Ziehl-Neelsen staining

technique
Mr.Muhammad Shafiq
Medical Technologist

Outline of Presentation

AFB in Pulmonary tuberculosis
AFB screening methods/ Procedures
Sensitivity and specificity relative to
other investigations and tests.
AFB screening
ZN staining
Fluorescence method.
Auramine O=Bright yellow
Auramine O-Rhodamine B=Yellow
orange


ZN staining

Staining for Mycobacterium
Tuberculosis
Screening for TB
Staining for Acid Fast Bacillus
ZN staining
History
Ehrlich-1882 discovered the AF
nature.
Franz Ziehl-1882.
F Neelsen-1883 modified

Ziehl-Neelsen stain









Advantages

Rapid, easy, cheap, fast, no
complex instrumentation.
accurate diagnosis even by
paramedical personnel using simple
and available equipment.
high specificity
Disadvantages

low sensitivity
Significance of sputum examination
Primary test in Pulmonary
Tuberculosis.
In diagnosis for rapid identification,
prompt isolation, and early
treatment.
To check effect of ATT drugs during
treatment.
To confirm the complete cure.

Cough 3 weeks
AFB X 3
Broad-spectrum antibiotic 10-14 days
If symptoms persist, repeat AFB smears, X-ray
If consistent with TB
Anti-TB Treatment
If 1 positive,
X-ray and
evaluation
If 2/3 positive:
Anti-TB Rx
If negative:
Role of ZN Smear in Diagnosis of
Pulmonary Tuberculosis
Prompt treatment of infectious
cases reduces spread of TB(3)
Smear positive patients usually seek
care.
Smear positive patients are 4-20
times more infectious.
Untreated, a smear positive patient
may infect 10-15 persons/year(
depends on congested society).
Smear positive patients are much
more likely to die if untreated.
In Diagnosis of Pulmonary
Tuberculosis
Patients feel ill and need prompt
medical attention.
Microscopy is appropriate technology,
indicating infectiousness, and priority
of treatment.
Serological and amplification
technologies(PCR etc ) currently of no
proven value.
ZN staining Vs X-ray :
X-ray causes over-diagnosis(1)
0
20
40
60
80
100
Diagnosed by X-
ray alone
Actual cases
Over diagnosis
Chest X-ray: Comparison
No chest X-ray is typical of
tuberculosis
10-15% of culture positive TB
patients not diagnosed by X-ray.
40% of patients diagnosed as having
TB on the basis of X-ray alone do not
have active TB.
X-ray is unreliable alone for diagnosis
and monitoring treatment of
Tuberculosis(2).
COLLECTION OF SPECIMEN
Specimen type
Sputum (3 specimens optimal)
Spot specimen on 1
st
visit.
Next day: Early morning specimen.
Spot specimen during second visit.



WHY THREE SPECIMENS?
Very small amount of sputum is
taken from container so it is
possible that that part of specimen
does not have significant amount of
bacillus(1ml sputum 5000-10,000
bacillus)------So possibility of
bacillus finding increases.
To rule out any mistake---- as
treatment ( having side effects) is
based on it.
Confirms the diagnosis.

Wide mouth, air tight, transparent, clean and
disposable
Non sterile
Container properties
Sputum smear making techniques
Direct Smear
Concentration method (and
sedimentation)
Hypochlorite centrifugation method

Centrifugation & Sedimentation
method
Liquefication and decontamination of sputum
is done with sodium hypochloride (NaOCl),
usually known as house hold bleach.
Briefly, sputum is mixed with an equal
amount of 5% NaOCl, and the mixture is
incubated at room temperature for 10-15
min. Distilled water is added to final volume
of 10 ml and the sample is concentrated,
either by centrifugation for 15 min or by
sedimentation overnight.
Concentration technique
increases the sensitivity of the
smear screening method
Smearing procedure

Film is made: from mucopurulent area (no sliva,
no blood, do not mix the specimen)
Use Diamond pencil to label the slides.

Smear making: by coiling pattern, 12 cm(
do not touch edge of the slide). Evenly spread the
sputum on the slide.Thickness of smear can be
judged by the hazy appearance of news print
through slide.

