adds FGIS as a TCK service provider; sections on Authorizing Official 

Personnel; Review Inspections; Proficiency Verification; and Fees. An attachment titled 

Official Procedure for Identifying and Enumerating Tilletia controversa Kühn (TCK) 

Teliospores in Grain Samples Using Light Microscopy was added.

3. POLICY

The Administrator has determined that the Federal Grain Inspection Service (FGIS) 

will, upon request, provide for analysis of grain samples for the presence of TCK smut 

spores under authority of the U.S. Grain Standards Act.

4. BACKGROUND

TCK, also called Dwarf Bunt, is a smut fungus found in the soil or on the surface of 

wheat kernels. The organism only infects plants that grow during the winter. High 

levels of the disease are usually found after early and persistent snow cover. Wheat 

plants infected with TCK are obviously dwarfed in contrast to healthy plants, and kernels 

are replaced with brown masses of spores known as smut balls. The spores are not 

harmful to humans or livestock. 

TCK testing is offered as Official Criteria under the United States Grain Standards 

Act. Using microscopy techniques, it is possible to ascertain the presence of spores. 

Differentiation of TCK from common bunt is based on identifying structural differences 

in the teliospores of TCK versus other smut species. Upon request, analysis of grain 

samples other than wheat for presence of TCK smut spores is permissible. 

5. REQUEST FOR SERVICES

Based on an agreement between the United States and the Peoples Republic of China 

(PRC), FGIS has arranged for TCK smut testing service (quantitative) to be provided 

through the Wheat Marketing Center (WMC) laboratory in Portland, Oregon for testing 

samples. Additionally, FGIS (League City (Texas) Field Office (LCFO) and Technology 

and Science Division (TSD)) may provide qualitative or quantitative TCK testing service 

to customers. Shipments to PRC are restricted to quantitative analysis only.
FGIS Directive 9180.47 April 23, 2021

Page 3

TCK testing may be performed on shiplot composite samples, unit train composite 

samples, bargelots, carlots, trucklots, and submitted samples. Interested parties 

wishing to receive TCK testing should contact their Official Agency or Field Office 

(hereafter OSP, official service provider) for service. 

6. RESPONSIBILITIES

a. Applicant for Service. The applicant will request TCK testing through their

OSP. The request must include the applicant’s name and telephone number and 

the destination of the lot, if known. 

b. Official Service Provider. The OSP will forward the sample to WMC or LCFO at the 

applicant’s expense with the required information. The OSP will issue the 

certificate and bill the applicant for service for samples tested by WMC. For 

samples derived outside the Pacific Northwest and Texas, FGIS will advise the 

OSP where to send the sample.

c. WMC. For TCK requests in the Pacific Northwest, WMC is responsible for

performing an original test, and reinspection analysis if requested; maintaining a 

file sample for 90 days; reporting the results to the OSP in a timely manner; and 

billing the OSP for the specified per sample analysis rate. The WMC will maintain 

all test records and documentation and provide them to FGIS upon request. 

d. FGIS  For TCK tests not performed by WMC, FGIS is responsible for performin

an original test and review (reinspection/appeal) analysis/Board appeal, if 

requested; maintaining a file sample for 90 days; reporting the results to the 

applicant for service in a timely manner; and billing the applicant for the specified 

per sample analysis rate. A quantitative analysis (actual number of spores per 50 

grams) is provided unless a qualitative analysis (a screening for the presence of 

TCK) is requested. The qualitative analysis will cease when the first teliospore is 

detected. FGIS will issue the certificate for testing performed by FGIS. 

7. GENERAL SAMPLING AND ANALYTICAL PROCEDURES

The TCK analysis requires a 650-gram minimum sample. Original service points must 

maintain an additional 650-gram sample as a backup file sample. Upon completion of 

official sampling and/or inspection, the service point will forward the official sample to 

WMC or the LCFO. The WMC or LCFO will maintain a file sample on each original and 

appeal inspection for 90 days. 

