4. The mucolytic reagent can be used within 24 hours of

preparation, if stored at 2–8oC.

Processing of Specimen

1. Take approximately 2.5 mL of the specimen in a clean

sterile 15–25 mL universal container.

2. Add 2.5 mL of the mucolytic Reagent and close the

container tightly with a screw cap fitted with an intact

liner.

3. Mix well by gently vortexing at every 5 minutes interval

for 20 minutes.

4. After 20 minutes, unscrew the cap of the container

carefully and add 5 mL of reagent C.

5. Close again the container tightly as in step 2.

6. Mix well and centrifuge for 25 minutes at 3000–4000 g.

7. After centrifugation unscrew the cap of the container

with the content carefully and discard the supernatant

gently in an activated 2% glutaraldehyde solution,

taking care as not to disturb the pellet at the bottom.

8. To the pellet at the bottom, add 1 mL distilled water

and resuspend the contents.

9. Use this suspended material for microscopy (acid fast

bacilli), acid fast bacilli culture or polymerase chain

reaction.

 

disposable container

Collect specimen directly into

container or transfer from

bedpan or plastic wrap stretched

over toilet bowl. Wax from

container may produce false

positive smear

Frozen specimen.Utility of

culturing stool for acid-fast

bacilli remains controversial

Contd...

Microbiology and Bacteriology 849

Additional Material Required

Sterile plating loops (10 µL), biosafety hood with

Bunsen burner, centrifuge at 3000–4000 g, activated 2%

glutaraldehyde solution. 5 mL measuring cylinder, vortex

mixer, 1 mL micropipette, 15–25 mL universal container.

Specimen Collection

Collect specimen prior to use of antimicrobial agent.

Wherever possible, indicate clearly that patient is on

antitubercular drugs. 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.

Procedure

The procedure mentioned below is for 2.5 mL of the

sputum sample. In case of variation in quantity of

specimen used, process using proportionate amounts of

reagent, mucolytic and disinfection reagent.

Preparation of Mucolytic Reagent

The mucolytic reagent must be prepared just prior to use.

1. Bring the reagents to room temperature.

2. Add one scoop full (~12 mg) of reagent B to 2.5 mL of

Reagent A with the provided spatula.

3. Mix to dissolve.

 

simultaneously eliminating all unwanted microorganisms.

Since mucous is sticky, acid fast bacilli trapped in mucoid

portion of sputum are released by mucolytic action of N-acetyl

L-cysteine. NaOH decontaminates other microorganisms,

and final wash with phosphate buffer ensures that specimen is at optimum pH for staining and culturing. Specimen

pretreatment and disinfection with LYFECTOL increases

relative acid fast bacilli concentration and ensures its more

sensitive detection during acid fast bacilli staining and

culture.

Storage and Stability

1. Store the LYFECTOL kit at 2–8oC, away from light.

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

mentioned on the label.

Smear on slides Smear specimen over 1.5 by 1.5

cm area of clear slide

Heat fix smears.Transport in

slide container taped closed and

labeled BIOHAZARD

Sputum 5–10 mL in sterile wax-free

disposable container. Collect an

early morning specimen from

deep, productive cough on at

least 3 consecutive days. Do not

pool specimens. For follow-up

of patients on therapy, collect

at weekly intervals beginning 3

weeks after initiation of therapy

For expectorated sputum,

instruct patient on how to

produce sputum specimen

as distinct from saliva or

nasopharyngeal discharge. Have

patient rinse mouth with water

before collecting sputum to

avoid contaminating specimen

with food particles, mouthwash

or oral drugs, which may inhibit

the growth of mycobacteria.

For induced sputum, use sterile

hypertonic saline. Indicate on

request if specimen is induced

sputum

24 hours pooled specimens;

saliva

Stool > 1 g in sterile, wax-free,

 

848 Concise Book of Medical Laboratory Technology: Methods and Interpretations

MUCOLYTIC, DISINFECTANT, SPECIMEN

PRETREATMENT AND BUFFERING SYSTEM

FOR AFB STAINING AND CULTURE

Lyfectol®

(Courtesy: Tulip Group of Companies)

Summary

Infection with Mycobacterium tuberculosis remains a major

public health problem. The epidemic of tuber-culosis and

multidrug resistant tuberculosis reflects the failure of public

health and social programs towards prompt treatment

of infected cases and screening of high-risk population.

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.

