This document discusses conventional laboratory methods for diagnosing tuberculosis (TB), including specimen collection, processing, and microscopy techniques. Key points: - Sputum and other respiratory samples are most commonly collected and must be handled safely due to the infectious nature of TB. - Samples undergo decontamination to reduce contamination using agents like N-acetyl-L-cysteine plus sodium hydroxide before centrifugation to concentrate the bacteria. - Microscopy techniques like Ziehl-Neelsen staining are used for presumptive identification by examining acid-fast bacilli in stained smears, though sensitivity is low. Culture remains the gold standard for definitive diagnosis.

1. Conventional methods of lab
diagnosis of TUBERCULOSIS
Dr. Dinesh Kr Jain, MD.,
Assistantprofessor,
Department of Microbiology,
SMS Medical college, Jaipur

2. TUBERCULOSIS -Introduction
• Global health concern for both developing and developed
countries.
• WHO estimates that a quarter of the world’s population
has TB infection.
• More complex due to persistence in aging populations and
the rise of drug-resistant strains.
• India has been engaged in Tuberculosis control activities for
more than 50 years.
• Yet TB continues to be India’s severest health crisis.
• TB kills an estimated 480,000 Indians every year and more
than 1,400 every day.
• Global annual incidence 8.4 million.

3. • Deaths from TB 1.4 million.
• India also has more than a million ‘missing’ cases every
year that are not notified .
• Most remain either undiagnosed or inadequately
diagnosed.
• Prompt diagnosis of active pulmonary TB is a priority
for TB control, treating the individual and for public
health intervention to reduce further spread..
• NATIONAL STRATEGIC PLAN FOR TUBERCULOSIS
ELIMINATION 2017–2025 in India under RNTCP with
four strategic pillars of “Detect – Treat – Prevent –
Build” (DTPB).

5. PATHOPHYSIOLOGY
C3b mediated
LAM
Internalization of bacilli
Cell mediated immune
response
T helper
cells cytokines
Tubercles :
Hard and soft

6. Detection and identification of
Mycobacteria:
• Laboratory safety:
– Tuberculosis ranks high among
laboratory-acquired infections.
– M. tuberculosis has a very low
infective dose infection rate is
approximately 50% with exposure
to fewer than 10 acid-fast bacilli).
– Hence inappropriate handling of
clinical specimens places laboratory
personnel at risk for infection.

7. • Biosafety level class II-
– Specimen preparation in which infectious aerosols or
splashes may be created are conducted in BSC level II.

8. • Biosafety Level class III (BSL3)-
– cultures M. tuberculosis or M. bovis.
– should have non permeable walls and work surfaces,
directional airflow(lowest pressure)
– double-door air lock to prevent the backflow of air.
– Air should be vented through high-efficiency particulate air
(HEPA) filters directly to the outside.
– Technicians must wear personal protective equipment,
including a respirator.

9. Specimen Collection
• Clinical specimens:
– Pulmonary specimens- sputum, bronchial washes,
bronchoalveolar lavage fluid, and bronchial biopsies.
– Extrapulmonary specimens: urine, feces, blood,
cerebrospinal fluid, tissue biopsies, wound, skin lesions
aspirates, blood.
Note :Specimens that may contain normal bacterial
flora should be processed as soon after collection as
possible to minimize the degree of overgrowth with
specimen contaminants.

10. • Respiratory specimens:
– Sputum samples (expectoration or by ultrasonic
nebulization) best obtained shortly after the patient
awakens in the morning.
– Two Specimen Strategy(Revised WHO policy)- For
microscopy, no. of specimens reduced from 3 to 2, in
settings with appropriate EQA & high quality microscopy
“Spot sputum” and “Early morning” specimen.
– Patients must be instructed to take a deep breath, hold
it momentarily, and then cough deeply and vigorously.