The OSP will use a Boerner divider to obtain a 650-gram minimum sample from 

a representative sample (vessel or unit-train composite) and place it in an airtight 

container (e.g., double walled, sealed plastic bag) for shipment to the lab. The sample 

bag will be marked with the carrier identification name or number and type of test 

requested, and be accompanied by documentation identifying the carrier, lot number (if 

applicable), original inspection certificate number (if available), Field Office or Official 

Agency address and phone number, the date, destination (mandatory if going to the 
FGIS Directive 9180.47 April 23, 2021

Page 4

PRC), and a legible copy of the application for inspection. Retain a file sample of at 

least 650 grams in an airtight container

For samples to be tested at WMC, send the sample(s) and documentation by overnight 

delivery (if appropriate), at applicant’s expense, to:

Wheat Marketing Center, Inc. 

1200 N.W. Naito Parkway, Suite 230

Portland, OR 97209-2800

ATTN: TCK TESTING

For samples to be tested by FGIS, send the sample(s) and documentation by overnight 

delivery (if appropriate), at applicant’s expense, to:

USDA-AMS-FGIS.

1025 E. MAIN STREET, #104

LEAGUE CITY, TX 77573- 2483 

281-338-2787

ATTN: TCK TESTING

Note: Sample analysis will take hours and possibly days. The lab will notify the 

OSP of the result(s) promptly upon completion of testing.

8. REVIEW INSPECTIONS

Sections 800.125 and 800.135 of the regulations permit a review inspection on either 

official grade/factors or official criteria. When requested, a review inspection for official 

grade/factors and official criteria may be handled separately even though both sets of 

results are reported on the same certificate.

Applicants may request a reinspection, appeal, and Board appeal. WMC or LCFO will 

perform reinspections on the basis of the file sample. The reinspection analysis should 

be performed with a different analyst if possible, using the file sample. 

The appeal must be performed at or under the supervision of the field office on a new 

extraction. The board appeal must be performed by the FGIS Technology and Science 

Division on the extraction from the appeal analysis, in the 15-ml tube containing the 

sample suspended in Shearer’s solution. 

Applicants may request either qualitative or quantitative review analysis unless the 

original or a review analysis was quantitative. Then, only a quantitative analysis is 

available. All review inspection results replace previous results.
FGIS Directive 9180.47 April 23, 2021

Page 5

9. CERTIFICATION

All shipments to the PRC shall be analyzed on a quantitative basis. Certify the results in 

the results section on the appropriate official certificate. The results must be certified on 

the official grade certificate unless separate certification is requested by the applicant. 

The applicant for service must make this request in advance of the lot being certified. 

Submitted sample results must be certified on a submitted sample certificate. 

Quantitative TCK testing:

The certification procedure/remark is based on the export destination. 

The following factor remarks must be used to describe the factor result:

Quantitative TCK testing:

Export shipments to the PRC: 

Tilletia controversa Kühn spores (exceed/do not exceed) 30,000 per 50 grams

of sample.

For all export (other than PRC) and domestic analyses:

(number of spores) Tilletia controversa Kühn spores per 50 grams of sample.

Qualitative TCK testing:

Tilletia controversa Kühn spores (present/not present).

10. FEES

FGIS will use billing code G2TCK, as stated in FGIS Program Directive 9180.74, found 

on the AMS website: https://www.ams.usda.gov/about-ams/fgis-program-directives

11. AUTHORIZING FGIS PERSONNEL

FGIS will authorize personnel to perform TCK testing. For Federal personnel, 

authorization requires a written test in FOL and a proctored practical test, to ensure 

procedures are followed according to the Official Procedure document. Authorization 

records will be housed in the FOL module of FGISonline, consistent with established 

practices for licensing official personnel to perform official duties. 
FGIS Directive 9180.47 April 23, 2021

Page 6

12. PROFICIENCY VERIFICATION

FGIS will establish and maintain a proficiency verification program to evaluate the 

proficiency of personnel performing TCK testing at both the WMC and FGIS sites. 

Proficiency will be evaluated on at least an annual basis. FGIS subject matter experts 

will randomly choose 10 percent of samples processed per service location for testing. 

Evaluation of proficiency will follow the statistical model of Whitaker et al (2001, Plant 

Pathology 50:755-760). 

13. QUESTIONS

Any questions should be directed to the Policies, Procedures, and Market Analysis 

Branch (PPMAB) at (816) 659-8403.