Reagent

LYFECTOL is a reagent for laboratory use only. LYFECTOL

is provided as a three component reagent.

a. Reagent A (2% NaOH solution)

b. Reagent B (N- acetyl L-cysteine)

c. Reagent C (Phosphate buffer pH 6.8).

Accessories: Spatula for approximate weighing (12 mg)

and transfer of reagent B.

LYFECTOL is used for decontamination and concentration of specimen containing normal microbial flora

such as sputum as per international recommendation.

Principle

Proper decontamination and concentration of specimen

containing normal microbial flora such as sputum are

crucial in detecting Mycobacterium tuberculosis.

LYFECTOL provides a liquefaction-decontamination

and specimen buffering procedure that maintains the

viability and pathogenicity of Mycobacterium tuberculosis,

 

—

Use maximum volume attainable

—

Gastric lavage fluid > 5–10 mL in sterile container.

Collect in the morning soon after

the patient awakens in order to

obtain sputum swallowed during

sleep

Collect fasting early morning

specimen on three consecutive

days. Use sterile saline. Adjust to

neutral pH with 10 mg of sodium

carbonate immediately following

collection

Laboratory should provide

collection tube containing

sodium carbonate

Specimen that has not been

neutralized

Lymph node Node or portion on sterile

container without fixative or

preservative

Collect aseptically, and avoid

indigenous microbiota. Select

caseous portion if available. Do

not immerse in saline or other

fluid or wrap in gauze

Specimen submitted in formalin

Skin lesion Submit biopsy specimen in

sterile container without fixative

or preservative. Submit aspirate

in syringe with Luer tip cap

Swabs in transport medium

(Amies or Stuarts) are

acceptable only if biopsy sample

or aspirate is not obtainable. For

cutaneous ulcer, collect biopsy

sample from periphery of lesion,

or aspirate material from under

margin or lesion

Dry swab

Contd...

 

peritoneal)

As much as possible

(10–15 mL/min) in sterile

container or syringe with Luer tip

cap. Collect bloody specimens

into SPS blood collection tubes

Disinfect site with alcohol and

collect by needle and syringe

Bone Bone in sterile container without

fixative or preservative

— Specimen submitted in formalin

Bone marrow As much as possible in SPS

blood collection tube or 1.5 mL

in pediatric Isolator tube

Collect aseptically. Mix SPS tube

contents immediately following

collection

Bronchoalveolar lavage or

bronchial washings

> 5 mL in sterile containers Avoid contaminating

bronchoscope with tap water,

Saprophytic mycobacteria may

produce false positive culture or

smear results

Bronchial brushings CSF Sterile container or Middlebrook

7H9 broth or Kirchner medium

> 2 mL in sterile container

 

Since mucous traps AFB and protects other organisms

from effective decontamination a combination of 2% NaOH

(decontaminant) and 0.5% N-acetyl-L-cysteine (mucolytic

agent) is preferably employed. Neutralization of strong

decontaminating solutions before using the sample

for AFB stain and culture is usually accompanied with

sequential buffered wash of the concentrated sample

because if the pH of the concentrate remains alkaline

or acidic it can destroy the culture medium and prevent

the growth of mycobacteria and staining efficiency of the

AFB smears. The buffered wash also helps in reducing

the specific gravity of specimen and sediments the

Mycobacterium more effectively.

Another important aspect post-decontamination is

the specimen concentration and relative centrifugal force

applied to the specimen. Improvement in correlation

between specimen showing a positive smear for AFB and a

positive culture has been demonstrated by increasing the

centrifugal force applied to pellet the specimen.

Microbiology and Bacteriology 847

Recommendations for sample collection for mycobacterial isolation and acid fast staining

Specimen type Specimen requirements Special instructions Unacceptable specimen

Abscess contents aspirated fluid As much as possible in syringe

with Luer tip cap

Cleanse skin with alcohol before

aspirating sample. Laboratory

may provide 7H9 broth/Kirchner

medium for transport of small

volumes of aspirates

Dry swab

Blood 10 mL SPS (yellow top) blood

collection tube or 10 mL isolator

tube

Disinfect site as for routine

blood culture. Mix tube contents

immediately after collection.

SPS is preferred anticoagulant

Heparinized blood is also

acceptable

Blood collected in EDTA, which

greatly inhibits mycobacterial

growth even in trace amounts

Coagulated blood

Body fluids (pleural, pericardial,

 

 long

chain mycolic acids, unique to the mycobacterial cell wall

are responsible for their:

• Acid fastness

• Failure to react with Gram stains

• Resistance to the actionof antibodies andcomplement.