11. – Container: wide mouthed, transparent, leak
proof, screw-capped, capacity of 50 ml falcon
tube.
– Good sputum specimen: recent discharge
material with minimal oral and nasal material,
volume 3-5 ml, presence of mucoid and
mucupurulent material.
• Bronchial lavages, washings, and brushings
are collected and submitted by medical
personnel
• Transported promptly to the laboratory
and refrigerated if processing is delayed.

12. • Gastric Lavage Specimens:
– Limited to senile, non ambulatory patients; children < 3
years, who fail to produce sputum by aerosol induction.
– Collected at the patient’s bedside before the patient
arises and before exertion empties the stomach.
– 5 to 10 mL of gastric secretions, expelled slowly down
the sides of the 50-mL conical collecting tube.
– Provide sterile receptacles containing 100 mg of sodium
carbonate to reduce the acidity improving the recovery
of organisms.
– Three specimens should be collected over a period of
consecutive days, processed within 4 hours.

13. • Blood specimens: Immunocompromised patients,
infected with HIV having disseminated
mycobacterial infection.
– use of the lysis-centrifugation blood culture system
(ISOLATOR; Wampole, Alere, Waltham, MA) has been
shown to increase the yield and shorten the time to
recovery of mycobacteria from blood cultures.
• Studies suggests as a routine, obtaining only a
single blood culture for the diagnosis and ordering
a repeat culture only if there is strong clinical
evidence of disseminated infection.

14. • Stool specimens :collected in a clean container
with a tightly fitting lid
– A direct smear is first prepared from a small quantity
of the specimen and stained for AFB.
– If the smears are negative for AFB, the specimen is not
processed further.
– If AFB are seen in the smear, 1 g of feces is suspended
in 5 mL of Middlebrook 7H9 broth or equivalent and
subjected to the same NaOH digestion–
decontamination as used for sputum specimens.

15. • Urine specimens: Early morning voided urine
specimens (40 mL) in sterile containers should be
submitted daily for at least 3 days.
– a clean-catch midstream urine Specimen.
– 24-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.

16. • CSF: At least 10 mL
• Other body fluids (10 to 15 mL minimum): pleural,
peritoneal, and pericardial fluids, should be
collected in a sterile container or syringe with a
Luer-tip cap.
• Wounds, Skin Lesions, and Aspirates: skin should
be cleansed with alcohol before aspiration of the
into a syringe.
• Tissues and needle biopsy material should be
placed in a small quantity of 7H9 or 7H11 broth as
a holding medium.

17. • Inadequate Specimens and Rejection Criteria:
(1) insufficient volume,
(2)contamination with saliva,
(3) dried swabs
(4) pooled sputum or urine,
(5) container has been compromised, broken or
leaking,
(6) length of time from collection to processing is too
long.

18. NOTE :
It is optimal to reject swab specimens because the
hydrophobic nature of the lipid-containing cell wall
of the bacteria inhibits transfer of the organisms
from the swab to the aqueous culture medium.
• Even if a swab is received in the laboratory and
material for culture cannot be recollected,
– the tip of the swab should be placed directly on the
surface of the culture medium or into a tube
containing approximately 5 mL of 7H9 broth and
incubated for 6 to 8 weeks.

19. SPECIMEN PREPARATION
• Sputum and specimens from non-sterile sites
should undergo prior treatment for digestion,
decontamination to reduce undesirable bacterial
overgrowth and concentration.
• Should be carried out in class II biosafety cabinets.
• After decontamination the mixture is centrifuged
at high speed to concentrate the mycobacteria.