14. ATTACHMENT
ATTACHMENT

Official Procedure for Identifying and 

Enumerating Tilletia controversa Kühn (TCK) 

Teliospores in Grain Samples Using Light 

Microscopy.

AMS-FGIS gratefully acknowledges the contributions of ARS, and in particular Dr. Gary 

Peterson of the Foreign Disease-Weed Science Research Unit for his generous 

contributions in training FGIS personnel on the procedures of TCK analysis and the 

preparation of these instructions. 
Table of Contents

1. Tilletia spp Infecting Wheat

a. Dwarf bunt

b. Common bunt

2. Protocols of Isolation, Enumeration and Identification of Tilletia Teliospores from 

Grain Samples

a. Sample Extraction

b. Microscopic Evaluation

c. Sample Analysis

Appendix I: Equipment

Appendix II: Materials and Supplies

Appendix III: Solutions

a. Shears Medium

b. Bleach Solution

c. Tween-20 Water

Appendix IV: Microscope Calibration

Appendix V: Logistic Index

Appendix VI: Example of Data Sheet

Appendix VII: 52μm Nylon Mesh Sieves

Appendix VIII: Warning on Shipping Grain Samples

Appendix IX: Quality Control Testing

USDA Disclaimer and EEO Statement
1. Tilletia spp Infecting Wheat

There are four Tilletia spp that infect wheat however two (T. laevis Kühn and T. indica

Mitra) are morphologically distinct and not relevant to this testing procedure.

Species Other Names Disease

T. controversa Kühn TCK Dwarf bunt

T. tritici Winter T. caries Tul., TCT Common bunt

T. laevis Kühn T. foetida Liro. Common bunt

T. indica Mitra

Neovossia indica,

Mundkur., KB

Karnal bunt

2. Protocol for Isolation, Enumeration and Identification of Tilletia 

Teliospores from Grain Samples

a. Sample Extraction

Step 1

If processing more than one sample, label a 600 ml beaker, a 500ml flask, two 50 

ml centrifuge tubes and one 15 ml centrifuge tube with the sample number. 

Step 2

Mix the sample by gently rotating kernels in a plastic bag. The bag should have 

sufficient air space for the kernels to roll around freely. Do not shake vigorously

(see Appendix VIII).

Step 3

Weigh 50 g sample into a weighing dish or 3-oz paper cup and transfer to a 500-ml 

Erlenmeyer flask.
Step 4

When ready to begin the extraction, add 100 ml of water containing Tween-20 

water (Appendix III). Do not allow the wheat to sit in the solution for extended 

periods of time.

Step 5

Swirl the flask by hand or on an orbital shaker at 200 rpm for 5 min.

 

Step 6

Immediately pour the contents of the flask into a 52-μm nylon micromesh pore-size

sieve set on an angle over the beaker. Rinse the contents of the flask with 10 ml of 

water, pouring the rinse into the sieve. There may be some wheat kernels remaining 

in the flask, but this is not of concern. Allow the sieve with grain to sit on the beaker 

until it has stopped dripping. 
Step 7

Swirl the beaker to keep the contents in suspension then pour equal amounts into 

each of the two 50-ml centrifuge tubes.

Step 8

Centrifuge at 600 x g for 5 min in a horizonal bucket rotor or 800 X g for 15 min 

in a fixed angle rotor. After the centrifuge comes to a stop, gently remove the 

centrifuge tubes and slowly pour off the supernatant into a 250 ml beaker. If the 

pellet dislodges from the centrifuge tube during decanting, the contents of the 250 

ml beaker can be centrifuged again.

Step 9

With a pipette, add 1.0 ml Shear’s mounting medium (see Appendix III) to each 

of the 50-mL conical centrifuge tubes. Using a disposable Pasteur pipet to aspirate 

the sample in and out of the pipette to resuspend the pellet and the transfer both 

suspensions to a single 15-ml centrifuge tube. If only one sample is being 

processed, add an equal amount of water to a second 15 ml centrifuge tube to 

balance the centrifugation.
Step 10

Centrifuge the sample in the 15 ml centrifuge tube at the same speed and time as 

before. Carefully pour off the supernatant into a 250 ml beaker.

Step 11

After pouring off the supernatant, there will be a small amount of supernatant that 

adheres to the inner wall of the centrifuge tube and settles back down on top of 

the pellet. Re-suspend the pellet using a Pasteur pipette.

