**Procedure: Microscope Preventative Maintenance**

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# Principle and Description

## Principle of bright-field microscopy

> 1\. The term \"bright field\" refers to the dark appearance of a
> stained or naturally colored, transparent or translucent specimen
> against a bright white background or field.

2.  A microscope consists of a compound magnifying system in which the
    observer looks at the first (primary) image with a lens that
    produces an enlarged secondary (virtual) image (Fig. 1). The two
    convex lenses are aligned in such a way that the observer sees an
    enlarged image of an object. The resolving power (also called
    resolution) of a microscope is the smallest distance between two
    separate dots that allows the viewer to differentiate two dots from
    a single dot.

### B. Use in clinical microbiology laboratories {#b.-use-in-clinical-microbiology-laboratories .unnumbered}

Observation and description of the microscopic morphology of bacteria,
fungi,parasites, and host cells in various stained and unstained
preparations

#### C. Basic requirements for optimal performance {#c.-basic-requirements-for-optimal-performance .unnumbered}

> 1\. Koehler illumination: sharply focused, clearly resolved, evenly
> illuminated field of view (see Appendix 1 to this procedure)

2.  Proper maintenance and care of instrument

3.  Good working knowledge of instrument

4.  Considerations when purchasing the microscope (see Appendix 2 to
    this procedure)

#### D. Description and function of component parts {#d.-description-and-function-of-component-parts .unnumbered}

> 1\. Eyepieces
>
> a\. Function: to enlarge primary image to form virtual image
>
> b\. Magnification: magnifying power of eyepiece; range, 5x to 20x
> (most common, 10 x or 12.5 x)
>
> c\. Position

Eyepieces are moved closer or farther apart by a pulling-pushing motion
or by moving a knurled ring to adjust for interpupillary distance.

> d\. Focusing

Some eyepieces can be focused independently of fine- or coarse-focus
adjustment knob. At least one focusable eyepiece is recommended to
compensate for differences in the strengths of users\' eyes

> Figure 1 Basic principle of microscopy (top) and microscope parts
> (bottom). The objective lens near the specimen (La) creates a
> magnified image of the specimen (small black arrow A\'B/). The
> eyepiece lens (Le) magnifies the image again to create a virtual image
> (large clear arrow A\"B\") (courtesy of A. G. Heinze Co., Inc.,
> Irvine, Calif., and Nikon Inc., Garden City, N.Y.)
>
> ![](media/image1.png){width="4.34375in" height="3.5in"}
>
> e\. Reticle: an engraved glass disk or glass-mounted microphotograph
> placed on the eyepiece diaphragm to assist in the following
>
> \(1\) Calibration

A micrometer scale is superimposed on the object image so that
measurements can be made.

> \(2\) Photomicroscopy

Cross hairs are superimposed on the object image to allow focusing of
the image at the photographic film plane.

> f\. Designations on the eyepieces
>
> \(1\) \"Plan\" or \"PIano\": correction for field curvature (e.g.,
> \"periplan\"). The image is in focus from the center to the periphery
> of the viewing field at the same time. These lenses are more expensive
> than standard lenses.
>
> \(2\) Wide field

Eyepieces encompass a large viewing area and are more comfortable when
viewing for long periods.

> \(3\) High eyepoint (sometimes designated with a pair of
> \"spectacles\") These lenses are designed for eyeglass wearers. The
> image is pro-jected some distance from the eyepiece. Certain eye
> defects, e.g., astigmatism, are not corrected for within the optics of
> the microscope. Therefore, one can use the microscope while wearing
> glasses. The high clearance from the top of the eyepiece reduces
> fatigue when viewing for long periods.
>
> 2\. Mechanical tube length

