NEHCRI Makassar Laboratory: TB Unit

Prepared by
Date
Revised
Date
Revised
Date
Approval by

: Muh. Nasrum Massi

: 03.10.2006
: Sabai Phyu
: 27.10.09
: Nasrum Massi
: 09.11.2010
: Sabai Phyu
(

Effective Date

: 11.11.2010

Protocol: 02

Protocol 02
Sputum Smear Microscopy using Ziehl Neelsen
Principle
:
The characteristic difference between mycobacteria and other microorganisms is the
presence of a thick, waxy (lipoidal) wall that makes penetration by stains extremely
difficult. Once the stain has penetrated, however, it cannot be readily removed even with
the vigorous use of acid alcohol as a decolorizing agent. Because of this property, these
organisms are called acid fast, while all other microorganisms, which are easily
decolorized by acid-alcohol, are non-acid-fast.
Materials
1. Glass slides
2. A pencil
3. A Bunsen burner
4. A forceps to hold smear slide
5. A sharp waste container with disinfectant (Vesphene)
6. A waste container for solid wastes
7. Immersion oil
8. lens paper
Equipment
1. Light microscopy
2. Hot plate
3. Staining tray
4. Timer
5. Drying rack
6. Slide box
Chemical Solutions
The acid-fast staining (Ziehl-Neelsen Method) uses three different reagent:
1. Carbol fuchsin 1%
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a. 20 gr Carbol fuchsin in 200 ml Ethanol 96% mix properly
b. 100 ml phenol mix thoroughly with 1700 ml DW
c. Mix (b) solution into (a) solution
d. Filter by Wharmant No. 3 into a 2 lt amber bottle
Label bottle with name of reagent as well preparation and expiry dates. Can be
stored at room temperature for six to twelve months and filter before use.
2. Decolourising agent:
Acid-alcohol (3% HCL + 95% Ethanol)
Concentrate hydrochloric acid (technical grade) 60 ml
95% ethanol (technical grade) 1940 ml
Wear the goggle when preparing this solution. Carefully add concentrated
hydrochloric acid to 95% ethanol. Always add acid slowly to alcohol, not vice
versa. The mixture will heat up. Store in an amber bottle. Label bottle with name
of reagent and dates of preparation and expiry. Can be stored at room temperature
for six to twelve months.
3. Counter stain: Methylene blue
Methylene blue chloride 6g
Distilled water: 2000 ml
Dissolve methylene blue chloride in distilled water by shaking and store in an
amber bottle. Label bottle with name of reagent and dates of preparation and
expiry. Can be stored at room temperature for six months.
Procedure of making smear on a glass slide:
1. Take the clean slide and put code in the one side of slide by pencil, according to
the hospital (X=BP4, Z=epidemiology samples and Y=Wahidin), number of
samples (1.0,2.0 ect) and sputum collection (A=when patient came, B= early
morning. C=when coming again in the next day)
2. Smear the specimen (100 ul) on the slide over an area approximately 2.0 by 3.0
cm using a micropipette tips. Make it thin enough to be able to read through it.
3. Discard the tips in a disinfectant containing receptacle, and use a new tip for each
specimen.
4. Place smear on the hot plate for 3 hours for fixing smear (Do not need to
pass slides through a flame three or four times.) Allow to cool before staining.
5. Place the slides on a staining rack in batches. Ensure that slides do not
touch each other. Make a gap about one finger width between two slides.
6. Flood entire slide with Ziehl-Neelsen carbolfuchsin
7. Heat the slide slowly until it is steaming. Do not boil and do not allow to dry.
8. Maintain steaming for five minutes by using low or intermittent heat as needed.
9. Rinse each slide individually in a gentle stream of distilled
water until all free stain is washed away.
10. Flood the slide with the decolourising solution for a maximum of three minutes.
11. Rinse the slide thoroughly with distilled water. Drain excess water from the slide.
12. Flood the slide with counter stain. Allow the smear to counter stain for 30 seconds
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13. Rinse the slide thoroughly with distilled water. Drain excess water from the slide.
14. Allow smear to air dry. Do not blot. Examine the slides under the microscope.
15. Use at first a 10x lens objective to focus the field of staining and then change to
100x objective lens for identifying AFB (immersion oil should be added to stained
smear area when 100x lens is used).
16. After examination of the slides, directly put into the Xylene solution for 15
minutes
17. Before examining the next slide, wipe the immersion lens with a piece of lens
tissue paper.
18. Keep all the slides for external quality control, according to established
procedures.
19. Analyse results on a weekly and monthly basis for percentage of positive results.
Investigate any sharp differences from the norm
Interpretation of smear result:
Report
Microscopy filed
1. Negative
: No AFB seen in 100 fields
2. Actual number : 1-9 AFB/100 fields
3. Positive 1+
: 10-99 AFB / 100 Fields
4. Positive 2+
: 1-10 AFB / 1 Field in at least 50 Fields
5. Positive 3+
: More than 10 AFB/ 1 Field in at least 20 Fields
Discarding the slides
All slides that will not use anymore directly put in the sharp container which
contain 2% Vesphene. Close the container cap loosely. The whole sharp container
will be placed in the another autoclavabale bag. Then close the bag and autoclave it
before sending it to the incinerator machine.
Cross check
Routine, every 4 months we follow slide cross check prgramme from BLK Makassar
as a representative NTP in South Sulawesi using LQAS method for total samples we
send for cross check.

REFERENCE:
1. James G. Cappuccino, Natalie Sherman. Microbiology A laboratory manual,
2001.
2. De Kantor I.N, Kim SJ, Frieden T, Laszlo A, Luelmo F, Norval P-Y, Rieder H,
Valenzuela P, Weyer K. Laboratory services in tuberculosis control Part II.
Microscopy. WHO 1998.
3. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC, Jr.
Micobacteria. Color Atlas and Textbook off Diagnostic Microbiology. Fifth
edition, 1997. lippincott.

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