Step 12

Draw the entire suspension into the Pasteur pipette then slowly return the 

suspension back into the 15-ml tube counting the individual drops. If there are 

less than 12 drops, add additional drops of Shears with a clean pipette to raise the 

count to 12 drops and mix. If the sample suspension is very viscus and opaque 

due to the amount of debris in the sample, add an additional 12 drops of Shears 

with a clean pipette and resuspend. If the debris pellet is very large, additional

Shears can be added and additional drops included. Record the total number of 

drops in the tube on the data sheet.

Note: Once the sample is suspended in Shears Medium, it can be stored at room temperature 

for more than a year with no changes. This is useful if you want to examine the sample at a 

later time, use for training or shipping to another laboratory or authority for results

confirmation.

Step 13

Clean-up: The extracted 50g wheat sample can be dumped in the trash or 

alternatively, first dump it into a fine-holed colander or colander lined with cheese 

cloth set in the sink. Grains left behind in the flask can rinsed into the colander. 

This prevents grain from lodging in the sink trap and germinating.

Sieves need to be back-washed with water, dipped in a dish washing detergent 

solution and transferred to a plastic box with lid containing a 33% Clorox solution 

for 15 minutes, rinsed and set out to dry. Do not leave the sieves in the Clorox 

solution past 15 minutes. The Clorox destroys or decolorizes the teliospores that 

may be left behind in the sieve, so they are not detected in future extractions.
b. Microscopic Evaluation

Step 1

Label a microscope slide with sample number. 

Step 2

Use a disposable Pasteur pipet to thoroughly aspirate to re-suspend the pellet.

Step 3

Using the Pasteur pipette, place one drop of the sample suspension on the slide 

and add a 22 x 22 mm cover slip. Seal edges of cover slips with clear nail polish 

to prevent drying out or movement of the coverslip when subjected to 

examination under oil immersion.

Apply of one drop (A); Apply cover slip (B); Seal with clear nail polish (C)

Notes: You will be counting all of the whole Tilletia teliospores encountered on the slide in 

addition to recording measurements for up to 30 teliospores. You may notice that many of the 

teliospores are cracked. As long as there is not a noticeable piece missing, it is counted and 

measured. For training purposes, slides with specimens of pure T. controversa and T. tritici

teliospores suspended in Shears Medium are helpful. Before you begin examining slides at 

100X, it is helpful to examine a slide with a drop from one of these pure specimens to train 

your eyes to recognize the size and color of teliospores. If you seal the specimen slide well, it 

can be kept and used many times. 

(A) Count and measure whole teliospore and (B) those with line cracks 

but (C) not those with pieces missing pieces
Step 4

Starting at a corner of the cover slip, scan the entire slide under the microscope, 

side to side, in slightly overlapping fields, at 100X or 200X. If you observe

something that looks like a teliospore, center it in the field and switch the 

objective to 400X magnification to confirm that the surface reticulations are 

typical of the two Tilletia species. 

Teliospore in extraction drop at 100X (A) and 400X (B) magnification

Note: If the debris on the slide is very dense, you can add additional Shears medium to the 

sample, mix and make a new slide. Just keep track of the number of additional drops added.

At the 400X magnification, center the teliospore in the view field. Rotate the 

400X objective away from the slide and place one drop of immersion oil on the 

slide cover slip. Rotate the 100X objective into place (in contact with the oil) and 

gingerly focus on the teliospore. Usually, you do not need addition immersion oil 

each time you switch to 1000X magnification on that slide. Never try to switch 

magnification directly from 100X or 200X to 1000X; you may miss the 1000X

focal plane and drive the objective into the slide and break it.

Note: When finished for the day, clean the oil objective with microscope lens 

cleaner. No not clean with alcohol, it will dissolve the adhesive that holds the 

lens in the objective.

. 

Adding oil to coverslip for 

1000X magnification
Step 5

On the video monitor, with the microscope magnification at 1000X (oil 

immersion), focus on the periphery of the spore. If you are using an AmScope 

microscope/camera combination with a laptop, make sure the magnification tab in 

the menu bar is set to 100%. What you want to see will be a dark, obtuse line 

(endospore wall) with what appears to be, depending on species, obtuse spikes or

short projections arising from it.