The four species in the Mycobacterium tuberculosis

complex are M. tuberculosis, M. microtic, M. africanum

and M. bovis. Laboratories can use biochemical tests for

differentiation between isolated strains.

Diagnosis of Mycobacterium Tuberculosis

Infection

The diagnosis of tuberculosis is often made on the basis

of clinical symptoms, chest X-ray and sputum AFB, since

available tests based on immunological principles for

Mycobacterium tuberculosis diagnosis have yet to overcome the problem of poor sensitivity and specificity

associated with them. For the time being, speedy and

appropriate laboratory diagnosis of tuberculosis infection

through AFB staining, culture and sensitivity have more

and more important role to play in sensitive detection

and appropriate treatment of patients with tuberculosis.

However, sample collection, preparation, processing

techniques and detection methods employed have a

profound effect on the sensitivity and specificity of the

results for the detection of Mycobacterium tuberculosis

infection by AFB and culture methods.

Specimen Selection

A critical factor in the ability of laboratories to isolate

Mycobacterium tuberculosis is obtaining appropriate

specimen for AFB smear and culture. Approximately 85%

of the TB cases are pulmonary. However, many patients

cannot produce sputum spontaneously and alternative

respiratory tract specimens such as induced sputum,

gastric lavage or fiberoptic bronchoscopy may be needed.

As the proportion of patients with extrapulmonary form

of tuberculosis is increasing, adequate specimen from

extrapulmonary sites need to be provided.

Sample Concentration and Decontamination

Specimens obtained from sterile sites such as CSF,

peritoneal or pleural fluids do not require decontamination.

However, most specimens for AFB smear and culture are

from the respiratory tract and do contain mixed microbial

flora. Successful recovery of mycobacteria depends

upon properly collected specimen and suppression of

contaminating bacteria.

 

Introduction

Humans are very susceptible to the tuberculosis

infection but are remarkably resistant to the tuberculosis

disease; which is dependent largely on the state of the

hosts immune system. Of all the mycobacterial species,

846 Concise Book of Medical Laboratory Technology: Methods and Interpretations

Mycobacterium tuberculosis remains the most common

cause of pulmonary tuberculosis and remains the most

virulent of all the mycobacterial species.

The disease, as now well known, is highly contagious.

Although the disease involves all susceptible individuals,

the incidence is higher among disadvantaged minorities.

Industrialization, increased crowded housing and

nutritional deprivation have influenced the spread.

With the emergence of HIV and resultant immunocompromise, TB has emerged as a major killer not only in the

third world countries but is also resurging in the Western

world. According to World Health Organization (WHO)

reports, each year an estimated eight million new cases

of tuberculosis occur, leading to three million deaths; and

almost a third of the world’s population is infected by the

causative organism, Mycobacterium tuberculosis.

According to a study, in India, the number of tuberculosis

patients is increasing at the rate of 1.5 million per year, and

a quarter of these are sputum positive. Thus, about 4% of all

Indians are infected with Mycobacterium tuberculosis.

With the emergence of the multiple drug-resistant

strains due to poorly administered therapeutic measures

and patient non-compliance, Mycobacterium tuberculosis

is challenging its containment, on the basis of empirical

treatment alone.

Brief Microbiology

The genus Mycobacterium is composed of slow growing

organisms, which are “acid fast”. Currently about 55 species

of Mycobacteria are recognized. They are non-motile,

slightly curved or straight rods (0.2–0.6 × 1–10 µm) and

may occasionally demonstrate branching. The organisms

are aerobic and have a gram-positive cell wall, although

they do not Gram stain well.

The mycobacteria contain a lipid rich cell surface which

includes true waxes and glycolipids 60–90 carbon,

 

Acid-fast bacilli can frequently be found in the mouths

of water taps, but some at least seem to be affected by 6%

H2SO4 when an attempt is made to grow them, but survive

treatment with weaker acid.

Anonymous Strains

Many of the so-called anonymous strains are classified by

their reaction to light. Photochromogens are those which

produce orange colonies when they are exposed to light

(day-light/artificial light). Scotochromogens are those

which produce colored colonies even in the dark. It is

therefore, important in dealing with this classification not

to grow them in an incubator that is opened frequently,

not to let them stand on the laboratory bench unless the

observation is actually being made. The yellow-orange

pigment develops after exposure to light in 6–24 hours. The

scotochromogens produce a yellow growth but this turns

more orange on exposure to light. The Battey type either

does not change, or if it does, the change is very slow.

