Specimen Preparation

Proper decontamination and concentration of specimen

containing normal microbial flora are crucial to detection

of Mycobacterium tuberculosis. Specimen obtained from

sterile sites such as CSF, peritoneal or pleural fluids

do not need decontamination. However, since most

specimens for Acid Fast Bacilli smear and culture are from

respiratory tract and the mucous traps Acid Fast Bacilli

and protects other organisms from decontamination and

concentration, decontamination and liquefaction is a

must. Most satisfactory for this purpose is a combination

of N-acetyl-L cysteine (mucolytic agent) and 2% NaOH

(decontaminant). Petroffs method of decontamination

can also be used.

Test Procedure

For direct sputum screening use 10 µL of purulent

sputum or use 10 µL of decontaminated and concentrated

specimen.

 

2. Stability of the kit is as per the expiry date mentioned

on the label.

Additional Material Required

Sterile plating loops (10 µL), biosafety hood with Bunsen

burner, activated 2% glutaraldehyde solution, distilled

water, microscope with oil immersion lens, cedar wood oil.

Specimen Collection and Preparation

Collect specimen prior to use of antimicrobial agent.

Wherever possible, indicate clearly that patient is on

antitubercular drugs.

CSF

Collect as much as possible in a syringe, clean skin with

alcohol before aspirating specimen.

Body Fluids

Disinfect the site and collect specimen with aseptic

precautions.

Sputum

Collect 5 to 10 mL in a sterile container from an early

morning specimen of deep productive cough. For induced

specimen use sterile saline. Have patients rinse mouth

with water to minimize specimen contamination with

food particles, mouthwash, or oral drugs.

Urine

As organisms accumulate in the bladder overnight, first

morning void provides best yield. Collect midstream clean

catch urine, first morning catheterization/suprapubic taps

in sterile containers.

 

h

Novachrom®

Summary

Infection with Mycobacterium tuberculosis remains a major

public health problem. The epidemic of tuberculosis and

multi drug resistant tuberculosis reflects the failure of public

health and social programs towards prompt treatment

of infected cases and screening of high-risk population.

While culture, isolation and sensitivity of Mycobacterium

tuberculosis from patient groups using standard methods

remain the gold standard for Mycobacterium tuberculosis

detection and effective and swift treatment worldwide, acid

fast bacilli staining is the first line microscopic procedure

performed towards this goal.

Reagent

NOVACHROM is a reagent for laboratory use only.

NOVACHROM Rapid Two Step Cold AFB Stain comprises

of:

a. AFB stain (A) — carbol fuchsin

b. AFB stain (B) — counterstain with decolorizer

Rapid Two Step Cold AFB Stain is provided as a ready

to use stain set. It is used for screening of Mycobacterium

tuberculosis from biological specimen such as sputum,

CSF and urine. It is also used in the identification of

Mycobacterium tuberculosis from isolated culture. It is a

modification of Kinyoun’s cold stain.

Principle

Carbol fuchsin forms acid insoluble complex with mycolic

and present on the Acid Fast Bacilli and renders red/pinkish

red color to Mycobacterium tuberculosis. Other elements

present in the smear take up counterstain (Methylene blue)

and are stained bluish. Rapid Two Step Cold AFB Stain avoids

the extra decolorization step associated with traditional

staining techniques as the decolorizing component is

incorporated within the counterstain. The staining system

is simple to perform and very much reproducible. It has a

sensitivity comparable to the traditional Ziehl-Neelsen hot

staining method and also as compared to Acid Fast Bacilli

culture results. It is a clinically proven, easy to use, time,

labor and cost saving. This simplicity of staining makes this

an ideal screening tool for Mycobacterium tuberculosis.

Storage and Stability

1. Store the kit at room temperature (25–30o

C), away from

light.

 ence monitoring the number of stainable organisms in

the sputum during treatment can provide an early and

objective measure of response.

It should be noted that in patients receiving

antimycobacterial therapy not all stainable organisms are

viable. Should the number of organism fail to decrease

after therapy is started, the possibility of drug resistance

must be considered. Additional cultures should be taken

and drug susceptibility studies obtained.

Two types of acid-fast stains are frequently used:

1. Carbol fuchsin based stains;

2. Fluorochrome based stains.

The carbol fuchsin stains, so called because of the

Reagent formed by mixing of the stain basic fuchsin with

the disinfectant phenol (carbolic acid). Carbolfuchsin

stained mycobacteria appear bright red/pinkish against a

bluish background.