AmScope software screenshot; select 100%

for measuring at 1000X oil immersion lens

Note: At 1000X very slight movements of the microscope stage are greatly magnified so it 

will take some practice to focus and move the teliospore to a position on the monitor where it 

can be measured. With a digital camera there is also slight delay in what you see when you 

move the slide on the microscope stage and what appears on the monitor.

Step 6

Using a plastic millimeter ruler, measure the depth of the reticulum from the 

center of this dark line to the top of the reticulum “spike”. Take four 

measurements from each spore, choosing points that closely correspond to 

north/south/east/west points on a compass. You may have to refocus as you 

change to different points on the teliospore. Measure to the nearest 0.5 mm. 

Record all measurements on the data sheet. Do not measure more than 30 

teliospores.
Measuring T. controversa

Measuring T. tritici

Note: If you have very few teliospores in the sample, such as one teliospore per microscope 

field or less, you should stop and measure each teliospore as you encounter them. If the 

teliospore count is greater than one teliospore per microscope field, it is faster to first count 

all the teliospores on the slide, then go back to the beginning and measure 30 teliospores.
c. Sample Analysis 

Step 1

Multiply the number of teliospores counted on the slide (C) by the number of 

drops in the tube (D) to obtain the number of teliospores in the 50g sample (T) 

C*D=T

Step 2

For each measured teliospore, determine the average of the four reticulum 

measurements and multiply by the conversion factor for your microscope/monitor 

system to determine the average reticulum depth in micrometers (Appendix IV). 

Step 3 

The mean reticulum depth for each teliospore is converted to a Logistic Index. 

This can be done by using the Logistic Indices Table in Appendix V, choosing the 

closest Indices value corresponding to the average micrometer value for each 

teliospore or by using the formula below: 

Logistics Index

Index = (ea+bR)/(1+ea+bR) 

a= -10.6914 b= 11.6214 R= reticulum depth e= 2.71828

Excel functions for calculating LI is presented in Appendix V

Step 4

The Logistics Index for each teliospore can be interpreted as the probability that 

the teliospore is Tilletia controversa rather than Tilletia tritici. A Logistic Index 

of greater than 0.50 is presumed to be T. controversa. 

Summing the logistic Indices for all of the teliospores yields the Absolute Index

which is interpreted as the expected number of Dwarf bunt teliospores in the 

sample.

 

Averaging the logistic Indices for all of the teliospores yields the Relative Index

which is interpreted as the proportion of Dwarf bunt teliospores in the 50g sample 
Appendix I: Equipment

1. Microscope:

The minimum requirement is a trinocular compound brightfield microscope with 10X, 

20X, 40X, and 100X objectives and 10X oculars equipped with a 5 to 10 mega pixel 

camera coupled with a monitor or laptop. 

ARS-FDWSRU tested and found a satisfactory system sold online by AmScope 

(https://www.amscope.com), Model T940B-9MP, which meets the above microscope 

specifications (except the 20X achromatic objective, which can be purchased separately; 

AmScope part number is A20X-V300, $24) 

including a 9MP camera and software to connect it to a PC laptop (not included). It also 

includes a spare halogen light bulb, stage micrometer, and immersion oil for 1000X 

magnification. At the time of this writing the list price was $570.99. Transport cases are 

also available.

2. Centrifuge

a. Horizonal centrifuge that takes 50 ml and 15 ml conical centrifuge tubes and 

reaches at least 600 X g

 Horizonal Bucket Fixed Angle

 

b. Fixed angle centrifuge that accommodates both 50 ml and 15 ml conical 

centrifuge tubes and reaches at least 800 X g. ARS-FDWSRU tested a small 

tabletop unit made by Southwest Science, Model SCL456 Clinical Centrifuge that 
handles both 50 ml and 15 ml conical centrifuge tubes and runs up to 1,800 X g.

At the time of this writing the list price was $568.00.

3. Orbital Shaker

If you are processing multiple samples at the same time you will want to use an

orbital shaker table that reaches at least 200 revolutions per minute. If you are 

only processing a single sample, you can forgo the shaker and swirl the flask by 

hand for 5-minutes.

