BY D R M A D H U RA C M A N D D R C H A KR AVA RTH I PA N U G A NT I

D E PT O F M I C ROB I OLOGY
B MC R I
 INTRODUCTION
Ziehl- Neelsen stain is a type of acid-fast
fast stain used to identify acid fast organisms, mainly
Mycobacteria.
It is a differential stain which differentiates between acid-fast
It acid bacilli and non acid-fast bacilli.
It is named after two German doctors who modified the stain: the bacteriologist Franz Ziehl and
It
the pathologist Friedrich Neelsen.
Other stains used are Kinyoun staining and Fluorochrome staining.
Other

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 HISTORY
Paul Ehrlich(1882) discovered that Mycobacteria
ycobacteria
stained with fuchsin in the presence of aniline oil as
mordant resist decolourization by mineral acids.

Franz Ziehl in the same year changed the mordant to

carbolic acid.
Friedrich Neelsen in 1883 increased the
concentration of carbolic acid and incorporated it
with the dye to form carbol-fuchsin.
Thus the standard stain for demonstrating acid-
fastness was formulated.

FRANZ ZIEHL FRIEDRICH NEELSEN

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 HISTORY

The application of heat in the technique gives it the name the hot
method of Acid-Fast staining
 This is a synonymous name for the Ziehl-Neelsen
Neelsen Staining
technique.

Joseph Kinyoun modified the Ziehl-Neelsen

Neelsen staining technique by
increasing the concentration of carbol-fuchsin
fuchsin and removing the
heating step .
Thus named the staining as Kinyuon staining.

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 PRINCIPLE
cobacteria are difficult to
due to the presence of large
unts of lipid (mycolic acid) in
ell wall making it waxy,
ophobic and impermeable.
hl-Neelsen staining method
carbol-fuchsin and heat,
h allows better penetration
e through cellwall.
mycolic acid and lipids in the
wall account for the property
id-fastness,which prevent the
tration of decolourising
ion.

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SAMPLE COLLECTION
PULMONARY EXTRAPULMONARY

1. Sputum 1. Lymph node

2. Laryngeal swabbing 2. Urine
3. Transtracheal aspiration 3. CSF
4. Bronchoalveolar lavage 4. Pleural fluid
5. Gastric lavage 5. Peritoneal fluid

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SPUTUM
Spontaneously produced sputum is the specimen of choice.
2 sputum samples should be collected, one at the time of first visit and another early morning
2
sample.
To raise sputum, patients must be instructed to take a deep breath, hold it momentarily, and
To
then cough deeply and vigorously.
Saliva and nasal secretions should not be collected.

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GASTRIC LAVAGE SPECIMENS
Gastric lavage is used to collect sputum from patients
Gastric
who may have swallowed sputum during the night.
 The procedure is limited to senile, nonambulatory
patients;
children younger than 3 years of age (specimen of
choice);
 The most desirable gastric lavage is collected at the
patient’s bedside before the patient arises and before
empties the stomach.

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 BODY FLUID SPECIMENS
At least 10 mL of CSF is recommended for recovery of mycobacteria.
At
Similarly, as much as possible of other body fluids (10-15
Similarly, (10 mL minimum), such as pleural,
peritoneal, and pericardial fluids, should be collected in a sterile container or syringe with a
Luer-tip cap.
An anticoagulant like trisodium citrate must be added [except for CSF]
An
Swabs are discouraged, because the recovery of organisms is decreased.
Swabs

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 URINE SPECIMENS
Early morning voided urine specimens (40 mL minimum) in sterile containers should be
submitted daily
for at least 3 days,
whole sample should be sent
The collection procedure is the same as for collecting a regular urine specimen .
The
The 24-hour
hour urine specimen is undesirable because of excessive dilution, higher contamination,
and difficulty in concentrating.
Catheterization should be used only if a midstream voided specimen cannot be collected.
Catheterization

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 PUS ,SKIN LESIONS AND ASPIRATES
 The skin should be cleansed with alcohol before aspiration of the material into a syringe.
 If the volume is insufficient for aspiration, pus and exudates may be obtained on a swab and
then placed in a transport medium, such as Amie’s or Stuart’s medium (dry swabs are
unacceptable).
Pus in syringe or universal container is better for isolation, than swab.
Pus

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Decontamination and concentration of
specimen
Specimens from non-sterile
sterile sites and sputum need prior treatment so that micro-organisms
micro
other than mycobacteria do not overgrow during prolonged incubation.
Also sputum samples contain an organic matrix which may trap mycobacteria cells.
Therefore liquefaction,decontamination and concentration improve the yield

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DECONTAMINATION AND CONCENTRATION OF
SPUTUM
Petroff’s method;
sputum is incubated with an equal volume of 4% NaOH at 37°C,
sputum 37
followed by frequent shaking until its clear,(which takes 20 mins)
followed
then centrifuged at 3000rpm for 20 min and
sediment is neutralized with N/10 HCL and used for preparation of smear.
sediment
NALC combined with 2% NaOH;
Acetyl Cysteine is used for liquefaction of sputum and NaOH kills the contaminating bacteria.
N-Acetyl
Then neutralized with buffer and concentrated by centrifugation.
Then