##### This is the distance between the insertion position of the objective and the top of the draw-tube into which the eyepiece fits; 160 mm is considered universal for standard microscopes, although infinity-corrected systems are now available. If an objective with a specified mechanical tube length is interchanged with one that has a different specified length (i.e., is designed for a different microscope), image quality may suffer. Do not interchange objectives on different microscopes unless you know the specifications are equivalent. {#this-is-the-distance-between-the-insertion-position-of-the-objective-and-the-top-of-the-draw-tube-into-which-the-eyepiece-fits-160-mm-is-considered-universal-for-standard-microscopes-although-infinity-corrected-systems-are-now-available.-if-an-objective-with-a-specified-mechanical-tube-length-is-interchanged-with-one-that-has-a-different-specified-length-i.e.-is-designed-for-a-different-microscope-image-quality-may-suffer.-do-not-interchange-objectives-on-different-microscopes-unless-you-know-the-specifications-are-equivalent. .unnumbered}

> 3\. Nosepiece
>
> a\. Function: rotates and houses the objectives
>
> b\. Housing: forward- or reverse-facing objectives (reverse-facing
> most com-mon)
>
> c\. Arrangement of objectives
>
> The sequence low power to intermediate power(s) to high power
> objec-tives reduces the risk of getting immersion fluid on high dry
> objectives.
>
> 4\. Obiectives
>
> a\. Function: formation of primary image
>
> b\. Magnification (magnifying power of objective)
>
> Range is 1.25x to 100x (most common, lOx, 40x, and lOOx).
> Magnifi-cation is marked on objective, usually with a characteristic
> colored ring for easy identification.
>
> c\. N.A.: measure of the light-gathering power of a lens (the widest
> angle of the cone of light that can enter the objective lens)
>
> \(1\) The higher the N.A., the better the resolving power (the ability
> of thesystem to allow the observer to distinguish individual tiny
> objects).
>
> \(2\) When the N.A. is \>1.00, a liquid immersion medium (e.g.,
> immersion oil) must be placed between the lens and the specimen slide.
> N .A. is a function of the refractive index of the material through
> which light passes from the object to the lens; using a material with
> a higher refractive index than air results in a higher N .A. because
> of increased concentration of light entering the lens.
>
> d\. Degree of optical correction for \<:;hromatic aberration
> (scattering by the lens of white light into colored light \[prism
> effect\] around the object, causing halos and decreasing resolution)
> and spherical aberration (distor-tion in shape of image due to bending
> of light rays)
>
> \(1\) Achromat: corrected for brightest part of the spectrum; no
> spherical correction unless designated \"plan\"
>
> \(2\) Fluorite (calcium fluorite): more highly corrected for
> aberrations;commonly used in fluorescent microscopy; transmits better
> than crown glass at shorter wavelengths
>
> \(3\) Apochromat: least common, most expensive, highest correction for
> aberrations
>
> 5\. Mechanical stage
>
> a\. Function: holds the specimen slide
>
> Adjusting knobs move the slide left, right, forward, and backward.
>
> b\. The mechanical stage may have calibrated graduated lines. Readings
> are taken from the x and y axes of the stage grids to facilitate
> relocation of a specific field of view.
>
> 6\. Condenser
>
> a\. Function: illumination of specimen and production of contrast
>
> \(1\) Light from the condenser converges, forming a cone of light on
> the specimen plane. The light then diverges from the specimen plane to
> form an inverted cone of light angled upward through the objective
> front lens.
>
> \(2\) The aperture diaphragm located beneath or within the
> condensercontrols the angle of the illuminating cone of light.
>
> b\. Degree of optical correction for chromatic and spherical
> aberration
>
> \(1\) Abbe: simplest and least expensive; no correction for optical
> aberrations
>
> \(2\) Aplanatic: corrected for spherical aberrations
>
> \(3\) Achromatic: corrected for chromatic aberrations
>
> \(4\) Achromatic-aplanatic: most expensive; corrected for both kinds
> of optical aberrations
>
> c\. N .A. of the condenser
>
> \(1\) Resolving power (ability of the system to allow the observer to
> distinguish between two small objects) is limited by the N.A. of the
> objective and of the condenser. The condenser N.A. should be equal to
> or greater than the N.A. of the highest objective.
>
> \(2\) Condensers with 0.9 N.A. are most common and do not accept
> immersion media. N .A. of the optical system of \> 1.0 cannot be
> achieved with this condenser.
>
> \(3\) Condensers with N.A. of \>1.0 accept immersion media to achieve
> an N .A. of \> 1.0 for better resolution. Immersion media must be
> placed between the condenser and specimen plane. Note: To optimize the
> resolving power of an objective with an N.A. of \> 1.0, use a
> condenser with an N.A. higher than the N.A. of the objective and also
> use immersion liquid between the objective and the specimen plane and
> between the condenser and the specimen plane.
>
> 7\. Other substage components
>
> a\. Condenser centering screws are used to correctly align the
> condenser on the same optical axis as the objective.
>
> b\. Two-sided mirror (one side concave and the other side flat)