Animal Inoculation

Sometimes for final identification it is necessary to

inoculate an animal to see if the organism will produce

disease. The human strain of M. tuberculosis will produce

disease in both guinea pigs and rabbits, but the rabbit will

show little sign of tuberculosis. With the bovine strain,

however, the rabbit is also highly susceptible. In some

specimens, the material may be directly inoculated, in

other, only after a primary culture has been grown.

OVERVIEW OF M. TUBERCULOSIS: DIAGNOS- TIC APPROACH, AFB STAINING, CULTURE AND

SENSITIVITY

 

M. lepraemurium

This is an organism found in rats in a disease somewhat

resembling leprosy. It can be passed on experimentally to

other animals of the same species.

Non-pathogenic Species

M. smegmatis

The smegma bacillus is an acid-fast bacillus found in the

smegma secretion around the genital and anal parts of human

beings and in some animals such as dogs. It can also be found

in other parts of the body, e.g. in the ear. The bacillus may be

found in the urine, but if the urine is carefully collected after

cleansing the external parts it can usually be avoided.

It is generally shorter and thicker than the tubercle

bacillus, and many strains of the smegma bacillus are

decolorized by alcohol, which distinguishes them from the

tubercle bacillus. They grow rapidly in culture.

M. butyricum

The butter bacillus. These bacilli may be found in grass,

water, butter, milk, manure, etc. They grow rapidly in

culture.

 

M. paratuberculosis

This is also known as bacillus of Johne’s disease and is

the cause of a chronic enteritis in cattle and sheep. The

primary isolation is difficult and medium has to contain

mycobactin—an extract from other mycobacteria.

M. ulcerans

This produces a chronic or subacute ulceration in both the

skin and the adjacent subcutaneous tissue, particularly of

legs and arms. Incubation temperature should be between

25 and 35°C and the best growth is at 33°C. It grows on

glycerin agar.

M. balnei

This bacillus has been isolated from swimming pools and

produces ulcerative lesions on the extremities. It grows

more rapidly than M. ulcerans, but will not grow above 35°C.

M. leprae

This is also known as Hansen’s bacillus and causes leprosy.

Smears are made from a scraping from the skin of suspected

lesions and from nasal smears. It has been found in sputum.

The usual method is skin clips from the affected areas.

Films are stained by Z-N stain, but it is customary to

use 5% sulfuric acid for decolorizing as M. leprae is not so

strongly acid fast as M. tuberculosis, but stained M. leprae

bacilli may resist decolorization with 20% sulfuric acid. The

bacilli are usually present in large numbers (in lepromatous

leprosy) and are generally found in packets like cigar

bundles within phagocytic cells called lepra cells. They

may stain uniformly but there is often marked beading. The

bacilli may also be stained fairly easily by Gram’s method.

Until recently, no claims of culture were substantiated,

but it is now believe that the organism may be isolated on

the footpads of mice.

 

Oxalic Acid Method

This is primarily used for laryngeal swabs, but is rather

unreliable in that it does not completely ensure the

destruction of untoward organisms.

The swab is simply left in oxalic acid for

30 minutes, and then smeared on the egg medium.

Trisodium Phosphate Method

This, too, is less preferable than Petroff’s method, because

it needs much longer incubation periods.

An equal quantity of sample and 10% trisodium

phosphate are incubated for 24 hours, after which the

container is centrifuged for 30 minutes and the supernatant

discarded.

The deposit is neutralized with 8% HCl using

bromothymol as indicator.

Other Pathogenic Mycobacteria

M. fortuitum

This bacillus has been isolated from soil and from

suppurative infections in men and animals and also in

Microbiology and Bacteriology 845

glandular infections. It grows rapidly and is not pathogenic

for guinea-pigs.

 

2. Other unwanted bacteria are killed, thus allowing the

tubercle bacillus to grow in pure culture.

Petroff’s Method

This is considered to be one of the most reliable methods.

1. Half fill a universal container with the sputum (or other

material) and add an equal quantity of 4% NaOH (For

lesser samples a proportionately equal quantity should

be added).

2. Invert the closed container twice or three times and

place in the incubator for 30 minutes, inverting it every

10 minutes.

3. Centrifuge at 3000 rpm for 30 minutes (The centrifuge

must be completely at a standstill before opening it

again after centrifuging tubercular samples.

4. Discard the supernatant.

5. Add a few drops of neutral red indicator to the deposit.

6. Neutralize with 8% HCl.

7. Inoculate neutralized deposit on the LJ or some other

egg medium.