20. • Liquefaction and Decontamination:
AGENTS COMMENTS
1. N-Acetyl-L-cysteine plus 2% NaOH Mild decontamination solution, free mycobacteria
entrapped in mucus.
2. Dithiothreitol plus 2%NaOHa Very effective mucolytic agent. More expensive than
NALC.
3. Trisodium phosphate, 13% plus
benzalkonium chloride (Zephiran)
Preferred if time of exposure to decontamination
solution cannot be controlled.
Specimens should be inoculated onto an egg-based
culture medium to neutralize the growth inhibition of
the Zephiran or by adding lecithin.
4. NaOH, 4% effects mucolytic action to promote concentration by
centrifugation.
5. Trisodium phosphate, 13% when exposure time can be completely controlled.
6. Oxalic acid, 5% Pseudomonas aeruginosa as a contaminant.
7. Cetylpyridium chloride, 1%, plus 2%
NaClb
for sputum specimens mailed from outpatient clinics.
Tubercle bacilli have survived 8-day transit.

21. N-Acetyl-L-cysteine plus 2% NaOH PROCEDURE :
• Specimens are mixed with the NALC– NaOH solution and
allow to stand at room temperature for 15–20 minutes.
• After the digestion–decontamination step, add phosphate
buffer (pH 6.8) up to the top ring in the tube.
• Mixture is centrifuged at 3,000 g for 20 minutes.
• Supernatant is decanted into a splash-proof receptacle
containing a phenolic disinfectant.
• Add a small quantity of phosphate buffer, pH 6.8 (1–2 mL)
and resuspend the sediment with a pasteur pipette.
• Prepare smears and inoculate the concentrate to
appropriate liquid and solid culture media.

22. • Centrifugation:
– specimens should be equipped with 50- or 250-mL
centrifuge cups with aerosol-free tops that can hold
50ml centrifuge tubes.
– relative centrifugation force of 3,000 g for 20 minute is
optimal for the recovery of mycobacteria.
– At RCFs of 3,000 g or higher, glass or plastic centrifuge
tubes may collapse so must be placed in sealed cups.
– refrigerated centrifuges are required as considerable
heat is generated at high speeds.
– NOTE: to maximally sediment the organism during the
centrifugation ,specific gravity of the suspending fluid
should be kept as low as possible and centrifugal force
should be as high as practical.

23. DIRECT SMEAR MICROSCOPY
• Presumptive identification.
• Useful in following response to treatment.
• Acid fast–stained smears of clinical specimens require at
least 104 acid-fast bacilli per milliliter for detection.
• Types of acid-fast stains :
1. Light microscopy- based on Carbol fuchsin –
a. Ziehl–Neelsen (hot stain)
b. Kinyoun (cold stain).
2. Fluorescence Microscopy -auramine O(with or without a
second fluorochrome), rhodamine.

24. 1. Ziehl–Neelsen stain:
• Hot stain procedure-presumptive diagnosis.
• High specificity, low and variable sensitivity(50-80%).
• Examine with 100× oil-immersion objective.
• M. tuberculosis appears as long slender, beaded, less
uniformly stained, red colored acid fast against blue
background.

25. • Before giving a negative report examine at least 100 oil
immersion fields.
• Adv: rapid, cheaper and easy to perform at peripheral
laboratories
• Disadv:
(1) less sensitive (detection limit of smear microscopy
10,000 bacilli/ml of sputum)
(2) cannot determine the viability of bacilli.

26. • RNTCP Grading for grading of sputum ZN smears
No of bacilli seen Grading No of OIF to be screened
>10/0IF 3+ 20
1- 10/0IF 2+ 50
10-99/lOO OIF 1+ 100
1- 9/lOO OIF Scanty 100
No AFB in lOO OIF Nil 100
useful for:
• Monitoring the treatment response ,
• Assessing the severity of the disease,
• Assessing the infectiousness of the patient (Higher the grade, more is
the infectiousness)
Hence, Smear-negative patients (<10,000 bacilli/ ml) are less infectious.

27. 2. Kinyoun Cold stain:
• differs from ZN staining:
o Heating is not required,
oPhenol concentration in carbol fuchsin is increased,
o Duration of carbol fuchsin staining is more.
• Typical acid-fast bacilli, purple to red, slightly curved, short
or long rods (2 to8 μm), beaded.