Southwest Science makes an orbital shaker that has had good reviews (not tested 

by ARS-FDWSRU). The model SBT300 costs $629, however the tabletop 

(SBT300-UNV top $63) and flask clamps for 500-ml flasks are sold separately 

(Model F-CLAMP-500 $25 ea.) for a total of $792 at the time of this writing.

4. Digital Balance

A digital balance for weighing grain samples with a minimum accuracy of 0.1g. 

This can be obtained from a scientific supply company such as Fisher Scientific 

or Lab Depot. 
Appendix II: Materials and supplies

Item Notes

Beakers 600 ml Kimble No. 14000

Beakers 250 ml

Glass Pasteur pipettes 5 3⁄4 inch

Bulbs for Pasteur pipettes

10 ml Plastic disposable pipettes

Pipette pump

500 ml Erlenmeyer flasks

Standard microscope slides, frosted end

Coverslips 22 X 22 mm

Lens cleaning solution (Non-alcohol)

Lens cleaner tissues

Microscope immersion oil

Tween-20 100 ml bottle

50 ml plastic capped conical centrifuge tubes

15 ml plastic capped conical centrifuge tubes

95% Ethanol (ACS reagent grade)

Glycerin

Potassium acetate

500 ml crew cap bottles or a 6 to10L carboy with 

spigot for Tween-20 water

Clear nail polish

Weighing dishes or 3 oz paper cups

Stage micrometer 1.0mm with 0.01mm divisions

52 μm nylon mesh screen See Appendix VII

3” PVC drainpipe See Appendix VII

Plastic storage box with lid for bleach solution to 

decontaminate sieves

Approximately 16 -

quart size 

Tally Counter for teliospore counting Free version for smart 

phones on-line
Appendix III: Solutions

a. Shears Mounting Medium

Distilled water 300 ml

Glycerin 120 ml

Ethanol 95% 180 ml

Potassium acetate 6.0 g

Dividing this final solution among several smaller bottles is recommended due to 

the risk of accidental contamination with teliospores.

b. Bleach Solution

Add 660 ml of standard Clorox bleach (5.25% sodium hypochlorite) to 

1,340 ml of water. Pour into a plastic box with lid for decontaminating sieves.

This 33% bleach solution should be changed weekly.

c. Tween-20 Solution

Tween-20 solution is mixed at a rate of 2 drops in 100 ml of water. For 10 L of 

this solution add 4 ml of Tween-20.
Appendix IV: Microscope Calibration

Using a 1.0 mm stage micrometer, which is divided into one hundred 0.01 mm marks, 

observe the stage micrometer at 400X then switch to the 1000X oil objective and bring a 

portion of the stage micrometer into focus on the viewing monitor. At that magnification

you may only see three wide vertical lines on the screen. Use your ruler to measure in

millimeters from the left edge of the first vertical line to the left edge of the next vertical

line. There is 0.01 mm or 10 mm between lines on the micrometer. Take the distance in

millimeters you measured between the lines and divide 10 mm by that measurement to

obtain the conversion factor. As long as the equipment remains the same, write the 

conversion factor on a piece of tape and attached it to the base of the microscope.

For example, on one scope that distance between the two lines is 76.0 mm. Divide the 

10 mm by 76 mm gives you 0.132. This is the conversion factor. If you measure the 

height of a reticulation on the monitor and it is 14 mm, then the reticulation depth is 14 

mm * 0.132 = 1.8 mm. 

1.0 mm Stage Micrometer

Appendix V: Tilletia controversa Logistic Indices 

Reticulum (μm) Logistics index Reticulum (μm) Logistics index

0.05 0.000 1.00 0.717
0.10 0.000 1.05 0.819

0.15 0.000 1.10 0.890

0.20 0.000 1.15 0.935

0.25 0.000 1.20 0.963

0.30 0.001 1.25 0.979

0.35 0.001 1.30 0.988

0.40 0.002 1.35 0.993

0.45 0.004 1.40 0.996

0.50 0.008 1.45 0.998

0.55 0.013 1.50 0.999

0.60 0.024 1.55 0.999

0.65 0.042 1.60 1.000

0.70 0.072 1.65 1.000

0.75 0.122 1.70 1.000

0.80 0.199 1.75 1.000

0.85 0.307 1.80 1.000

0.90 0.442 1.85 1.000

0.95 0.586 1.90 1.000

LI >0.50 is presumed to be T. controversa 

Logistics Index

LI = (ea+bR)/(1+ea+bR) 

a= -10.6914 b= 11.6214 R= reticulum depth e= 2.71828

The LI can be programed into Excel using the formula syntax below:

