To conclude, the objective of adapting different types

of technology and instruments is to shorten the times

for isolation, identification and susceptibility testing of

bacteria and other microorganisms has been particularly

relevant for mycobacteria. Hopefully, alternative methods

to the standard procedures now used, could be developed

soon enough for routine use, to provide cultures and

susceptibility information in a shorter time interval.

Till such time the AFB staining, culture and sensitivity

remain the gold standard for accurate and early diagnosis

of tuberculosis, improvements and standardization of

techniques for these classical methods is important for

better laboratory diagnosis and clinical information

support. Significant cost savings might be effected by a

reduction in hospitalization and return of the patient to a

productive career.

Troubleshooting LB

Problem: Growth not obtained on LJ control slant after diluting the culture suspense to 1:10000 of the Standard 0.5 McFarland turbidity

Possible causes Solutions

1. During scraping of culture growth, the Select only the cultural colony without scraping the media (egg yolk base)

media (egg yolk base) is being scrapped

which gives turbidity matching standard

0.5 McFarland

Contd...

Microbiology and Bacteriology 863

Problem: Collapse of slants

Possible causes Solutions

1. Improper transportation, i.e. kits Discard the slant and use fresh slant for testing

subjected to violent jerks and vibrations

or not handled with care during storage,

shipping and incubation

2. Contaminated slants If the slants are found contaminated because of various reasons, discard the slants and use

fresh slants to perform the test

3. Contaminated untreated sputum sample Pretreat sputum sample prior to inoculation

used

IN DETERMINATION OF ADENOSINE DEAMINASE ACTIVITY IN SERUM, PLASMA AND

BIOLOGICAL FLUIDS

Courtesy: Tulip Group of Companies

ADA-MTB®

 

1. If ratio of the growth in antibiotic containing media as

compared to control is less than 0.01 the isolate will be

termed as sensitive.

2. If ratio of the growth in antibiotic containing media as

compared to control is more than 0.01 the isolate will

be termed as resistant.

Example:

No. of colonies on antibiotic

containing media Ratio = _______________________________

 No. of colonies on control media

Sensitive if ratio is less than 0. 01

Resistant if ratio is more than 0.01

Border line if ratio is equal to 0.01

Remarks

1. Discolored, dislodged or contaminated medium

should not be used.

2. Good laboratory practices and hazard precautions

must be observed at all times.

3. Treat the specimen and used slants by immersing in

2% activated glutaraldehyde for at least 2 hours before

incineration and disposal.

Other Markers

Adenosine deaminase, a surrogate marker, for the

diagnosis of tuberculosis has also shown promise. It

is based on the measurement of activity of Adenosine

deaminase, an enzyme produced by lymphocytes. The

test has excellent sensitivity for TB meningitis and for

examining pleural infections.The sensitivity and specificity

is reported well above 90%, the test is easy to perform and

relatively inexpensive.

 

Additional Material Required

Sterile plating loops (10 µL), incubator at 37± 0.5oC,

biosafety hood with Bunsen burner, activated 2%

862 Concise Book of Medical Laboratory Technology: Methods and Interpretations

glutaraldehyde solution, vortex mixer, 0.1–0.5 mL

micropipettes, sterile micropipette tips.

Inoculum Preparation for Sensitivity Testing

a. Take a loopful aseptically from the Mycobacterium

tuberculosis colony grown on Lowenstein-Jensen slant.

b. Transfer it aseptically to the screw capped bottle

containing 0.1 mL of sterile distilled water and glass

beads, for inoculum preparation.

c. Close cap tightly and subject the contents of the bottle

to mechanical shaking (vortex) for 10 minutes.

d. Keep standing for 10 minutes before opening the

bottle.

e. Dilute this in saline to match McFarland 0.5 Standard.

This contains approximately 1.5 × 108

 org/mL.

f. Further dilute to 1: 10000 with saline. This is seed

culture. (10000–12000 org/mL).

g. Mix well and use this as inoculum.

h. Discard the container with glass beads in 2% activated

glutaraldehyde solution.

