SINGIDA REGIONAL HOSPITAL Doc.

Code: SOP # Page 1 of

LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY.. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

Name & title Position Signature Date

Prepared by ASIA SONDA Head Microbiology
(Lab. Technologist).
Section.

Reviewed by HERJINDER Quality Officer.

JASWANT.

(Lab. Technologist).

Approved by FOIBE M. SUMARI Lab. Manager.

(Lab. Technologist).
 SINGIDA REGIONAL HOSPITAL Doc.Code: SOP # Page 2 of
LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL-NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

This SOP has been read and understood by;

Name Signature Date

 SINGIDA REGIONAL HOSPITAL Doc.Code: SOP # Page 3 of
LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL-NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

Annual Review.

Details of Amendment
Revision no. Effective date Details of amendment
 SINGIDA REGIONAL HOSPITAL Doc.Code: SOP # Page 4 of
LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL-NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

Copies Distributed to;

Name Date
Quality Office. 25/03/2013.
BACTERIOLOGY/MICROBIOLOGY 25/03/2013.
section.

Purpose
This procedure provides instructions for step-by step staining of sputum smears,
examination of stained slides for detection and reporting of Acid Fast Bacilli (AFB).

Principle
Mycobacteria retains the primary stain (carbolfuchsin) even after exposure to
decolorizing acid alcohol, hence the term ʿʿacid fast ʾʾ. A counter-stain is employed to
highlight the stained organisms for easier recognition.
Bacilli that retains the carbolfuchsin after decolonization are referred to as acid fast
bacilli ( AFB). The counter-stain stains the background blue and provides a good
contrast against the red AFB.

Materials
Reagent(s) Supplies Equipment(s)
 1% Carbol Fuchsin  Staining rack.  Bunsen
solution.  Drying rack. burner/spirit lamp.
 20% Sulphuric acid  Forceps.  Bright field
solution.  Beaker. Microscope.
 0.1% Methylene  Slide storage box.
blue/Malachite  Alcohol sand jar.
green.  Clean grease free
 Immersion oil. new slide.
 Diamond point
pencil.
 Plastic disposable
 SINGIDA REGIONAL HOSPITAL Doc.Code: SOP # Page 5 of
LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL-NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

loops/Applicator
stick.

Sample
Fixed sputum smear on a clean grease free new slide.

Special Safety Precautions

Wear gloves, lab coat, and all necessary safety precautions.
The Ziel-Neelsen staining technique applies to ordinary (bright field) microscope.

Quality Control
Prepare bathes of control slides from suitable sputum specimens.
These are sputum smears of known negatives and positive (both of high and low
positivity). A low positive of 1+ i.e. 10 – 99 AFB/100 field.

Check every newly prepared staining solution with unstained control slides and
report the result in QC log.

Every day include one positive and negative control slides in the patients slides
and stain together , examine first the QC slides and if the results are the same
then examine the patients slides. Report the result in QC log sheet.

Specimen quality collection

A good sputum specimen consists of recently discharged material from the
bronchial tree.

Satisfactory quality implies the presence of mucoid or mucopurulent material

and is of greater significance than volume.
 SINGIDA REGIONAL HOSPITAL Doc.Code: SOP # Page 6 of
LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL-NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

Induce sputum resembles saliva and it is important that these specimens not be
discarded as un suitable.

Decontaminated sputum must be concentrated by centrifugation.

Procedure
Follow the activities in the table below:

A)Smear preparation.

Step Action
1 Label a new , clean, unscratched slide at one end with the relavant
patient number.

2 Transfer an appropriate portion of the specimen to the slide by using

an Applicator stick/ Plastic disposable loop. Use blood specked,
opaque, grayish or yellowish cheesy mucus for smear preparation
when it is present.

3. Smear the specimen on the slide over an area approximately 2X1 cm.

4 Allow the smears to air dry for 15 minutes (Do not use heat for
drying.

5 Fix smears to the slide by one of the following methods;

-Pass slides through a flame 3-4 times with the smear
uppermost. Do not over heat and allow to cool before
staining.