 

Morphology

It is a slender or slightly curved bacillus, in direct smears

measuring about 2.5 to 3.5 by 0.3 µm although both shorter

and longer forms may be seen. In cultures, short forms are

found especially on solid media, but longer forms may be

found in liquid media. The bacillus may occur singly, or in

pairs, or in larger or smaller masses.

The tubercle bacillus (TB or Koch’s Bacillus) does not

stain easily by the ordinary dyes, but it stains well with a

strong dye with a mordant, such as carbol fuchsin when the

stain is hot but it takes a longer time when the stain is cold.

When once it has been stained it resists decolorization

with 20% H2SO4 or HNO3 (Nitric acid); it also resists

decolorization with alcohol, and so it is both acid and

alcohol fast. The method generally used for staining is

Ziehl-Neelsen’s method or one of its modifications. The

bacilli may stain evenly, or they may show beading or

barred staining or sometimes may have terminal granules.

The bacilli are gram-positive but by this method they are

stained only with great difficulty and Gram’s method is of

no use for their identification. A more recent method is the

use of auramine-phenol fluorescent stain.

Cultures

The tubercle bacillus will not grow on ordinary media.

Primary cultures are usually made on some form of egg

844 Concise Book of Medical Laboratory Technology: Methods and Interpretations

medium of which Lowenstein-Jensen (LJ) medium is

probably the most widely used. Dorset’s egg medium

or Petragnani’s media may also be used. After the

primary culture has been established, it is possible to

make subcultures on media without egg, such as Dubos,

Proskauer and Beck, Kirschner and Youman’s media.

All the varieties, viz. hominis, bovis and avium may be

found in man but in India, the bovine seems to be rare in

man, and the avian is not a common finding.

Cultural Characteristics

M. tuberculosis is aerobic, and the optimum temperature

for the human and bovine varieties is 37oC, and for

the avian 40–44oC. It grows slowly. It does not grow on

ordinary media but for the primary isolation requires an

enriched medium, a medium including eggs is used most

commonly. Glycerol stimulates the growth of the human

and avian varieties but not the bovine. On LJ medium,

the general appearance of the growth is dry, irregular,

tough and tenacious, a buff to light orange in color, but

if the surface of the medium is moist the appearance of

the colony is smoother. The growth is said to be eugonic

that is growing well. The bovine variety is dysgonic that

is growing with difficulty and the colonies are smaller,

discrete, rather smooth, slightly moist, and grayish yellow

in color. The avian grows rapidly, the colonies are moister,

more luxuriant and individual colonies have a smooth

shiny surface, yellowish to faint pink in color.

Laboratory Diagnosis

In many specimens the finding of acid-fast bacilli typical

in shape and staining, is accepted as sufficient for calling

them tubercle bacilli, but it must always be remembered

that there are many other acid-fast bacilli, which may be

found particularly in stomach-wash, urine, feces, and even

in sputum. Also when bacilli are few in number, they may

not be found in direct smear. Concentration methods such

as sodium hydroxide and trisodium phosphate may help

but on the whole considerable labor is involved without a

great increase in positive findings. Therefore, cultures are

being increasingly used even if 3 to 4 weeks elapse before a

positive result is given, and most workers report a negative

only after 6 to 8 weeks.

The specimens most commonly examined for tubercle

bacilli are sputum, stomach wash, laryngeal swabs,

urine, CSF, pleural or peritoneal fluid, pus and tissue. The

specimens are collected in clean, sterile vessels, and most

are treated with 4% NaOH or 6% H2SO4 (v/v) before the

culture is made. The treatment homogenices the specimen

and also destroys organisms other than mycobacteria.

Concentration Methods

Except in very severe cases, the organism is not always

present in large numbers and for this reason, techniques

have been devised for concentrating the organisms present

to facilitate their detection and isolation. Concentration

serves two other purposes.

1. The mucoid material is broken down and the sample

homogenized.

 

Strains of Mycobacteria

a. Pathogenic species

• M. tuberculosis:

 – hominis

 – bovis

 – avium

• M. fortuitum

• M. paratuberculosis

• M. ulcerans

• M. balnei

• M. leprae

• M. lepraemurium.

b. Non-pathogenic species

• M. smegmatis

• M. phlei

• M. butyricum.

c. Anonymous strains

• Photochromogens

• Scotochromogens

• Battey types

• Rapid growing saprophytes.