Test Procedure

1. Bring the primary/secondary drug containing

Lowenstein-Jensen media panel for MTB sensitivity

tests slants to room temperature.

2. Apply 100 µL from the seed stock to each slant of

primary/secondary drug containing LowensteinJensen media panel for MTB sensitivity tests also

control LJ.

3. A fresh disposable loop should be used for each slant.

4. Close the cap tightly and incubate at 37± 0.5° C.

5. Observe for the growth after 2 weeks till 8 weeks, every

week.

Interpretation of Results

As and when there is sufficient growth on control

(>100 colonies) compare the growth with the antibiotic

containing media.

 

1. p-Aminosalicylic

acid

PA 7.0 ± 0.1 0.5 µg/mL

2. Ciprofloxacin CP 7.0± 0.1 2 0. µg/mL

3. Amikacin AM 7.0 ± 0.1 20.0 µg/mL

4. D-cycloserine DC 7.0 ± 0.1 30.0 µg/mL

5. Kanamycin KA 7.0 ± 0.1 20.0 µg/mL

6. Ethionamide ET 7.0 ± 0.1 20.0 µg/mL

7. LJ Control LJ 7.0 ± 0.1 —

8. Sterile distilled water with glass beads for inoculum preparation

Principle

Due to increase in drug resistant strains of Mycobacterium

tuberculosis and increasing failure rates of antitubercular

drug regimens, it is desirable to start antitubercular

therapy only after sensitivity assay of the most suitable

drug against particular isolate infecting the patient.

Storage and Stability

1. Avoid jerks and vibration while storage, shipping and

incubation.

2. Store the LJ kits at 2–8oC, away from light.

3. Stability of the unopened media is as per the expiry

date mentioned on the label.

4. Upon opening, the medium must be put into use

instantly.

 

Summary

Inadequate chemotherapy, irregularity of treatment and

use of improper antitubercular regimen lead to high

failure rates of antitubercular treatment. As a result, the

prevalence of chronic patients discharging drug-resistant

organisms increases. Alarming figures of drug resistance in

newly detected patients are being reported, mainly from

developing countries. This calls for testing of antibiotic

sensitivity in vitro prior to starting therapy.

Reagent

SENSICULT: LJ Primary/secondary drug panels are

Reagents for laboratory use only. Primary/secondary

drug containing Lowenstein-Jensen media panel for

MTB sensitivity tests is a set of ready to use LowensteinJensen solid medium slants incorporated with individual

antitubercular drugs of recommended specified strength.

Contents

I. Primary drug Lowenstein-Jensen medium panel

contains Lowenstein-Jensen medium with the

following antibiotics/antitubercular drugs.

Drug Symbol pH Concentration

1. lsoniazid IN 7.0+ 0.1 1.0 ng/mL

2. Ethambutol EB 7.0± 0.1 2.0 µg/mL

3. Rifampin RP 7.0± 0.1 4.0 µg/mL

4. Streptomycin ST 7.0 ± 0.1 4.0 µg/mL

5. Pyrazinamide PY 5.5 ± 0.1 100. µg/mL

6. Control for

Pyrazinamide

PC 5.5 ± 0.1

7. LJ Control LJ 7.0 ± 0.1 -

8. Sterile distilled water with glass beads for inoculum preparation

II. Secondary drugs Lowenstein-Jensen media panel

contains Lowenstein-Jensen medium with the

following antibiotics/antitubercular drugs.

Drug Symbol pH Concentration

 

A simplified method for preparing drug susceptibility

plates has also been developed. This method uses filter

paper disks containing the primary antitubercular drugs,

and the test for susceptibility is run in a similar fashion

as the Kirby Bauer method for routine drug susceptibility

tests.

