To provide you with an understanding of the appearance and Purpose various forms of acid-fast bacilli (AFB) that appear in clinical
Prerequisite Module 6
Learning At the end of this module, you will be able to Objectives ? Describe the method for observing 100 high power
? Correctly use microscope objectives
? Recognize the appearance of AFB in a stained smear
? Appropriately quantify results in the Laboratory
? Describe the procedure for storing slides for External
Quality Assessment (EQA).
Content Outline ? Required materials
? 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: Viewing of Exercises prepared and stained smears from Laboratory Practical
session # 4
Appendix ? Appendix 1- Microscope specifications
? Appendix 2 - Microscopy recording form
Module 7: Microscopy Page 1 of 10
Module 7: Microscopy
Examination of sputum smears for acid-fast bacilli (AFB) requires a good
microscope and a motivated, trained technician.
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
The microscope should be placed on a stable level bench, well away from the
Module 7: Microscopy Page 2 of 10
Diopter ring adjustment
Nose piece Power switch Objectives Voltage regulator Mechanical stage Slide holders Condenser diaphragm Coarse focus knob Centering screws Fine focus knob Filter Field diaphragm Stage movement X-Y Lamp axis
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 ring Used to focus the image by turning it 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 These are the bunch of lenses of various
magnification powers, used to view the object.
Module 7: Microscopy Page 3 of 10
Microscope Part Function Mechanical stage The horizontal platform to place the object for
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 on the
diaphragm 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
Fine focus knob Focusing knob that allows a fine adjustment of the
Power switch Controls the power supply to microscope
Voltage regulator Controls the amount of voltage supplied to the lamp.
Stage movement knobs Used to move the slide in x and y direction for
complete coverage of object, in our case it is the
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
2. Place a specimen slide on the stage. Be sure the slide is not placed
3. Focus the specimen with the 10? objective by turning the coarse
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
Module 7: Microscopy Page 4 of 10
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
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
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.
? Systematically examine the smear under the 100X objective.
? 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
? 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.
Module 7: Microscopy Page 5 of 10
WHO/IUATLD GRADING 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+).
AFB seen Reporting scale
No AFB seen in at least 100 fields Negative
1-9 AFB per 100 fields Actual number
10-99 AFB per 100 fields (1+)
1-10 AFB per field in at least 50 fields (2+)
More than 10 AFB per field in at least 20 fields (3+)
? 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
? 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.
? Ensure that three slide spaces are left for each new diagnostic case.
? 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
Module 7: Microscopy Page 6 of 10
? 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.
? NEVER use petroleum, benzene, acetone, or xylene to clean objective
? After cleaning, cover the microscope with a vinyl or cotton cloth cover and
store it in a place free from moisture and dust.
Key messages ? Use the WHO/IUATLD grading scale for the smears.
? Systematically scan the slide by moving across the
smear in a horizontal direction.
? Examine each field before moving onto the next field.
? 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.
Module 7: Microscopy Page 7 of 10
Module Review: Module 7
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?
Module 7: Microscopy Page 8 of 10
? 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 and parcentered 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
? 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
? 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
? 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
? 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.
? Stage finger assembly is to be slide friendly that does not damage or
? Microscope must have ergonomic design.
*UL: Underwriters Laboratories Inc.
*CSA: Canadian Standards Association
*CE: Conformance European
Module 7: Microscopy Page 9 of 10
(For Laboratory Practical Session# 5)
Participant's Name_______________________ Date____________________
Negative Characteristics of AFB Smear Slide number appearance
Colour Appearance Size Arrangement
Module 7: Microscopy Page 10 of 10