The flat side of the mirror is always used with a condenser. The concave
side of the mirror is used without the condenser for objectives with
very low N.A.

> c\. Built-in light sources (bulbs)
>
> \(1\) Tungsten

Turn light source down for standby. Turning it completely off and on
allows heat buildup.

> \(2\) Tungsten-halogen

Turn light source completely off for standby.

> d\. Filters
>
> \(1\) Blue

Changes yellow light source into a more natural \"white\" light source;
good for some color photography

> \(2\) Neutral density

Reduces brightness without changing color of the background; two can be
used together to further reduce brightness; especially good for
photography

> \(3\) Other filters with light-modifying characteristics are used for
> different purposes. Consult technical °. representativ_s for filter
> information related to an individual application.
>
> 8\. Coverslip

In biomedical applications, objectives are corrected for use with a
coverslip. Objectives are corrected for spherical aberration only when
used with a coverslip of the proper thickness. A no. 1112 coverslip is
approximately 0.16 to 0.19 mm thick. Note: Thickness of the coverslip
must not vary more than i:.0.05 mm from the thickness indicated on
objective.

> a\. A coverslip is mounted on top of the specimen to enhance
> resolution and protect the specimen.
>
> b\. The appropriate thickness of the coverslip is specified by the
> number engraved on the objective (0.17 or 0.18 mm).
>
> c\. No coverslip is necessary when the objective has a dash (-) on it.
>
> 9\. Immersion liquid

With objectives having an N .A. of \> 1.0, use immersion liquid, usually
oil, to expand the angle of light entering the lens and increase the
resolution. \"Oil\" or \"oel\" printed on an objective indicates that
immersion liquid must be used. Medium-viscosity immersion oil is
adequate, but for best resolution, use the oil recommended by the
manufacturer. Note: Certain special objectives with an N.A. of \<1. 0
require an immersion medium. The objective will be labeled to indicate
this.

###### E. Other designations, features, and definitions {#e.-other-designations-features-and-definitions .unnumbered}

> 1\. Flat field, the correction for field curvature, is indicated by
> the prefix \"plan\" or \"pIano\" with the type of objective, e.g.,
> planachromat.
>
> 2\. An objective may have a built-in adjustable correction system,
> also called a \"correction ring,\" that allows the operator to modify
> the objective to accommodate a coverslip thickness other than that
> designated on the objective.
>
> 3\. An immersion oil objective may have a locking mechanism to lock
> the objective up out of the way to protect it and to prevent immersion
> oil from spreading to other objectives.
>
> 4\. Objectives are usually marked with different-colored rings or
> different numtlers of rings for quick magnification reference.

######## II. Environmental Concerns {#ii.-environmental-concerns .unnumbered}

####### A. Placement in the laboratory {#a.-placement-in-the-laboratory .unnumbered}

> 1\. Place on a level surface where vibration from centrifuges or other
> pieces of equipment does not occur. Vibration-free bases are available
> to put on a countertop underneath the microscope (see Appendix 3 to
> this procedure).

2.  Windows should have adjustable coverings to vary the amount of
    sunlight directed to the work area.

3.  Place the microscope facing away from windows.

4.  Place the microscope in a location where it does not have to be
    moved frequently.

5.  Allow sufficient counter space for report recording;

> 6\. Use a pneumatic chair to allow different people to adjust the
> chair heightquickly for comfort while using the microscope.

###### B. Special precautions {#b.-special-precautions .unnumbered}

> 1\. Remove oil from lens after each use.