 

A B C

1 Avg. Ret. μm (ea+bR) Logistic Index

2 R =EXP(((-10.6914+(11.6214*A2)))) =(B2)/(1+B2)

Example: Excel Output

Avg. Ret. Depth (ea+bR) Logistic Index

1 0.95 1.417549958 0.58635808

2 1.1 8.102235409 0.89013688

3 0.5 0.007591698 0.0075345

Appendix VI: Example of Data Sheet 

Identification: SS Minnow Date Loaded: 2/31/2021 

Calibration: 0.22 Date Tested: 3/1/2021 
No. drops (D) in sample: 24 Teliospores (C) in drop: 63

Reticulum (R) Measurements (no more than 30 teliospores)

Teliospore R1 R2 R3 R4 Avg R R*.22=μm L I*

1 6 4 5 5 5 1.1 0.89

2 5 5 4 4 4.5 0.99 0.586

3 5 5 5 5 5 1.1 0.89

4 7 6 7 7 6.75 1.485 0.996

5 6 5 4 4 4.75 1.045 0.819

6 5 5 5 5 5 1.1 0.89

7 6 5 5 5 5.25 1.155 0.935

8 4 4 4 4 4 0.88 0.442

9 5 4 5 5 4.75 1.045 0.819

10 4 6 4 4 4.5 0.99 0.586

11 5 4 4 4 4.25 0.935 0.586

12 5 4 5 5 4.75 1.045 0.819

13 6 5 6 6 5.75 1.265 0.979

14 5 7 5 5 5.5 1.21 0.963

15 5 4 4 4 4.25 0.935 0.586

16 5 4 4 5 4.5 0.99 0.586

17 6 4 5 6 5.25 1.155 0.953

*See Appendix IV

Total teliospores in 50g (C*D=T): 1,512

Relative Index (AVG LI): 0.78 (78% of the sample teliospores are T. 

controversa)

Absolute Index (SUM LI): 13.3 (13 of the 17 teliospores measured were T. 

controversa)

Technician: A. Wadyko

Note: Example datasheet is suitable as an official record for an FGIS or certified 

laboratory.
Appendix VII: 52μm Nylon Mesh Sieves

The frames for the 52-μm nylon mesh sieves are fabricated from 3-inch white PVC, 

Schedule 40 drainpipe. The pipe is cut into equal numbers of 1⁄4 inch and 3-inch-long

pieces. A power radial miter saw with a 60 tooth/inch or greater blade works well. 

Remember to wear safely goggles and dust mask when cutting PVC pipe. Cut slowly. It 

is best to clamp the pipe to the rear fence of the saw when cutting rather than holding in 

place with your hand.

You may need to slide the cut pieces across some sandpaper or a belt sander to remove 

any burrs from the surface.

There are several sources for the 52-μm nylon mesh screen material. One source is 

Component Supply Company (https://componentsupplycompany.com/productpages/nylon-screening-mesh.php) which sells the screen material by the 1⁄2 yard. One 

yard will make over 50 sieves.

Components are assembled using J-B Clear Weld. Pre-cut the 52-μm nylon mesh screen 

material into 4 X 4-inch squares. The glue cures rapidly so do one sieve at a time. In a 

ventilated area apply the cleaner to one surface of the 1⁄4 inch pipe and the 3-inch pipe 

with a cotton swab or acid brush. Center the piece of screen onto the 3-inch pipe and 

press down and out to prevent wrinkles. Place the glue side of the 1⁄4 inch pipe over the 3-

inch to sandwich the screen between. Press down for 30 seconds, set a flat weight (brick, 

book etc.) on top and leave for an hour. Trim the excess screen from the outside with a 

razor blade.