Use sand alcohol jar to clean the loop
Air dry( do not heat dry) can take 15-20 min.
Fixed by flaming( 2-3 sec thrice)
Homogeneously distributed
specimen on slide is required
Staining
Methods
Cold method
Kinyoun
Tan Thiam HoK
Hot method
Ziehl-Neelsen

ZN I & ZN II type
Principle
Powerful solution containing Phenol
and subsequent heating---results in
penetration of Dye through cell wall
containing mycolic acid and waxes of
bacillus.
Decolorization does not permit the
release of stain and bacillus can seen
as red rod like ACID FAST BACILLI
Recipe



1-Carbol Fuchsin( strong) 1%

Basic fuchsin 10g
Ethanol 100% 100ml
Phenol 5%( in water) 1 L
Dye is dissolved in alcohol and add to phenol solution
Use after filter
2-Sulphuric Acid 25% solution

250ml concentrated sulphuric acid in 750ml of water.
Mix gently.
or
* to make 3%HCL solution
3 ml of conc. HCl in 97ml of 95 % ethanol
3-Methylene blue( Loefflers) 0.3%
saturated solution of methylene blue in alcohol
300ml
KOH, 0.01% soln in water 1000ml

Procedure (ZN I)


Cover the whole slide with filtered carbol fuchsin.
Heat until steam rises (should not boil)
Allow the preparations to stain for 5 min.
Wash with water.
Cover with 25% sulphuric acid
Wash the slide with water after 2-3 minute. Pour the
acid again if required. Film is faintly pink or colorless
Wash with water
Counter stain for 15-30 sec
Wash, blot and Place vertical to dry
Staining precautions.

Use one slide for one specimen
On staining rack do not place slides
touching each other.
Do not over and under heat
Use filtered stain solutions
Do not use staining jars
Smear
Carbol fuchsin
Decolorized
Counterstained
Acid fast bacteria Non acid fast bacteria
Microscopy
Focus with 10objective
Open full iris for 100 .
Move up the full condenser.
Then screen with 100objective using
immersion oil.
See 100 oil fields
edge to edge longitudinal once
edge to edge width twice


Result
Mycobacterium tuberculosis

Acid fast Bacillus Bright Red straight or
slightly curved occurring singly or in groups and may
appear beaded.
Size 1-4m 0.2-0.6m
Non sporing, noncapsulated,

Blue
Background
Pus cells
Dust cells (suggestive of true pulmonary specimen)
Squamous epithelial cells (salive if in excess)
Red Blood cells
Non acid fast bacteria and fungi
Reporting
2cm=100 oil fields (1000)
no AFB seen in 100 fields Report as
negative

100 Fields 1-9 AFB Number
100 10-99 +
1
a
1-10 ++
1
b
>10 +++
a
see 50 fields
b
see 20 fields
Sensitivity and Specificity
Smear positivity depends on Number
of bacillus in specimen
If 5000-10,000 bacillus in 1ml of sputum

ZN staining is specific for
Mycobacterium Species.
Factors effecting sensitivity of AFB
screening
Highly dependent on technical
factors.
Quality of sputum, morning sputum
10-100% more positives
Number of samples
Quality of smear, staining, quality of
microscopes.
Effort in examining number of fields

ZN II (modification)

Staining time is increased 10-15 min
and 5% H
2
SO
4
is used
For leprosy bacilli.
For Cryptosporidium oocysts.
For Actinomyces, Nocordia.
For bacterial Spores.
As Brucella differential stain.

Quality Assurance
As per laboratory SOPs
New lot of staining solutions are
checked for results before use on
known specimens
In routine solutions are checked once
a month.
Routine evaluation of personal
experties in sample handling,
staining, microscopy.
Random re-evaluation of old cases.
References
1. NTI, Indian J of Tuberculosis. 1974
2. Thomas k. Tuberculosis case finding
and chemotherapy. WHO.1979.
3. Rouillon A. tubercle.1976;57:275-299.
4. www,cdc.gov
5. www.who.com
6. Diagnostic Microbiology by Mackey &
Meckartney
7. Clinic Microbiology by Jewts
8. Microbiology Manual by Monica

Safety During Work
&
Disposal of Waste



Direct smear microscopy can be safely performed on
the open bench.

Steps where care required
When patient gives the sample, he/she
may contaminate the container and paper
request form.
Increased risks when Laboratorians have
contact with coughing patients.
While handling and opening the sputum
container.
Using hot wireloop for taking sample or
making smear.
flaming the contaminated wireloop.
Fixing freshly un dried smear by flaming.


