+-------------------+--------------------------------------------------+
| ![](media/image1. | Module 7                                         |
| jpeg){width="1.50 |                                                  |
| 83333333333333in" | **Microscopy**                                   |
| height="1.135     |                                                  |
| 4166666666667in"} |                                                  |
+-------------------+--------------------------------------------------+

+------------------+---------------------------------------------------+
|                  |                                                   |
+------------------+---------------------------------------------------+
| **Purpose**      | To provide you with an understanding of the       |
|                  | appearance and various forms of acid-fast bacilli |
|                  | (AFB) that appear in clinical specimens           |
+------------------+---------------------------------------------------+
| **Prerequisite** | Module 6                                          |
|                  |                                                   |
| **Modules**      |                                                   |
+------------------+---------------------------------------------------+
| **Learning       | At the end of this module, you will be able to    |
| Objectives**     |                                                   |
|                  | -   Describe the method for observing 100 high    |
|                  |     power fields                                  |
|                  |                                                   |
|                  | -   Correctly use microscope objectives           |
|                  |                                                   |
|                  | -   Recognize the appearance of AFB in a stained  |
|                  |     smear                                         |
|                  |                                                   |
|                  | -   Appropriately quantify results in the         |
|                  |     Laboratory Register                           |
|                  |                                                   |
|                  | -   Describe the procedure for storing slides for |
|                  |     External Quality Assessment (EQA).            |
+------------------+---------------------------------------------------+
| **Content        | -   Required materials                            |
| Outline**        |                                                   |
|                  | -   Reading the smear                             |
|                  |                                                   |
|                  | -   Microscope components and operation           |
|                  |                                                   |
|                  | -   Appearance of AFB                             |
|                  |                                                   |
|                  | -   WHO/ IUATLD grading scale                     |
|                  |                                                   |
|                  | -   Storing smears                                |
|                  |                                                   |
|                  | -   Cleaning the objectives                       |
+------------------+---------------------------------------------------+
| **Handout and    | Exercise: Laboratory Practical session \# 5:      |
| Exercises**      | Viewing of prepared and stained smears from       |
|                  | Laboratory Practical session \# 4                 |
+------------------+---------------------------------------------------+
| **Appendix**     | -   Appendix 1- Microscope specifications         |
|                  |                                                   |
|                  | -   Appendix 2 - Microscopy recording form        |
+------------------+---------------------------------------------------+

Module 7: Microscopy

Examination of sputum smears for acid-fast bacilli (AFB) requires a good
microscope and a motivated, trained technician.

### 

**REQUIRED MATERIALS**

The following materials are required to perform the microscopy of AFB
smear:

1)  Binocular microscope with oil immersion objective

2)  Electric power or daylight

3)  Immersion oil

4)  Lens paper or fine tissue paper

5)  Lens cleaning solution

6)  Laboratory register

7)  Slide storage boxes

8)  Red and blue writing pens

### Microscopy

A binocular microscope is recommended for reading AFB smears. It must
have an oil immersion 100X objective with 8--10X magnification
eyepieces, and a mechanical stage. (For complete microscope
specifications, refer to Appendix 1.)

It should have a backup mirror. It is best to read in subdued lighting.
If sunlight is the light source, place the microscope in front of a
window but not in direct sunlight.

The microscope should be placed on a stable level bench, well away from
the staining area.

**MICROSCOPE COMPONENTS**
![](media/image2.jpeg){width="3.7562992125984254in"
height="4.275590551181103in"}

Study the components of the microscope and know how each part functions.