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 MATERIALS

1. Primary dye : Concentrated Carbol fuchsin

2.Decolourizer : Sulphuric acid, alcohol, acid alcohol
3.Counterstain : Methylene blue, malachite green
4. Filter paper strips, Bunsen burner, or, preferably, an electric staining rack

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Components of Conc. Carbol fuschin
Basic fuschin
100% ethanol
Phenol crystals
Distilled water

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Sulphuric Acid(20%) decoloriser
Concentrated Sulphuric Acid-250ml
Distilled water-1 liter
Alcohol-95%

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Malachite green counterstain
Malachite green
Distilled water

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 ZN METHOD
Cover the heat fixed slide with ZN carbol fuchsin
Heat the slide until the steam rises, but without boiling.
Allow the preparation to stain for 5 min .
Do not allow the stain to dry on the slide, if necessary pour on more carbol fuchsin to cover it.
Wash with water and cover the slide with 20% sulphuric acid ,decolourization generally requires conta
with acid for at least 10 min.
Wash the slide with water to remove all traces of acid.
Cover with 95% alcohol for 2min ,this step is optional.
Wash with water , counterstain with Loeffler’s methylene blue or dilute malachite green for 15-20
15 sec
Wash, blot with clean paper,dry and mount

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 Mycobacteria typically appear as slender
rods, curved, bent or filamentous, 1 to 10
µm long.

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ZN STAIN
ADVANTAGE DISADVANTAGE

presence of Mycobacteria in a clinical not specific for Mycobacteria.

specimen,
cannot differentiate M. tuberculosis from
monitor the progress of patients on non-tuberculous Mycobacteria.
antimycobacterial therapy,
lacks sensitivity.
clarify a need for appropriate infection
control procedures, physical morphology of the organism is
distorted.
confirm the presence of acid-fast bacilli in
culture.

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 KINYOUN STAIN
. Flood the entire fixed slide with carbol fuchsin.
. Allow the smear to stain for 5 min.
. Rinse the slide with water.
. Flood the slide with 3% acid-alcohol
alcohol to decolorize for 2 min.
. Rinse the slide with water; drain excess water from the slide.
. Flood the slide with counterstain.
. Counterstain for 2 min.
. Rinse the slide thoroughly with water; drain excess water from the slide.
. Air dry. Do not blot.
0. Examine with a 100x oil immersion objective (1,000 total magnification) using a light microscope

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KINYOUN STAIN

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 GABBET’S METHOD
Reagents
◦ Basic fuchsin - phenol
◦ Gabbet’s methylene blue

Procedure:
The slide is stained with carbol fuchsin-phenol
phenol at room temperature for 10 mins.
The slide counterstained with Gabbet’s methylene blue for 2mins and seen under oil immersion.
immersion

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 FIUOROCHROME STAIN
Flood the slide with the fluorochrome stain(auramine phenol ) and stain for 10 min.
Flood
Rinse the slide with water; drain the excess water from the slide.
Rinse
Flood with 1% acid-alcohol and decolorize for 5min.
Rinse the slide with water; drain the excess water from the slide.
Rinse
Flood the slide with the counterstain with 0.1% potassium permanganate for 15 secs.
Flood
Rinse with water; drain the excess water from the slide. Air dry; do not blot.
Rinse
Examine the smear with a fluorescent microscope with a 25 or 40 objective (250 or 450 total
Examine
magnification)
stained slides may be directly restained with the carbol fuchsin stain after immersion oil is
Fluorochrome-stained
remove with xylene.

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 RESULT
Fluorochrome stain
◦ Positive: yellow to orange fluorescence
against a black background.
◦ Negative: no fluorescence

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 ZN Sputum Smear Evaluation as per RNTCP Guidelines
umber of bacilli Result RNTCP Grading (Report )

0/field + ve 3+

10/field + ve 2+

-99/ 100 field + ve 1+

9/ 100 field + ve Scanty

o of AFB in 100 - ve Negative

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GANISMS CONC OF SULPHURIC ACID TO DECOLOURISE

tuberculosis 20%

leprae 5%

cardia 1%

cyst OF CYCLOSPORA and ISOSPORA, 0.5%

YPTOSPORIDIUM,
rmati c head,
oklets of T.saginata,
lex of E.granulosus,
ionella micdadei

CTERIAL SPORES 0.25%

cella abortus 0.5% (Acetic acid )

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NOCARDIA

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CRYPTOSPORIDIUM

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ISOSPORA

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CYCLOSPORA

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 GRADING OF SMEARS FOR M.LEPRAE
10 bacilli in 100 fields 1+

10 bacilli in 10 fields 2+

10 bacilli/ fields 3+

0-100 bacilli/ fields 4+

00-1000 bacilli/ fields 5+

More than 1,000 bacilli, clumps and 6+

lobi in every field

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 FITE’S STAIN
Used to detect Mycobacterium leprae and Nocardia in tissue sections

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