Two procedures using carbol fuchsin based stains are in

common use:

a. Three component Ziehl-Neelsen, or “hot stain”, and

b. Three component Kinyoun or “cold stain”.

The Kinyoun stain is a modification of the classical

Ziehl-Neelsen “hot stain”. The classical Ziehl-Neelsen

“hot stain” requires application of heat to the fixed smears

flushed with the stains during staining process, whereas

the Kinyoun stain does not require the application of heat

and is less tedious to perform and standardize.

Recent advances in staining techniques have been

reported where the cold Kinyoun stain has been further

modified to accommodate the decolorizer within the

counter stain. The novel two component two step stain is

time, labor and cost saving, more user friendly and easy to

standardize. It also has good correlation with the classical

Ziehl-Neelsen “hot stain” and AFB cultures.

Contd...

Microbiology and Bacteriology 851

RAPID TWO STEP COLD AFB STAIN

(Courtesy: Tulip Group of Companies)

 

850 Concise Book of Medical Laboratory Technology: Methods and Interpretations

Problem: False positive results in LJ media

Possible causes Solutions

1. Sputum collected in a contaminated Collect the sputum in a clean sterile container

container

2. Contaminated container used for Clean sterile container is to be used during treatment of sputum to avoid contamination

treatment of the sputum

Problem: Turbidity in phosphate buffer

Possible cause Solutions

1. Prolonged storage of phosphate buffer Gently warm phosphate buffer in a water bath at 25–30°C before usage

at 2–8°C

Problem: No liquefaction of mucoid sputum

Possible causes Solutions

1. Mucolytic reagent used is prepared and Freshly prepared mucolytic reagent should be used. If stored at 2–8°C, should be used

stored at 2–8°C for more than 24 hours within 24 hours

The AFB Smear

The sensitivity of AFB smear for specimen from

extrapulmonary sites is lower than from sputa. The lipidcontaining cell walls of mycobacteria have a unique

characteristic in binding carbol fuchsin stain so tightly

that it resists destaining with strong decolorizing agents

such as strong alcohols and strong acids. This “acid-fast”

staining reaction of mycobacteria, along with their unique

beaded and slightly curved shape, is a valuable aid in the

early detection of infection and monitoring of therapy.

It has been estimated that there must be 10,000 acid-fast

bacilli per milliliter of sputum to be detected by microscopy.

Patients with extensive disease will shed large numbers

of mycobacteria and show a good correlation between

a positive smear and a positive culture. In patients with

minimal or less advanced disease, the correlation of positive

smears to positive cultures may range from 30 to 80%.

Acid-fact stains performed on a weekly basis are

also useful in following the response of patients to drug

therapy. After drugs are started, cultures will become

negative before smears, indicating that the bacilli are

injured sufficiently to prevent replication but not to the

point of preventing binding of the stain. With continued

drug treatment, more organisms are killed and fewer shed,

 

Remarks

1. Treat the unused specimen and contaminated

containers by immersing in 2% activated glutaraldehyde for at least 2 hours before incineration and

disposal.

2. Good laboratory practices and hazard precautions

must be observed at all times.

3. Discolored or contaminated reagent should not be

used.

4. The reagent containing the phosphate buffer may

appear turbid on prolonged storage at 2–8°C. Gently

warm at 25–30°C before usage to remove such a

appearance.

Effect of centrifugal force on positive smears/cultures

for mycobacteria

Specimen Relative Centrifugal Force (g)

1260 3000 3800

Positive smear 1.8% 4.5% 9.6%

Positive cultures 7.1% 11.2% 11.6%

Correlation of positive

smear/cultures

25% 4 0. % 82%

Thus, proper decontamination and preparation of

specimen is crucial to AFB detection by culture and AFB

staining.

Troubleshooting

Problem: False negative results in LJ media

Possible causes Solutions

1. Mucoid sputum exposed to 2% NaOH Contact time of 2% with sputum should be for only 20 minutes since prolonged

for longer duration period of contact may kill or injure the mycobacteria

2. Saliva used as specimen Thick yellowish green mucoid sputum collected from an early morning deep productive

cough should be used as a specimen

Contd...