28. 3. Auramine-rhodamine Fluorochrome stain:
• Screening procedure.
• Principle- fluorochrome dyes forms complex with mycolic acids
in acid-fast cell walls.
• Detection is enhanced by the brightness against a dark
background.
• Examine with 25× objective using a mercury vapor burner and
BG-12 filter or a strong blue light.
• Mycobacterium spp. fluoresce yellow to orange.
• Rapid & 10% more sensitive than light microscopy.
• Detection limit- 1000 AFB/ml sputum.
• Less time consuming (screens at low magnification).
• Potential disadvantage: lower sensitivity for detection of rapid
growers.

29. M. tuberculosis stained with
fluorochrome stain
widely used by RNTCP in laboratories having higher
sample load.

30. 4. Light Emitting Diode (LED) Fluorescence
Microscopy –
• New recent approach in microscopy
• Recommended by WHO
• Uses less expensive light source (LED)
• Less power consumption
• Bulbs have long half life & not hazardous if broken
• Disadvantage is hazardous toxicity due to the dye
used.

31. CULTURE
• GOLD STANDARD for definitive diagnosis of
TB
• More sensitive than smear microscopy
• Detection limit: 10- 100 viable organisms/ml
of specimen
• Establishes viability of organisms
• Use to differentiate between MTB and NTM
• Drug Susceptibility Testing (DST) can be
performed

32. Indications :
1. Smear negative pulmonary & EPTB
2. Childhood TB
3. Follow up TB cases who failed standard treatment
4. For surveillance of drug resistance
5. High risk individuals who are symptomatic
- HIV positive cases
- H/o exposure to MDR-TB cases
- Lab workers

33. Conventional culture
Types of media used:-
1.Solid:
- Egg based :- L J Media (Most commonly used)
- Petragnani medium
- Dorset medium
- American Thoracic Society (ATS) medium
- Agar based : Middlebrook (MB) 7H10 & 7H11
- Potato based : Pawlowsky medium
- Blood based : Tarshis medium
- Serum based : Loeffler medium
2.Liquid/Broth media : - Kirschner’s medium
- Middlebrook 7H9 broth
- Dubos’ medium

34. CONVENTIONAL CULTURE MEDIA
• SOLID MEDIA:
 Egg based media:
• Lowenstein-Jensen :most commonly used
Malachite green, glycerol,asparagine,potato
starch, coagulated eggs, Mineral salt
solution(potassium dihydrogen
phosphate,magnesium sulfate,sodium citrate)
Used for differentiating different species of
Mycobacterium & for drug suseceptibility
testing

35. Gruft modification of LJ media:
• Selective media containing antimicrobial
agents,Used to suppress bacterial and
fungal contamination, consists of
penicillin and nalidixic acid

36. INOCULATION
 Always inoculate 2 plain LJ slopes along with
one PNB(p-nitro-α-acetylamino-β-hydroxy-
propiophenone) slope
 At the concentration of 500ug/ml, PNB
inhibits the growth of M.tuberculosis,
therefore can differentiate between MTB
from NTM(non-tuberculous mycobacteria)

37. IDENTIFICATION OF MTB
• Formation of typical
rough, tough and buff
colored colonies
between 10-28 days
after incubation at 37˚C
• No late production of
pigment on LJ media
• No growth on PNB
media

38. Principle of calculating results
GROWTH GRADING
Confluent growth +++
More than 100 colonies ++
20 to 100 colonies +
1 to 19 colonies Record actual number of colonies
No growth Negative

39.  Colony morphology if smooth, moist pigmented-
presumptive identification as NTM(Non Tubercle
Mycobacteria)
 If one slant having contamination ,the other
showing good growth of MTB,then reporting is
done according to the growth not as a
conatmination