2.  Turn objective to lowest-power lens before removing slide and for
    standby.

> 3\. Do not turn power on light source past maximum specified by
> manufac-turer.
>
> 4\. Use only lens paper. Do not use facial tissues or Kim Wipes (these
> hav fibers that can scratch the lens surface).
>
> 5\. If slides are always placed on stage in same orientation, it will
> be easier to find specific areas for reexamination. Be certain that
> slides are seated properly; if not, location of position on slide will
> not be correct.
>
> 6\. Use only manufacturer\'s recommended cleaning solutions.
>
> 7\. Cover microscope when not in use.
>
> 8\. Place slide with specimen side up.
>
> 9\. Align microscope with each use (Koehler illumination) for best
> image quality .
>
> 10\. Never touch light bulbs with fingers. Natural skin oils may burn
> and dark the bulb surface, resulting in a premature decrease in light
> intensity. Use lens paper to touch the bulb when inserting it into the
> light source housing.
>
> 11\. Use coarse-focus knob initially to approximate distance of
> objective above the slide. Then use the fine-focus knob when viewing
> through the objective.
>
> 12\. When viewing wet-mount (KOH) slides, reduce light and increase
> contrast (see section IV.A.17 below).
>
> 13\. Always carry the microscope with one hand supporting the base and
> the other hand around the arm. Never move the microscope by the
> nosepiece.
>
> 14\. Do not introduce bubbles into the immersion oil, since a
> microscope bubble under the objective lens will cause flare and lower
> contrast, thus affecting image quality.

15. Always keep immersion oil bottle capped and free of dust and debris.

16. Do not exchange objectives between microscopes unless you know that
    their mechanical tube length specifications are equivalent or that
    the m_croscope has been calibrated with those objectives.

######## III. Materials {#iii.-materials .unnumbered}

###### A. Supplies {#a.-supplies .unnumbered}

> 1\. Lens-cleaning fluid

Used to clean optical surfaces without harming the antireflex coatings
of the lenses or softening the sealers and cements in the optics. A
variety of cleaning fluids are available (see Appendix 3 to this
procedure). Use whatever the manufacturer recommends.

> a\. Ethyl ether (extremely flammable) and xylene (toxic) do not damage
> the optics. However, these chemicals must be stored safely and used
> with proper ventilation.
>
> b\. Do not use alcohols (methyl, ethyl, or isopropyl), acetone, or any
> other ketones by themselves, as they may dissolve the sealers in and
> around the lens.
>
> 2\. Lens paper

Lens paper is manufactured specifically to keep abrasiv\_ particles out
of the paper. Do not use facial tissues or Kim Wipes. .

> 3\. Immersion oil

Place a drop of oil on the microscope slide between the specimen and the
oil immersion lens to increase resolution for a sharper image. Use a
nondrying, nonhardening oil for ease of cleaning. Observe the
manufacturer\'s recommendation for oil that will best complement the
optical quality of its lens.

> 4\. Light bulbs

Maintain a supply of bulbs for each different model of microscope.

###### B. Equipment {#b.-equipment .unnumbered}

> 1\. Air brush (available from photography supply stores) or canned
> compressed air (available from art supply stores, computer and
> electronic supply stores, and other sources)

Use air to blow particles away from the surfaces of the scope. Be
careful around the stage area, as specks of broken glass may be present.
Wear safety goggles when cleaning with air, especially if canned
compressed air is used.

> 2\. Microscope cover After use, replace dust cover to protect
> microscope from dust.

######## IV. Initial Aligning Procedures {#iv.-initial-aligning-procedures .unnumbered}

A.  Koehler illumination (Appendix 1)

> Koehler illumination is a precise way of aligning the light pathway
> onto the specimen plane to evenly illuminate the entire field of view.
> This procedure ensures the highest resolution for the optical system,
> enabling visualization of as much detail as possible.
>
> 1\. Place a cleaned stained specimen (cells, bacteria, or diatoms;
> subject identity is not critical, but best to have distinct form for
> ease of focus) slide right side up in the stage slide holder.
>
> 2\. Open the field diaphragm all the way.
>
> 3\. Open the condenser diaphragm all the way. If the condenser has an
> auxiliary lens, swing it out of the light path. Otherwise, you will
> not be able to image the diaphragm.
>
> 4\. Rack the condenser all the way up.
>
> 5\. Rotate the nosepiece to the lOx objective position (8x to 20x
> objective is acceptable).