+---------------------+------------------------------------------------+
| **Microscope Part** | **Function**                                   |
+=====================+================================================+
| Eyepieces           | > Pair of lenses used to view the magnified    |
|                     | > image from the objective lens.               |
+---------------------+------------------------------------------------+
| Diopter adjustment  | > Used to focus the image by turning it        |
| ring                | > clockwise or anticlockwise to obtain a image |
|                     | > view in the same plan for the both eyes.     |
+---------------------+------------------------------------------------+
| Binocular tube      | > The part holding the eyepieces and dividing  |
|                     | > the light between them. It is used to adjust |
|                     | > the distance between the eyes so that a      |
|                     | > single, overlapping image is obtained by     |
|                     | > both eyes.                                   |
+---------------------+------------------------------------------------+
| Nose piece          | > The mechanical and revolving part that holds |
|                     | > the objective lenses.                        |
+---------------------+------------------------------------------------+
| Objective lenses    | > These are the bunch of lenses of various     |
|                     | > magnification powers, used to view the       |
|                     | > object.                                      |
+---------------------+------------------------------------------------+
| Mechanical stage    | > The horizontal platform to place the object  |
|                     | > for viewing.                                 |
+---------------------+------------------------------------------------+
| Slide holder        | > Mechanical arm that is used to hold the      |
|                     | > object or slide for a smooth and uniform     |
|                     | > movement.                                    |
+---------------------+------------------------------------------------+
| Condenser with      | > The lens system that concentrates the light  |
| diaphragm           | > on the object to be magnified. It contains   |
|                     | > an iris diaphragm meant to reduce glare from |
|                     | > dispersed light.                             |
+---------------------+------------------------------------------------+
| Filter              | > A blue-colored glass that makes the light in |
|                     | > the visual field to appear as natural        |
|                     | > daylight.                                    |
+---------------------+------------------------------------------------+
| Field diaphragm     | > Controls the amount of light form light      |
|                     | > source.                                      |
+---------------------+------------------------------------------------+
| Lamp                | > Light source in the base of the microscope   |
|                     | > stand.                                       |
+---------------------+------------------------------------------------+
| Coarse focus knob   | > Focusing knob that allows a coarse           |
|                     | > adjustment of the image.                     |
+---------------------+------------------------------------------------+
| Fine focus knob     | > Focusing knob that allows a fine adjustment  |
|                     | > of the image.                                |
+---------------------+------------------------------------------------+
| Power switch        | > Controls the power supply to microscope      |
+---------------------+------------------------------------------------+
| Voltage regulator   | > Controls the amount of voltage supplied to   |
|                     | > the lamp.                                    |
+---------------------+------------------------------------------------+
| Stage movement      | > Used to move the slide in x and y direction  |
| knobs               | > for complete coverage of object, in our case |
|                     | > it is the smear.                             |
+---------------------+------------------------------------------------+

**OPERATION OF THE MICROSCOPE**

Learn how to correctly operate the microscope. This task is as important
as performing the smear and staining procedures. It will ensure that the
microscope's lenses and other working parts are handled carefully,
preventing damage to any of its important parts.

Review the details of operation in the instruction manual of each
microscope.

1.  Plug the microscope into the electrical source and switch on the
    light at low intensity.

2.  Place a specimen slide on the stage. Be sure the slide is not placed
    upside down.

3.  Focus the specimen with the 10× objective by turning the coarse
    adjustment knob.

4.  Adjust the distance between the ocular lenses until both the right
    and left images become one.

5.  Field diaphragm- adjust Kohler illumination, if required. Refer to
    module 9 for details.

6.  Fine-focus the image with the right eye looking into the right
    eyepiece by turning the fine adjustment knob.

7.  Focus the image with the left eye looking into the left eyepiece by
    turning the diopter ring.

8.  Put one drop of immersion oil on the smear .

9.  Change to the 100× objective. Be sure the condenser is raised as
    high as possible to maintain the intensity of the light. Open the
    condenser iris to 70-80% of the aperture diameter. Focus the
    specimen slide by turning only the fine-focus adjustment knob.

10. Never adjust the stage upward while looking through the eyepiece*.*
    If you do so, you will push the objective through the slide. This
    may damage the slide and the objective.

11. Use only the 100x objective (immersion objective) for observation
    through immersion oil. All other objectives must be used without
    immersion oil and kept dry.

12. To view next slide, entire procedure does not need to be repeated.
    Turn away the 100 X objective and take out the former slide, add a
    drop of immersion oil on new stained smear and insert on to stage,
    then turn 100 X objective.

# Applying immersion oil

-   Make sure that the smear is facing upwards when the slide is placed
    on the mechanical stage.

-   Focus the smear using low power objective 10X.

-   Put one drop of immersion oil on the stained smear, letting it fall
    freely onto the slide.

-   Never allow the oil applicator to touch the slide. Touching the
    slide with the applicator could lead to contamination of the oil
    with AFB and could transfer AFB to a negative slide.

# 

# 

# READING THE SMEAR

-   Carefully rotate the 100x objective over the smear and focus it.

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-   Systematically examine the smear under the 100X objective.

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-   Scan smears by moving across the smear in a horizontal direction.

-   Stop and observe each field before moving onto the next field.