40.  Agar based media:
• Middlebrook 7H10
• Middlebrook 7H11:
• Defined salts,vitamins,cofactors
• Oleic acid
• Albumin
• Catalase
• Glycerol
• 0.1% casein hydrolysate
Selective 7H11(Mitchison’s medium):consists of
middlebrook7H11 along with carbeniciilin,
amphotericinB, polymyxinB, trimethoprim lactate

41. 2. Liquid/broth medium: Middlebrook 7H9
Supplemented with:
OADC enrichment growth medium:
oleic acid,albumin,dextrose and catalase
PANTA antibiotic mixture:
PolymyxinB, AmphotericinB, Nalidixic
acid, Trimethoprim and Azlocillin

42. RAPID CULTURE METHODS
1. Direct growth observation methods
 Septicheck AFB system (biphasic media)
 MODS (Microscopic Observed Drug Susceptibility) assay
 Thin Layer Agar (TLA)
2. Colorimetric methods
 TK medium
 Nitrate Reductase Assay (NRA)
 Colorimetric Redox Indicator (CRI) assay :- Alamar Blue assay
3. Automated Methods
 BACTEC 460 TB (Semi-automated)
 BACTEC MGIT 960
 MB/BacT ALERT
 VersaTREK (ESP Culture SYSTEM II)

43. Direct growth observation methods
1. Septicheck AFB
2. MODS Assay
3. Thin Layer Agar (TLA)

44. Septicheck AFB
 Biphasic culture system
 Design :- Bottle with MB 7H9 broth in 20% CO2 atmosphere
 Paddle containing 3 types of solid media i.e.
MB 7H11 agar on one side & chocolate agar and 7H11
with NAP (Nitro acetylamino propiophenone) on reverse
 Solid media is inoculated by inverting the bottles periodically
 Growth in 12-21 days
 Simultaneous detection of M.tuberculosis, NTM & even
contaminants

45. MODS Assay
( Microscopic Observed Drug Susceptibility )
MODS :- A manual liquid culture (supplemented MB 7H9 broth)
- Culture medium in sealed multi-well plastic plates
- Processed sputum inoculated in drug free & drug
containing media
-Uses an inverted light microscope (micro-colonies)
 Characterstic corded growth pattern of MTBC
 Recommended by WHO
 But still in late stage of development & evaluation
 Adv. - Faster growth & direct DST results to
RIF & INH simultaneously in 7 days
- Inexpensive, can be implemented
on large scale
Disadv.- Requires adequate lab infrastructure (BSL2/3)
- Risk of bacterial contamination

46. Thin Layer Agar (TLA) Culture
Solid culture technique
 Uses thin layer of supplemented MB 7H11 agar medium
 Microscopic detection of MTBC micro-colonies (cording)
 Simultaneous DST for INH & RIF directly from processed
sputum samples
 Growth detection in 7days & DST in 10-15 days
 Adv.- Inexpensive
- Useful in smear negative samples
- Highly specific for INH & RIF resistance
 Disadv.- Not as fast & as sensitive as liquid cultures
- Requires BSL-2/3

47. Colorimetric Methods
1. TK medium
2. Nitrate Reductase Assay (NRA)
3. Colorimetric Redox Indicator (CRI)
Assay

48. TK Medium
 A novel colorimetric system
 Early identification of mycobacterium by
changing its color, even before the bacterial
colonies appear
 Time to growth detection- 2wks
 Simple & Inexpensive
Interpretation
 Red- Negative
 Yellow- Positive for MTBC
 Green- contaminants

49. Nitrate Reductase Assay (NRA)
(Griess method)
Solid culture technique
 Based on ability of MTBC to reduce nitrate to nitrite
 On adding Griess reagent to the medium, color changes
which indicates MTBC growth
 Recently evaluated for DST to 1st & 2nd line anti TB drugs
 Adv. - Simple, inexpensive method for DST
Faster results (before colonies on solid media)
 Disadv.- Culture is killed by reagent used in assay, so
multiple cultures be inoculated if further testing required