Note: Don’t substitute the grey CPVC or black ABS pipe for the white PVC. We experimented 

with stainless steel and polyester screens and found that over time the decontamination with 

Clorox solution roughed up the surface of the screen threads which resulted in teliospores 

hanging up in the screens.
Appendix VIII: Warning about Shipping Grain Samples

In 2000, the Wheat Marketing Center (WMC) in Portland, OR was established as the 

official T. controversa laboratory for testing PNW wheat exports to China. ARS 

provided initial training at WMC and FGIS. WMC is certified by FGIS annually after an 

inspection of the facility to affirm the testing protocols, record keeping, and personnel 

training requirements were being met. As part of that review, quality control (QC) testing 

of 10% of the China export file samples retained at WMC were retested by the National 

Grain Center AMS, FGIS in Kansas City, MO and results compared to those obtained by 

WMC. During the first quality control testing in Kansas City, the number of teliospores 

reported by FGIS average less than 20% of those reported by WMC.

In an investigative study conducted by ARS, T. controversa grain samples were analyzed 

at ARS-FDWSRU, Fort Detrick, MD, then shipped by commercial carrier to and back 

from ARS, Raleigh, NC, and retested again. Results show a reduction of 70 to 80 % of 

teliospores after shipping. Similar results were obtained when dry grain was placed in an 

Erlenmeyer flask and shaken vigorously for a few minutes then retested. The conclusion

of the study was that the brittle teliospores were being ground-up between the seeds 

during shipping. The problem with the QC evaluation was remedied by conducting the 

annual QC testing on site at the WMC facility in Portland.

In general, this means we cannot expect comparable results, if for example, a grain 

sample is tested in League City, TX then sent by courier to College Station, TX or 

Kansas City, MO for confirmation testing. Likewise, if you sample grain in a rail car in 

Tulsa, OK and mail it to League City, TX for testing, the teliospore numbers may not 

accurately reflect the contamination level in the rail car. However, the extracted sample 

suspended in Shears medium can be shipped with no damage to the possible teliospores 

in the sample.
Appendix IX: Quality Control Verification

Quality control verification testing is conducted as part of the annual laboratory 

inspection for certification by USDA FGIS. The inspection requires FGIS to test 10% of 

the samples evaluated by the testing facility. Samples are selected randomly from the file 

samples the facility is required to retain in cold storge. The facility will provide FGIS 

with copies of their test results for the selected samples.

FGIS personnel will perform the testing analysis at the facility being certified.

The analysis is based on the equation developed by Dr. Thomas Whitaker of ARS which 

estimates the variability associated with TCK export sampling (Whitaker et al, 2001).

The equation says the standard deviation is approximately 1/2 (0.551) the TCK 

concentration. Ninety-five percent of the sample results will vary from a low of T1.96*s, to a high of T+1.96*s. Our best estimate of the lot TCK concentration, T, is the 

average, A, of the 2 sample test results, X1 and X2. The average is: A = (X 1+ X 2)/2 = T,

and the expected random variation among samples from a lot at T is 0 to 2T. So it is 

acceptable for X 1 and X 2 to differ by 2T (Hi - Lo or 2T - 0). Take the difference (D) 

between X 1 and X 2 and compare D to 2A or 2T. If D <= 2T, then results are acceptable. 

If D > 2T, then results are unacceptable, and some action is required.

Below is an example of a QC analysis conducted by ARS for FGIS from samples tested 

at WMC:

WMC Number WMC TCK ARS TCK D

Average 

(T) 2T

Accept if 

D ≤ 2T 

120061264 5 6 1 5.5 11 Yes

120071040 1534 552 982 1043 2086 Yes

120070127 2651 558 2093 1605 3209 Yes

120071182 1536 384 1152 960 1920 Yes

120060054 54 10 44 32 64 Yes

120060141 0 0 0 0 0 Yes

120061078 1 0 0 0.5 1 Yes

120061249 1 0 0 0.5 1 Yes

Analysis shows that the numbers of TCK spores found in the ARS over-sight sample 

extractions are not statistically different from those obtained by WMC.

* Whitaker, T.B., Wu, J., Peterson, G.L., Giesbrecht, F.G., and Johansson, A.S. 2001. Variability associated with 

the official USDA sampling plan used to inspect export wheat shipments for Tilletia controversa spores. Plant 

Pathology 50:755-760
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