As discussed, the direct mycobacterial susceptibility

test is inoculated from digested and concentrated sputum

found to be positive for acid-fast bacilli. The indirect

susceptibility test is inoculated from colonies isolated

from a primary culture. The direct test will usually give

good results only if large numbers of mycobacteria are

present in the specimen. The advantage of the direct

susceptibility test is an earlier report (3 to 4 weeks) in

contrast to the indirect test, which may take up to 6 to 8

weeks. The disadvantage of the direct susceptibility test is

that it usually requires a large number of mycobacteria for

successful growth and is often overgrown by large numbers

of contaminating bacteria.

Other novel methods of susceptibility testing have been

developed based on the mycobacteriophage technique,

using the luminescent luciferase activity. Other researchers

have localized specific Mycobacterium tuberculosis

mutations responsible for drug resistance. These sites have

been used as amplification targets and promise to provide

a rapid method for testing the susceptibility of patient

isolates to these drugs.

PRIMARY/SECONDARY DRUG CONTAINING

LOWENSTEIN-JENSEN MEDIA PANEL MTB

SENSITIVITY TESTS

(Courtesy: Tulip Group of Companies)

Sensicult®

 

The absolute concentration method determines if 1%

or more of an inoculum will grow after being cultured on

media containing critical concentrations of a drug on the

plate. It requires growth of the patient strain on drug free

medium to demonstrate the viability, but does not compare

the colony numbers on drug free and drug containing

media so that the inoculum must be carefully standardized.

The resistance ratio is similar to the absolute

concentration method except that the patient strain is

compared with the growth of a standard laboratory strain.

Results are reported as the ratio of the MIC of the patient

strain to that of the laboratory strain. A patient strain with

a ratio of 8:1 is considered resistant, while 4:1 is suggestive

of resistance. This method is more tolerant to variation in

concentration of drugs within different batches of media.

The proportion method compares the growth of a patient

strain in the presence and absence of a drug. If 1% or more

of the inoculum produces colonies on media that contains an

agent at the critical concentration compared with controls,

the isolate is considered to be resistant. This method is the

most popular and is relatively simple to perform and interpret.

Susceptibility Testing of Mycobacteria

Eleven drugs are used in the treatment of tuberculosis.

Five are considered “primary” and include streptomycin,

isoniazid, rifampin, pyrazinamide and ethambutol, while

the remaining six, Ethionamide, ciprofloxacin, kanamycin,

D-cycloserine, para-aminosalicylic acid and amikacin are

considered “secondary” and used only when resistance

develops to the primary drugs.

Although drugs have been incorporated in inspissated

egg-based media for conducting susceptibility tests,

many laboratories internationally now prefer using

Middlebrook 7H11 or 7H10 as a base medium, adding

the drugs after cooling the agar to 45°C. Adding the drugs

to the agar medium after autoclaving decreases the loss

of activity that can occur in egg-based medium such LJ

during inspisstion. An additional loss of drug activity may

occur in egg-based media with binding of some agents to

egg albumin and other proteins.

Drug Concentrations for Proportion Method

Susceptibility Testing using Various Culture Media*

Drug concentration (µg/mL)

Drug 7H10 7H11 Lowenstein-Jensen

Isoniazid 0.2,1.0 0.2,1.0 0.2,1.0

p-Aminosalicylic acid 2.0 8.0 0.5

Streptomycin 2.0 2.0 4.0

Rifampin 1.0 1.0 40.0

Ethambutol 2.0 7.5 2.0

Ethionamide 5.0 10.0 0.0

Kanamycin 5.0 6.0 20.0

Capreomycin 10.0 10.0 20.0

D-Cycloserine 20.0 30.0 30.0

Pyrazinamide 50.0 — 100.0

Microbiology and Bacteriology 861

 

Susceptibility Testing Methodology

Susceptibility tests can be performed directly, from a

smear positive specimen, or indirectly, from the growth

of colonies from the specimens. The former has the

advantage of measuring the sensitivity prior to cultivation

on laboratory media. The direct method also produces

results more rapidly but; because of uncertainty of the

species of Mycobacterium, and due to less control of the

viable inoculum size, the results require confirmation with

an indirect test, the direct test is not generally utilized.