50. Colorimetric Redox Indicator (CRI)
Assay
 Based on reduction of a colored indicator solution by
viable mycobacteria
 When indicator is added to a liquid culture medium on a
microtitre plate after the MTBC strains have been exposed to
different antibiotics
 Colorimetric change- Number of viable mycobacterium
- INH & RIF resistance
 Results in 7-14 days
 Different indicators are :-
- Tetrazolium salts
- Redox indicators Alamar blue
& Resazurin

51. AUTOMATED CONTINUOUS
MONITORING SYSTEM
1. Semiautomated and radiometric:
 BACTEC 460 TB
2.Automated and non radiometric:
 MGIT SYSTEM(Mycobacterial growth tube
indicator):detects growth and resistance to
antitubercular drugs(ATDs); max. turnaround
time 6 weeks/42 days
 BacT/ALERT:detects growth
 ESP CULTURE SYSTEM II(VersaTREK):detects
growth and resistance to ATDs

52. Advantages of non-radiometric broth system :-
 No radioisotopes used
 Have continuous monitoring
 Less labor intensive & safe
 No potential for cross contamination by the
instrument
 Electronic data management

53. BACTEC TB460
• First broth based Semiautomated radiometric system
• PRINCIPLE:
• Uses 14C labelled palmitic acid as carbon source in
the medium –microorganisms produce radioactive
14CO2 -- measured as growth index (GI) by BACTEC
instrument (GI >10 is positive)
• Advantages:
• More sensitive & early growth detection(7-14days)
• differentiate MTBC from NTM using PNB
• Disadvantages:
potential to cause cross contamination
need to dispose of radioactive material

54. Mycobacteria growth tube
indicator(MGIT)
PRINCIPLE:
 A fluorescent compound(ruthenium pentahydrate) is embedded in
silicone on the bottom of tube(modified middlebrook 7H9 broth) which
is sensitive to dissolved oxygen in the broth (i.e. the presence of O₂ in
uninoculated medium serves to quench the emission of fluorescent
light.)
 Actively growing mycobacterium consumes the dissolved
O₂,fluorescence is unmasked and can be detected :
 manually using BD BACTEC microMGIT fluorescence reader or under
long wave UV light(Wood’s lamp)or
 by automated system, BACTEC MGIT 960

55. MGIT

56. • MGIT tube contains 7ml of broth base
• After adding supplement/PANTA mixture(0.8ml),
tube is inoculated with 0.5ml of specimen
• Tubes entered into BD BACTEC MGIT instrument
are continuosuly incubated at 37℃ and are
monitored every 60min for increasing fluorscence

57.  Growth can be detected by
observing a
nonhomogeneous turbidity
or small grains or flakes in
culture medium
 A bright orange color is
seen in the bottom of the
positive tubes
 Culture vials which remain
negative for a min. of 42
days & show no signs of
positivity are removed from
instrument as negatives

58. To report negative results
1. Inoculation in BHI (to check contamination)and
incubated for 48 hrs: no growth
2. Stained for AFB : If AFB negative & tube shows no
turbidity
3. Subculture on LJ Media: no growth on LJ slant for
8weeks
 If only contamination grows and AFB smear remains
negative-contamination
 If contamination present and AFB smear positive-
decontaminate it with 4%NAOH

59. MB/BacT ALERT Mycobacterium
detection system
PRINCIPLE:
• Bottom of each broth is fitted with a gas
permeable sensor that changes from dark green
to bright yellow when CO₂ is produced in broth by
metabolizing mycobacteria
• Contains enhanced Middlebrook 7H9 broth,
antibiotic mixture(PANTA),OADC
• Positive cultures in 16 days
• Not used for blood

60. MB/BacT ALERT
Bottles are placed bottom
down within individual
cells in the incubation
chamber,and reflected
light is used to monitor
the production of
microbial-generated CO₂