In Lab
MT contaminate inanimate objects and hands

& AEROSOLE FORMATION
Opening:risk
Smear making:risk
Pipettes
Raymond A. Lamb, LLC
VWR International
Beckman Coulter, Inc.
Aerosol Producers
Vortex
Tubes
Activity Risk factors Administrative controls Practices and procedures BS
L
Staining
specimen for
AFB without
culture
Centrifugation
and manipulation
of specimen may
produce
infectious
aerosols.
Kill tubercle
bacilli.
Treat specimen with
equal volume of 5%
hypochlorite solution;
process in BSC; use
aerosol- containing
safety cups for
centrifugation.
2
Preparing
specimens for
centrifugation
and AFB
culture
Suspect
specimens
contain limited
numbers of AFB
and many are
negative; set-up
procedures
involve potential
for
aerosolization.
Train personnel in
applicable safety
procedures.
Conduct all work in
the BSC on a towel
moistened with a
tuberculocidal agent;
use aerosol-
containing safety cups
for centrifugation.
2
Centrifugation
of specimens
suspected of
containing live
tubercle bacilli
Centrifugation
and manipulation
of specimen may
produce
aerosols.
Use
biocontainment
devices.
Use aerosol-
containing safety cups
for centrifugation;
open in BSC.
3
Bio-safety level-2

is suitable for work involving agents of moderate
potential hazard to personnel and the environment. a)
laboratory personnel have specific training in handling
pathogenic agents and are directed by competent
scientists; b) access to the laboratory is limited when
work is being conducted; c) extreme precautions are
taken with contaminated sharp items; and d) certain
procedures in which infectious aerosols or splashes may
be created are conducted in a biological safety cabinet
(BSC) or other physical containment equipment.




Preventive Measures

Well ventilated area (Exhaust fan).
Sunlight is appritiateable (----kills TB).
Frequent hand washing with disinfectant .

In laboratories where only smear microscopy
is performed, personal respiratory protection
(e.g., respirators) is not needed. (No surgical
mask.if required then only N95).



Biosafety Cabinets Class II (BSC II): cabinet that uses a laminar air flow in
addition to exhaust to protect both the specimen /culture and the worker from
contamination.

Centrifuge
Avanti J-E, Beckman Coulter, Inc.
Aerosolve
Cannister



Rotor
Beckman Coulter, Inc.
SAFE DISPOSAL OF WASTE MATERIALS

Infectious waste can be divided in
two types
Disposable and re-usable materials
Infectious TB lab waste is a risk to
health care workers and the
community
Waste must be sterilized or
disinfected before it leaves the
laboratory
FLOW CHART FOR DISPOSAL OF MT contaminated
items
CONTAMINATED
items
DISPOSABLE
(slides, containers,contaminated paper
or wooden sticks
5% PHENOL
DEEP HOLE
BURNING & BURY
INCINERATION
RE-USABLE(
forceps,wireloops,slide holder)
5% PHENOL
Autoclave
Autoclave
wash
Sand alcohol jar ---Wire loop
Burner----wire loop
Incinerator/Autoclave----disposable items,
containers, others
Bleach/Phenol 5%----Disinfect containers,
contaminated items, floors, accidental
spills, slides, AFB working area
IMPORTANT POINTS TO REMEMBER:




1.Follow the Good laboratory practice(GLP)
2. Biosafety level 2 facilities and procedures are
sufficient for laboratories performing direct
AFB smears on samples that have been
treated to inactivate the tubercle bacilli.
3. laboratorians themselves should make
infection control a high priority( no
carelessness in the handling of MT).
4. Contaminated hands/gloves endanger all lab
staff.







Cont
5. No eating, drinking, smoking, use of cell
phones in the lab.
6. Prevent leaking specimen containers
and contaminated work forms.
7. Prevent aerosols and spills
8. Frequent and appropriate hand
washing
9. Use of disinfectants and proper waste
disposal
References
2000. TB Microscopy in Pacific Island countries Japan International
Cooperation Agency and The research Institute of Tuberculosis

Date of Publication. Acid-fast Direct Smear Microscopy,WHO,
IUATLD, CDC, KNCV, JATA, APHL

Draft publication. External Quality Assessment for AFB Microscopy

1995. Mycobacterium tuberculosis: assessing your laboratory.
ASTPHL and CDC.

2002. Zambia AFB microscopy Training Manual. Central Board of
Health.

1993, Channel BTE CO, Inc. About Tuberculosis, Booklet #37788.
Southfield, MA.

1998. Laboratory Risk Assessment: What, Why and How. Centers for
Disease Control and Prevention

WHO. 1999. Guidelines for Prevention of Tuberculosis in Healthcare
facilities in resourcelimited settings
PIMS