Three methods make use of critical concentrations to

define drug resistance and can be performed directly or

indirectly:

¾ Absolute concentration method

¾ Resistance ratio method

¾ Proportion method.

 

In a recent Indian study, a total of 3181 samples were

processed for isolation of tubercle bacilli; and 707 samples

were culture positive. The pattern of drug resistance is

shown in the following table:

Pattern of drug resistance for mycobacterium

tuberculosis

Drug Percent resistance

Isoniazid 30.41

Rifampin 58.55

Streptomycin 46.95

Ethambutol 3.67

D-Cycloserine 24.32

Kanamycin 14.42

Ethionamide 60.67

Amikacin 15.84

Ciprofloxacin 7.49

860 Concise Book of Medical Laboratory Technology: Methods and Interpretations

In India, it has been observed that private practitioners

use different drug regimens to treat tuberculosis and very

few regimens match with the standard (recommended by

WHO).

The problem of acquired drug resistance (ADR) is

truly man made. Poor administered tuberculosis control

program, inadequate dosages, monotherapy, insufficient

durations of treatment, irregularity in drug intake, frequent

defaults are some of the common reasons for emergence

of ADR. In addition, HIV is quickening the pace at which

Tuberculosis is spreading. Therefore, Tuberculosis is

becoming the leading killer disease of HIV-positive people.

Clinicians should ensure that Mycobacterium tuberculosis

susceptibility tests are carried out for patients:

¾ Who fail to respond after 3 months of treatment

¾ Who do not convert to having negative smears after

3 months of treatment; with regimens that include INH

and Rifampin, and 5 months for treatment without INH

and Rifampin

¾ Whose smears demonstrate increasing number of AFB

after an initial decrease

¾ Patients whose cultures do not become negative after

4–6 months

¾ Patients who relapse

TB susceptibility testing has three main goals:

¾ It provides data as to what drug should be used for

treatment

¾ Screens for drug resistance

¾ Measures incidence and prevalence of drug resistance

within the community.

 

Troubleshooting

Problem: Growth on the slant within first week of incubation

Possible causes Solutions

1. Improper decontamination of sputum Proper decontamination and concentration of specimen containing normal microbial flora

specimen due to which contaminants are crucial to detection of Mycobacterium tuberculosis. Therefore, proper decontamination

over grow of sputum specimen should be carried out using Lyfectol

2. Fast growing organism of Mycobacterium Confirm the type of organism grown with biochemical tests

species

Problem: Collapse of slants

Possible causes Solutions

1. Improper transportation, i.e. kits Discard the collapsed slant and use fresh slant for testing

subjected to violent jerks and vibrations

or not handled with care during storage,

shipping and incubation

2. Contaminated slants If the slants are found contaminated because of various reasons, discard the slants and use

fresh slants to perform the test

3. Contaminated untreated sputum sample used Pretreat sputum sample prior to inoculation

Susceptibility Testing of

Mycobacterium tuberculosis

Resistance to antitubercular agents was recognized soon

after their introduction in early 1960s, and standardized

methods for antimicrobial susceptibility have been

developed. Routine laboratory susceptibility testing of

primary TB isolates has not been generally suggested

unless drug resistance in a particular community exceeds

5%. However, with the resurgence of TB drug resistance,

CDC USA has recently recommended that susceptibility

tests should be performed on all primary isolates.

 

However, the radiometric system is more labor

intensive, requires disposal of radioactive material and

still cannot detect some Mycobacterium tuberculosis

isolates that can only be detected on agar slants. Some

laboratories prefer to use LJ slants as a backup to

Radiometric media. Considering the cost aspects and

the fact that Mycobacterium tuberculosis is largely a

problem of the third world, use of radiometric media is

still restricted and use of solid and liquid media is widely

practiced.