6.  Turn on the light source, and adjust it to a low, comfortable light
    intensity.

> 7\. Adjust the binoculars to your interpupillary distance.
>
> 8\. Use the focusing eyepiece, if available, to compensate for any
> dioptic discrepancy (differential ability to focus) between each eye.
> Adjust both eyepieces to equal height.

9.  Look through the fixed eyepiece, and focus on the specimen, using
    the coarse and fine adjustment knobs.

10. Use the adjustable eyepiece to focus the image sharply.

11. Gradually close the field diaphragm until you see a multisided
    polygon(image of diaphragm) around the field of the specimen.

12. Lower the condenser slightly until the diaphragm edge is as sharp as
    possible. (A magenta color may be seen.)

13. Adjust fine-focus knob so the specimen is in sharp focus.

14. If the diaphragm image is not centered, then center.it by gently
    turning the centering screws located on the condenser. \'

15. Open the field diaphragm until the image of the diaphragm just goes
    out of the field of view. Do not open any further.

16. Do not disturb the condenser height.

> 17\. Set the optimal contrast by gradually closing the aperture
> diaphragm. (Rule of thumb is to remove the eyepiece, look down the
> tube at the back focal plane of the objective, and adjust the aperture
> to two-thirds open. Control contrast by adjusting the condenser
> diaphragm.
>
> 18\. Most microscopes will not need further alignment for higher
> magnification.

B.  Calibration of the ocular micrometer See Appendix 4 to this
    procedure.

######## V. Routine Quality Control Procedures {#v.-routine-quality-control-procedures .unnumbered}

See Appendix 5 for a sample form.

####### A. After daily use {#a.-after-daily-use .unnumbered}

> 1\. Wipe oil from objective, condenser, and stage with lens paper.

2.  Turn off the microscope light source.

3.  Replace microscope cover.

####### B. Each month {#b.-each-month .unnumbered}

> 1\. Use airbrush to blow away dust. Clean objectives, eye pieces,
> condenser, and field diaphragm with lens-cleaning fluid. Apply fluid
> with lens paper. Do not put fluid directly onto the part.
>
> 2\. Remove slide holder from stage, and clean.
>
> 3\. With a water-moistened tissue, wipe dust off of the body of the
> microscope and the window of the illuminator in the base (include blue
> filter if used).
>
> C. Every 6 months
>
> Thoroughly clean, lubricate, and inspect the microscope. Service
> personnel may do this.

######## VI. Routine Operation {#vi.-routine-operation .unnumbered}

> A. Turn on the microscope light source.
>
> B. Adjust binoculars and eyepieces to your personal preference.
>
> C. Adjust power source for a comfortable light intensity. Be sure that
> Koehlerillumination has been achieved.
>
> D. Secure slide in stage slide holder.

E.  Rotate nosepiece for desired magnification objective, and raise or
    lower stage.

F.  With one hand, focus on specimen by using coarse- and fine-focus\'
    knobs.

> G. With the other hand, move the slide by turning the stage drive.
>
> H. When reading is finished, rotate objective away from slide.
>
> I. Release tension on slide holder, and remove slide.
>
> J. If oil was used, wipe oil from objective with lens paper.
>
> K. Turn light down or off.
>
> L. Eyestrain should not develop if the microscope is set up properly
> and the chair is at the correct height for the user.
>
> .
>
> Table 1 lists some common problems and suggestions for solving them