-   Read at least 100 high power fields before reporting a negative
    result. (Note: Fewer than 100 fields may be read if the slide is
    found positive for AFB.)

-   Usually examining 100 fields takes about 5 minutes.

# Appearance of AFB in the smear

AFB stain red or pink against a blue background with the Ziehl-Neelsen
staining method. They are usually thin and rod-shaped, but occasionally
may appear as coccoid (beaded), filamentous (thread-like), or clumped
(in group) forms.

**WHO/IUATLD GRADING SCALE**

### Reporting scale

Follow the scale when reporting smears:

-   If no AFB are seen in at least 100 fields, report as negative for
    AFB.

-   If 1--9 AFB are seen in 100 fields, report actual number of AFB
    seen.

-   If 10--99 AFB are seen in 100 fields, report as (1+).

-   If 1--10 AFB/field in at least 50 fields report as (2+).

-   If more than 10 AFB/field in at least 20 fields, report as (3+).

+----------------+-----------------------------------------------------+
| ## R           | ## AFB seen                                         |
| eporting scale |                                                     |
+----------------+-----------------------------------------------------+
| **Negative**   | # No AFB seen in at least 100 fields                |
+----------------+-----------------------------------------------------+
| **Actual       | **1-9 AFB per 100 fields**                          |
| number**       |                                                     |
+----------------+-----------------------------------------------------+
| **(1+)**       | **10-99 AFB per 100 fields**                        |
+----------------+-----------------------------------------------------+
| **(2+)**       | **1-10 AFB per field in at least 50 fields**        |
+----------------+-----------------------------------------------------+
| **(3+)**       | **More than 10 AFB per field in at least 20         |
|                | fields**                                            |
+----------------+-----------------------------------------------------+

# Recording results 

-   Record results in the laboratory register immediately after reading
    smears.

-   Use a red pen to recording POSITIVE results in the laboratory
    register.

Return the completed Request for Sputum Examination form to the
requesting doctor or clinic.

The laboratory register is not only an important record of laboratory
results but also helps indicate laboratory performance.

Such performance indicators include:

-   Positivity rates among suspects and follow up patients

-   Consistency with case series

-   Case yield of first smears

-   Ability to detect low positive case smears

# STORING SMEARS

-   Store ALL slides in slide boxes in the order they were recorded in
    the laboratory register. This will allow easy sampling of slides for
    external quality assessment using blinded slide rechecking.

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```
-   Ensure that three slide spaces are left for each new diagnostic
    case.

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-   Remove oil from smears either by placing the slide face down on
    toilet tissue or wrapping it in the tissue and leaving it this way
    overnight. Gravity helps remove most of the oil when the slide is
    kept straight up in the slide rack overnight.

-   Xylene is not necessary to remove oil at the peripheral level.
    Xylene should never be used to clean the microscope or dilute
    immersion oil.

# CLEANING THE OBJECTIVES

-   After reading the smears, gently wipe the objective lens with lens
    paper or fine tissue paper.

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```
-   NEVER use petroleum, benzene, acetone, or xylene to clean objective
    lenses.

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```
-   After cleaning, cover the microscope with a vinyl or cotton cloth
    cover and store it in a place free from moisture and dust.

+-----------------+----------------------------------------------------+
| **Key           | -   Use the WHO/IUATLD grading scale for the       |
| messages**      |     smears.                                        |
|                 |                                                    |
| ![](m           | -   Systematically scan the slide by moving across |
| edia/image3.wmf |     the smear in a horizontal direction.           |
| ){width="1.1701 |                                                    |
| 388888888888in" | -   Examine each field before moving onto the next |
| height="0.83194 |     field.                                         |
| 44444444445in"} |                                                    |
|                 | -   Read at least 100 high power fields before     |
|                 |     reporting a negative result.                   |
|                 |                                                    |
|                 | -   Store all examined smears in the order they    |
|                 |     appear in the laboratory register.             |
+-----------------+----------------------------------------------------+