 

1. Discolored, dislodged, contaminated or turbid

medium should not be used.

2. Improper decontamination and concentration

procedure will yield erroneous results.

3. Good laboratory practices and hazard precautions

must be observed at all times.

4. While observing growth in liquid medium, care needs

to be taken to differentiate between Mycobacterium

growth and specimen material’s own turbidity.

5. Treat the specimens and used slants by immersing

in 2% activated Glutaraldehyde for at least two hours

before incineration and disposal.

6. Preparation of Kirchner medium has to be carried out

just prior to inoculation of specimen or culture.

7. In specimens from patient, already on antitubercular

drugs, the initial growth may be further delayed.

Microbiology and Bacteriology 859

8. Growth on the Lowenstein-Jensen slant/Kirchner

medium within the first week postinoculation usually

indicates atypical Mycobacterium or contamination

due to insufficient decontamination of specimen.

9. All culture growth should be characterized based on

morphology, AFB stain and biochemical tests.

Radiometric media: Developed in 1970, represent

a significant improvement in the rapid isolation of

Mycobacterium tuberculosis. Detection time is directly

proportional to the number of metabolically active

bacteria present and the metabolic rate is influenced

by the type of specimen, number of organisms, therapy

status of patient, decontamination procedures and the

incubation temperature.

The average time for reporting the isolation of

Mycobacterium tuberculosis using radiometric technique

is reportedly 22 ± 9 days as compared to 31 ± 9 days for

solid media.

 

Test Procedure

Kirchner Medium

1. Bring the Kirchner medium to room temperature.

2. Label the Kirchner medium appropriately.

3. Draw 10 µL of the decontaminated and concentrated

specimen from the reconstituted pellet with a sterile

calibrated loop.

4. Inoculate in Kirchner medium aseptically.

5. Close the Kirchner medium cap tightly and incubate at

37 ± 0.5° C.

6. Observe for growth every third day till 8 weeks.

Lowenstein-Jensen Slant

1. Bring the Lowenstein-Jensen medium slant to room

temperature.

2. Label the Lowenstein-Jensen medium slant

appropriately.

3. Draw 10 µL of the decontaminated and concentrated

specimen from the reconstituted pellet with a sterile

calibrated loop and plate it on the Lowenstein-Jensen

medium slant aseptically.

4. For quantitative evaluation prepare bacterial

suspension to match McFari land 0.5 standard, dilute

this 1:10000 and Seed 100 µL on the LowensteinJensen medium slant aseptically (seed stock consists

of approximately-15000 organisms/mL).

5. Close the Lowenstein-Jensen slant cap tightly and

incubate at 37± 0.5oC.

6. Observe for growth weekly till 8 weeks.

Interpretation of Results

a. Mycobacterium tuberculosis colonies on LowensteinJensen slant may be detected from third week onwards

up to 8 weeks. The colonies are characterized by rough

granular buff colored growth, which has initial size of

1–3 mm and full-grown size of 5–8 mm.

b. Mycobacterium tuberculosis growth in Kirchner

medium is characterized by fluffy growth to small

granules. The granules sediment to the bottom.

c. Since both the media differ in their composition,

growth of Mycobacterium tuberculosis in either

medium should be considered as a positive culture

result. The growth needs to be identified.

Remarks

 

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.

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 AFB smear and culture are from respiratory tract and

the mucous traps AFB 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.

Preparation of Kirchner Medium

1. Reconstitute the Kirchner selective enrichment with

1 mL of sterile distilled water provided with the kit.

2. Transfer reconstituted selective enrichment aseptically

to the Kirchner medium base, which is now ready to

use as ‘Kirchner Medium’.

 

Storage and Stability

1. Store the kit at 2–8°C, away from light.

2. Stability of the unopened media is as per the expiry

date mentioned on the label.