+-----------------------------------+-----------------------------------+
| # Light source f                  | Loose plug connection at wall     |
| lickers on and off {#light-source | socket                            |
| -flickers-on-and-off .unnumbered} |                                   |
|                                   | Loose plug connection at          |
|                                   | transformer or power supply to    |
|                                   | microscope                        |
|                                   |                                   |
|                                   | Improperly installed light bulb   |
|                                   |                                   |
|                                   | If dark spots on bulb, bulb may   |
|                                   | be about to blow out              |
|                                   |                                   |
|                                   | Dirty bulb contacts: buldup of    |
|                                   | crusty deposits must be gently    |
|                                   | filed away                        |
|                                   |                                   |
|                                   | Frayed or broken wiring           |
|                                   |                                   |
|                                   | Faulty on-off switch              |
+-----------------------------------+-----------------------------------+
| **Light source does not turn      | Microscope not plugged in         |
| on.**                             |                                   |
|                                   | Light bulb burned out             |
|                                   |                                   |
|                                   | Faulty switch mechanism           |
+-----------------------------------+-----------------------------------+
| **Specimen unevenly illuminated** | Light source not centered;adjust  |
|                                   | for Koehler illumination.         |
+-----------------------------------+-----------------------------------+
| **Specimen poorly illuminated     | Objective not quite in alignment  |
| even though light is turned all   | with cIick-stop position          |
| the way up**                      |                                   |
|                                   | Diaphragms almost closed          |
|                                   |                                   |
|                                   | Condenser too low; adjust for     |
|                                   | Koehler illumination.             |
+-----------------------------------+-----------------------------------+
| **Fine-focus control does not     | Turn fine-focus control knob to   |
| work.**                           | halfway point within its range.   |
|                                   | At 40x,adjust focus with the      |
|                                   | coarse adjustment.                |
+-----------------------------------+-----------------------------------+
| **Specimen goes out of focus more | Remove slide holder; clen it and  |
| than usual during high-power      | the stage of oil and possible     |
| observations**                    | debris                            |
+-----------------------------------+-----------------------------------+
| **Specimen can be focused at lOx  | Check that slide surface with     |
| but not at higher power.**        | specimen is right side up.        |
|                                   | Coverslip and/or mounting medium  |
|                                   | may be too thick.                 |
+-----------------------------------+-----------------------------------+
| **Deterioration of 40x objective  | Examine objective lens (see       |
| image quality**                   | Appendix 6). If scratches, nicks, |
|                                   | cracks, or oil seepage into the   |
|                                   | lens is apparent, have lens       |
|                                   | serviced professionally           |
+-----------------------------------+-----------------------------------+
| **Oil immersion lens (SOx, 60x,   | Examine front surface of          |
| or lOOx) fails to give clear      | objective for oil or fingerprints |
| image.**                          | (see Appendix 6). Clean lens with |
|                                   | lens paper moistened with         |
|                                   | cleaning fluid. Air bubble may be |
|                                   | trapped between oil and lens if   |
|                                   | lens is brought directly down     |
|                                   | onto specimen; sweep objective    |
|                                   | into oil from the side.           |
+-----------------------------------+-----------------------------------+
| **Artifacts moving across field   | May be caused by \"floaters\" in  |
| of view**                         | the observer\'s eye. If not,      |
|                                   | adjust interocular distance.      |
|                                   | Adjust condenser diaphragm to     |
|                                   | vary contrast                     |
+-----------------------------------+-----------------------------------+
| **Eyes strain to focus at the     | Adjust eyepieces. One may be      |
| same time**                       | screwed all the way in or out.    |
|                                   | Readjust focus for each eye. If   |
|                                   | eyepieces have removable lenses,  |
|                                   | be certain that they are          |
|                                   | assembled correctly.              |
|                                   | Alternatively, the binocular head |
|                                   | may be out of collimation (the    |
|                                   | two separate optical pathways do  |
|                                   | not converge at the same spot on  |
|                                   | the slide). Requires professional |
|                                   | realignment.                      |
+-----------------------------------+-----------------------------------+
| **Unable to focus at any power**  | Draw a line on slide surface with |
|                                   | wax marking pencil. Focus on the  |
|                                   | lineand then move the slide to    |
|                                   | the area of interest.             |
+-----------------------------------+-----------------------------------+

######## VII. Trouble-shooting {#vii.-trouble-shooting .unnumbered}

###### Magnification of eyepieces and objectives

> Magnification is determined by size of organisms or cells to be
> viewed.

,.