-Allow the slides to fix on an electric slide warmer (65 - 75°c

for 2 hours.
 SINGIDA REGIONAL HOSPITAL Doc.Code: SOP # Page 7 of
LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL-NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

B)Staining method.

Step Action
1 Place the numbered slides with smear upwards on a staining rack
over a sink about 1 cm apart.

2 Flood entire slide with filtered 1% carbol fuchsin staining solution for
5-10 minutes.

3 Heat the slides slowly until it is If no Bunsen burner;

steaming (Do not boil) by keeping Prepare a torch by dipping its
the Bunsen burner/Torch a little cotton wool end in a burning
below the slide and moving it spirit and light it.
continuously forth and back THEN, proceed to step 3 left.
alone the line, repeat twice at
the interval of 3 – 5 minutes.
4 Tilt the slide using forceps to drain off the carbol-fuchsin staining
solution, and then rinse the slide well with clean water from a
beaker or running tape water, and rinse the slide well with clean
water from a beaker or running tape water.
5 Flood the slide with the decolorizing solution for 2 minutes.

6 Tilt the slide using forceps to drain off Acid solution, and then gently
rinse the slide well with clean water from a beaker or running tape
water.

7 Flood the slide with counterstain (methylene blue) solution for 1

minute.

8 Rinse the slide well with clean water from a beaker or running tape
water, and then tilt the slide using forceps to drain off excess water
 SINGIDA REGIONAL HOSPITAL Doc.Code: SOP # Page 8 of
LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL-NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

from the slide.

9 Using forceps take the slide from staining rack, drain off water, and
stand the slide on the edge to air dry on the drying rack (Do not
blot).

c) Reading, reporting, and recording.

1 Reading:
-Use the bright field using 100 times oil immersion objective to examine both controls
and patients slides.
-Check first the positive control and then the suspect’s slide.
-AFB appears bright yellow against the dark background material.
-Store the slides in a slide box following the laboratory register number as they will be
needed for EQA.
-Do not write the result on the slide.

2 Reporting:
-Result should therefore be quantified.
-A number of AFB found is an indication of the degree of infectivity of the patient as
well as the severity of the tuberculosis disease.
-Report that AFB seen and grade of positivity.
-For negative result report AFB negative.

Number of Field. Report.

AFB
(According
to
IUATLD/W
HO scale
results).
No AFB Per 100 No acid fast bacilli observed
immersi (No AFB per 100 fields).
 SINGIDA REGIONAL HOSPITAL Doc.Code: SOP # Page 9 of
LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL-NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

on fields
1 – 9 AFB Record exact figure
(1 to 9 AFB per 100 fields).
Per 100
10 – 99 immersi 1+
AFB on fields (10 to 99 AFB per 100 fields).

2+
1 – 10 AFB Per 100 (1 to 10 AFB per field in 50 fields)
immersi
on fields 3+
More than (More than 10 AFB per field in
10 AFB 20 ields).
Per field

Per field

-Use red pen for positive results

-It should not be assumed that AFB are tubercle bacilli.

3 Recording:
-The results have to be reported in the microscopy register.

Reference
Laboratory Manual for Health Centre Worker. Ministry of Health, Dar es salaam,
June 1993.

Laboratory Services in Tuberculosis Control. Part II microscopy. WHO, 1998.

 SINGIDA REGIONAL HOSPITAL Doc.Code: SOP # Page 10 of
LABORATORY 20 10

Section: BACTERIOLOGY/MICROBIOLOGY. Date of Preparation: 27/02/2013.

Subject Title: SOP FOR ZIEL-NEELSEN STAINING.
Effective date: 25/03/2013. Revision no.1

Laboratory Manual for for Acid Fast microscopy, US department of Health,

Education, and Welfare, CDC; 1979.

Association of Public Health Laboratories, External Quality assessment for AFB

smears microscopy. Washington; 2002.

Lumb R, Bastian I, Laboratory diagnosis of Tuberculosis by sputum microscopy,

Adelaide: Institute of Medical and Veterinary Science; 2005.