3. Avoid jerks and vibrations while storage, shipping and

incubation.

4. Upon opening, the media must be put into use instantly.

Additional Material Required

Sterile plating loops (10 µL), incubator at 37 ± 0.5°C,

biosafety hood with Bunsen burner, activated 2%

glutaraldehyde solution, 0.2 mL micropipettes.

Specimen Collection and Preparation

Collect specimen prior to use of antimicrobial agents.

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.

858 Concise Book of Medical Laboratory Technology: Methods and Interpretations

Body Fluids

Disinfect the site and collect specimen with aseptic

precautions.

 

Reagent

COMBICULT is a reagent for laboratory use only. LowensteinJensen medium is provided as a ready to use slant Kirchner

medium is provided as a three-component medium.

a. Kirchner medium base

b. Kirchner selective enrichment containing antibiotic

cocktail Polymyxin B, Amphotericin B, Carbenicillin,

Trimethoprim (PACT)

c. Sterile distilled water for reconstitution of Kirchner

selective enrichment.

Lowenstein-Jensen medium is a standard non-selective

inspissated egg based solid medium for the isolation of

Mycobacterium tuberculosis from biological specimen

such as sputum, CSF, urine. Lowenstein-Jensen medium

supports the growth of Mycobacterium tuberculosis.

The glycerol present in the Lowenstein-Jensen medium

enhances the growth of Mycobacterium tuberculosis.

Accurate amount of malachite green not only has an

inhibitory effect on growth of organisms other than

Mycobacterium but also provide the desired color contrast

for easy identification of Mycobacterium colonies.

Kirchner medium is a liquid medium enriched with

serum. Kirchner medium has polymyxin B, amphotericin

B, carbenicillin, trimethoprim as inhibitory antibiotic

cocktail for most of the bacteria and fungus other than

Mycobacterium. Being a buffered medium, it allows direct

inoculation of larger inoculum up to 500 µL, and also

keeps up the acid base balance during the growth phase.

Principle

The gold standard for primary isolation of Mycobacterium

tuberculosis is the use of liquid media in conjunction

with solid media. Most Mycobacterium species grow

more quickly in liquid media than solid media. Liquid

media also support higher detection rates especially

with specimen material containing smaller number of

bacilli. Simultaneous inoculation of solid media and

liquid media yields significantly higher recovery rates for

Mycobacterium tuberculosis growth as compared to when

each media is used independently.

 

Summary

Infection with Mycobacterium tuberculosis remains a major

public health problem. The epidemic of tuberculosis and

multidrug resistant tuberculosis reflects the failure of public

health and social program’s towards prompt treatment

of infected cases and screening of high-risk population.

Culture, isolation and sensitivity of Mycobacterium

tuberculosis from patient groups using standard culture

methods remain the gold standard for Mycobacterium

tuberculosis detection and effective and swift treatment

worldwide.

 

Troubleshooting

Problem: Growth on the Slant within First Week of Incubation

Possible causes Solutions

1. No proper decontamination of sputum Proper decontamination of sputum specimen is to be carried out using Lyfectol

specimen due to which contaminants (decontamination reagent) to ensure that all unwanted organisms are killed

over grow

2. Fast growing organism of Mycobacterium Confirm the results with biochemical tests

species

Microbiology and Bacteriology 857

Problem: Collapse of Slants

Possible causes Solutions

1. Improper transportation, i.e. kits subjected Discard the slant and use fresh slant

to violent jerks and vibrations or not

handled with care

2. Contaminated slants If the slants are found contaminated because of various reasons, discard the slants and use

fresh slants to perform the test

3. Contaminated untreated sputum sample Pretreat sputum sample prior to inoculation

used

COMBIPACK OF SOLID AND LIQUID MEDIUM

FOR MYCOBACTERIUM TUBERCULOSIS

ISOLATION

(Courtesy: Tulip Group of Companies)

Combicult®