> 1\. Total magnification equals eyepiece magnification x objective
> magnification x intermediate microscope magnification (if any). For
> example, lOx (eyepiece) x Ix (intermediate magnifi-cation) x 40x
> (objective) = 400x.
>
> 2\. Empty magnification occurs when increased magnification no longer
> improves detail. Maxi-mum magnification for a complete system should
> not exceed approximately 1,000 times the N.A. of the objective being
> used. For example, for a 40x objective with an N.A. of 0.65, maximum
> magnification equals 0.65 x 1,000 = 650x.
>
> 3\. Choose the appropriate combination of objectives for magnification
> needs, e.g., lOx, 40x, and 1O0x, or 4x, 25x, and 40x. .
>
> 4\. Choose specialized objectives for special applications, such as
> fluorite glass objective for fluorescence work. Fluorite glass allows
> better light transmission in the near-UV range. Apo-corrected
> objectives are ideal for color photography because they allow the
> least chromatic aberration across the entire field, they reproduce
> colors most accurately, and they have even focus across the entire
> field.

####### B. Optical quality versus expense {#b.-optical-quality-versus-expense .unnumbered}

> Optimal optical quality is necessary for photomicroscopy. Be prepared
> to pay a premium for good lenses. :

####### C. Manufacturer\'s customer service {#c.-manufacturers-customer-service .unnumbered}

> 1\. Knowledgeable technical personnel
>
> 2\. Availability of parts
>
> 3\. Short service call turnaround time
>
> 4\. Good reputation: check with other users for questions about
> customer service.

Steps for inspecting objective lens with microscope eyepiece

A. Ca!efully unscrew objective from nosepiece.

B. Gently remove one eyepiece to use as a magnifier.

C. Grasp the objective in one hand with the front lens face up.

D. Hold the eyepiece in the other hand with the top lens facing down.

> E. Bring the eyepiece very close to your eye, and focus on the
> objective_lens. Angle the objective so that room light can reflect off
> of its surface. The two lens surfaces will be about 1 in. apart. Try
> to avoid letting them touch each other.
>
> F. nspect the objective for scratches, nicks, cracks, deterioration of
> the seal around the lens, or oil seepage into the lens.

Calibration of microscope with an ocular micrometer

I. Principle The identification of protozoa and other parasites depends
on several factors, one of which is size. Any laboratory doing
diagnostic work in parasitology should have a calibrated microscope
available for precise measurements. Measurements are made with a
micrometer disk that is placed in the ocular of the microscope; the disk
is usually calibrated as a line divided into 50 U. Depending on the
objective magnification used, the divisions in the disk represent
different measurements. The ocular disk division must be compared with a
known calibrated scale, usually a stage micrometer with a scale of 0.1-
and O.O1-mm divisions (1).

II\. Materials

######### A. Supplies {#a.-supplies-1 .unnumbered}

> 1\. Ocular micrometer disk (line divided into 50 U) (any laboratory
> supply distributor:Fisher, Baxter, Scientific Products, VWR, etc.)
>
> 2\. Stage micrometer with a scale of 0.1- and O.O1-mm divisions
> (Fisher, Baxter, Scientifc Products, VWR, etc.)
>
> 3\. Immersion oil
>
> 4\. Lens paper

###### B. Equipment {#b.-equipment-1 .unnumbered}

> 1\. Binocular microscope with 10 x, 40 x, and 100 x objectives. Other
> objective magnifications (50 x oil or 63x oil immersion lenses) may
> also be used. :
>
> 2\. Oculars should be lOx. Some may prefer 5x; however, smaller
> magnification may make final identifications more difficult.
>
> 3\. Single lOx ocular to be used to calibrate all laboratory
> microscopes (to be used when any organism is being measured) ill.
> Quality Control
>
> A. Recalibrate the microscope a minimum of once each year. If the
> scope receives heavy use, twice a year is recommended.
>
> B. Often the measurement of RBCs (approximately 7.5 ILm) is used to
> check the calibrations of the three magnifications (x100, x400,
> xI,OOO).
>
> C. Latex or polystyrene beads of a standardized diameter can be used
> to check the calculations and measurements (Sigma, J. T. Baker, etc.).
> Beads of 10 and 90 ILm are recommended.