+-----------------+----------------------------------------------------+
| > ![](m         | Module Review: Module 7                            |
| edia/image4.wmf |                                                    |
| ){width="1.0in" |                                                    |
| >               |                                                    |
| height="0.96041 |                                                    |
| 66666666667in"} |                                                    |
+=================+====================================================+
| > Find out how  |                                                    |
| > much you have |                                                    |
| > learned by    |                                                    |
| > answering     |                                                    |
| > these         |                                                    |
| > questions.    |                                                    |
+-----------------+----------------------------------------------------+
| > How many AFBs |                                                    |
| > are required  |                                                    |
| > for a 1+, 2+, |                                                    |
| > and 3+ smear? |                                                    |
| >               |                                                    |
| > \_\_          |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
+-----------------+----------------------------------------------------+
| > How many      |                                                    |
| > fields need   |                                                    |
| > to be         |                                                    |
| > examined when |                                                    |
| > reading a     |                                                    |
| > smear for     |                                                    |
| > AFB?          |                                                    |
| > \_\_\_\_      |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
+-----------------+----------------------------------------------------+
| > Which smears  |                                                    |
| > must be       |                                                    |
| > stored after  |                                                    |
| > examination?  |                                                    |
| >               |                                                    |
| > \_\_\_        |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
+-----------------+----------------------------------------------------+
| > When and how  |                                                    |
| > are           |                                                    |
| > microscope    |                                                    |
| > objectives    |                                                    |
| > cleaned?      |                                                    |
| >               |                                                    |
| > \_\_\_        |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
| \_\_\_\_\_\_\_\ |                                                    |
| _\_\_\_\_\_\_\_ |                                                    |
+-----------------+----------------------------------------------------+

Appendix 1

**[Microscope specifications]{.underline}**

-   Microscope must be completely UL\*, CSA\* and CE\* tested, listed,
    and approved to insure fire and/or shock safety. Only UL listed
    components or line cords are not acceptable.

-   Must have10x/18mm eyepieces.

-   Must have auto compensating Siedentopf style binocular with diopter
    scale for interpupillary distance (must have visible diopter
    scales).

-   Must have 4-position reversed nosepiece of metal construction with
    internal ball bearing stops. External clip system not acceptable.

-   Must have 4x HI-Plan, 10x HI-plan , 40x HI-plan, and 100x oil
    HI-plan [parfocal]{.underline} and [parcentered]{.underline}
    infinity corrected objectives.

-   Mechanical stage must be of built-in design with metal rack and
    pinion X-Y drives. No polymer belts, metal cables, timing belt
    systems or non-metallic components are acceptable in the drive
    mechanism. Coaxial controls must be low mounted for ease of use.

-   Pre-aligned Abbe condenser with graduated iris diaphragm wheel with
    markings to show where iris aperture should be set for each
    objective magnification.

-   Focus drive must be a self-tensioning, three ball design of all
    metal construction. Fine focus must have graduations of 100
    divisions and 3 microns per division. Focusing knobs on both sides
    must have these markings.

-   All gears throughout the microscope: mechanical stage, focus,
    condenser rack and pinion must be made of metal, brass, stainless
    steel or aluminum -- no plastic components.

-   Illumination system must be designed for 12v/35w tungsten halogen
    2,000 hour average life bulb.

-   Microscope must have hinged lamp door that is angled to help prevent
    breakage. Sliding "drawer" type bulb covers not acceptable for
    safety reasons.

-   Must have blue filter fixed into its mount, not loose. In Koehler
    kits, lollipop filters have "locking slots" to prevent them from
    falling out when tilted.

-   Microscope base temperature must not exceed 37 degrees centigrade
    using a 12v/20w halogen lamp at full voltage for 6 hours.

-   Power supply must be voltage sensing 85-265 volts with surge
    suppression and soft start lamp control.

-   Lamp intensity must be conveniently located in stand armrest and
    controlled via an illuminated rotating wheel.

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-   Stage finger assembly is to be slide friendly that does not damage
    or break slides.

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-   Microscope must have ergonomic design.

**\*UL:** Underwriters Laboratories Inc.\
\***CSA:** Canadian Standards Association\
\***CE:** Conformance European

  ----------- ------------- ---------- ----------------- ------------ ------ -------------
  Slide       Smear         Smear                                            
  number      appearance    Results                                          

                            Negative   Characteristics                       
                                       of AFB                                

                                       Colour            Appearance   Size   Arrangement

  1                                                                          

  2                                                                          

  3                                                                          

  4                                                                          

  5                                                                          

  6                                                                          

  7                                                                          

  8                                                                          

  9                                                                          

  10                                                                         
  ----------- ------------- ---------- ----------------- ------------ ------ -------------

Appendix 2

**Microscopy**

**Recording Form**

**(For Laboratory Practical Session# 5)**

Participant\'s Name\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
Date\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Workshop Place\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_