D. Record all measurements in QC records.

######## IV. Procedure {#iv.-procedure .unnumbered}

> A. Unscrew the eye lens of a lOx ocular, and place the micrometer disk
> (engraved side down).within the ocular. Use lens paper to handle the
> disk; keep all surfaces free of dust or lint.
>
> B. Place the calibrated micrometer on the stage, and focus on the
> scale. You should be able to distinguish the difference between the
> 0.1- and O.O1-mm divisions. Make sure you understand the divisions on
> the scale before proceeding.
>
> C. Adjust the stage micrometer so that the \"0\" line on the ocular
> micrometer is exactly lined up on top of the 0 line on the stage
> micrometer.
>
> D. When these two 0 lines are lined up, do not move the stage
> micrometer any farther. Look to the right of the 0 lines for another
> set of lines superimposed on each other. The second set of lines
> should be as far to the right of the 0 lines as possible; however, the
> distance varies with the objectives being used (Fig. AI).
>
> E. Count the number of ocular divisions between the 0 lines and the
> point where the second set of lines is superimposed. Then, on the
> stage micrometer, count the number of O.l-mm divisions between the 0
> lines and the second set of superimposed lines.
>
> F. Calculate the portion of a millimeter that is measured by a single
> small ocular unit.
>
> G. When the high dry and oil immersion objectives are used, the 0 line
> of the stage micrometer will increase in size, whereas the ocular 0
> line will remain the same size. The thin ocular 0 line should be lined
> up in the center or at one edge of the broad stage micrometer 0 line.
> Thus, when the second set of superimposed lines is found, the thin
> ocular line should be lined up in the center or at the corresponding
> edge of the broad stage micrometer line.
>
> Example: If a helminth egg measures 15 ocular units by 7 ocular units
> with the high dry objective, then multiply the measurements by the
> factor 2.0 ILm (for that objective). The egg then measures 30 by 14
> ILm and is probably Clonorchis sinensis.
>
> Example: If a protozoan cyst measures 27 ocular units with the oil
> immersion objective, then multiply the measurement by the factor 0.8
> ILm (for that objective). The cyst then measures 21.6 ILm.

######## V. Results {#v.-results .unnumbered}

> A. For each objective magnification, a factor will be generated
> (locular unit = certain number ofmicrometers).
>
> B. If standardized latex or polystyrene beads or an RBC is measured
> with various objectives, thesize for the object measured should be the
> same (or very close), regardless of the objectivemagnification.

######## VI. Reporting Results {#vi.-reporting-results .unnumbered}

> A. Post the multiplication factor for each objective either on the
> base of the microscope or on anearby walI or bulIetin board for easy
> reference.
>
> B. Once the number of ocular lines per width and length of the
> organism is measured, then, dep\"ending on the objective
> magnification, the factor (locular unit = certain number of
> millimeters) can be applied to the number of lines to obtain the width
> and length of the organism.
>
> C. Comparison of these measurements with reference measurements in
> various books and manuals should confirm the organism identification.

######### **VII. Procedure Notes** {#vii.-procedure-notes .unnumbered}

> A. The final multiplication factors will be only as good as your
> visual comparison of the oculular a and stage micrometer a lines.
>
> B. As a rule of thumb, the high dry objective (4ax) factor should be
> approximately 2.5 times more than the oil immersion objective (lOa x)
> factor. The low-power objective (lOx) factor should be approximately
> 10 times the oil immersion objective (lOax) factor.

**VIII. Limitations of the Procedure**

> A. Mter each objective has been calibrated, the oculars containing the
> disk and/or these objectives cannot be interchanged with corresponding
> objectives or oculars on another microscope. \--
>
> B. Each microscope used to measure organisms must be calibrated as a
> unit. The original oculars and objectives that were used to calibrate
> the microscope must also be used when an organism is measured.
>
> C. The objective containing the ocular micrometer can be stored until
> needed. This single ocular can be inserted when measurements are
> taken. However, this particular ocular containing the ocular
> micrometer disk must also have been used as the ocular during
> microscope calibration.