61. VersaTREK
PRINCIPLE:
• Each culture bottle(liquid culture broth,
growth &antibiotic supplement,cellulose
sponges)is placed in a special drawer in the
incubation module with a attached sensor& is
continuously monitored(24min) for any
change in gas pressure due to metabolic
activity of microorganisms
• Positivity-17 days

62. Versa TREK

63. Culture identification
MPT 64 antigen detection(SD Bioline test):
• Rapid immunochromatographic test
• Used for detection of MPT64, a 28Da
antigen,specific for M.tuberculosis complex
• Used to differentiate MTBC from NTM using
the mouse monoclonal anti-MPT64 antibody

64. • 200ul of liquid culture
for each sample is
taken in micropipette
and dispense in the
well of test kit
• Observe the kit after
10min
• Two lines in the kit
gives positive test

65. BIOCHEMICAL IDENTIFICATION

66. Differences between M. tuberculosis and nontuberculous
mycobacteria (NTM)
Property M.tuberculosis NTM
Niacin test Positive Negative (except
M.simiae, occassionally
M.africanum, M.marinum.
M.chelonae)
Nitrate test Positive Variable for species
identification of NTM
Pyrazinamidase Variable for species
identification of NTM
Tween 80 hydrolysis Variable Variable for species
identification of NTM
Optimum temperature for
growth
37°C Most species grow at 37"C
except: M. ulcerans at
32°C. M.marinum at 30°C ,
M. chelonae at 25°C,
M.xenopi at 42°C

67. Catalase test
• semi quantitative
catalase test
• heat stable catalase
test
Bubble rise <45mm of the
tube
Negative
Bubble rise >45 mm of the
tube (positive)
Most of the species are
positive
Growth in presence of p-
Nitrobenzoic acid (PNB)
Does not grow grow
Arylsulfatase test negative Only rapid growers give
positive test
Growth on MacConkeyagar
(added with 5% NaCl)
No growth Only M. fortuitum and M.
abscessus grow

68. Pigment Production
Runyon group Property Species
I. Photochromogenes Produce pigments only
in light
M.marinum, M. asiaticum
M.simiae, m.kansasii
M.genavense
II. Scotochromogenes Produce pigments
both in dark and light
M.scrofulaceum, M.szulgai,
M. gordonae ,M.celatum ,
M. flavescens
III. Non
photochromogenes
Do not produce
pigments
M.avium-complex,
M.xenopi; M. ulcerans.
M.paratuberculosis,
M.malmoense.
IV. Rapid growers Grow within one week M chelonae, M fortuitum,
M. smegmatis, M.abscessus
M. tuberculosis produce light buff color pigment.
Runyon's classification of nontuberculous mycobacteria

70. Niacin Accumulation
• All mycobacteria produce niacin.
• M. tuberculosis, M. simiae, and occasional
strains of M. africanum, M. bovis, M. marinum,
and M. chelonae produce excess niacin
• They lack the enzyme necessary to further
convert the niacin to niacin ribonucleotide.
• Niacin, as its water soluble, gets accumulated in
the culture medium.

71. • Tubes containing filter paper strips impregnated with
cyanogen bromide are used.
• Fluid is extracted from the surface of LJ medium after
colonial growth of unknown Mycobacterium species.
• The development of a yellow color in the test medium
incubated with a reagent strip is indicative of niacin
accumulation and a positive test.

72. Reduction of Nitrates to
Nitrites
• M. tuberculosis, produce nitroreductase,
which catalyzes the reduction of nitrate to
nitrite
• Sulfanilic acid and N-naphthyl
ethylenediamine are added to an extract of
the isolate.
• Red color- positive test
• Also a key test in the identification of M.
kansasii and M. szulgai

73. Tween 80 Hydrolysis
• Tween 80 is the trade name of a detergent
• Useful in identifying mycobacteria that possess a lipase
that splits the compound into oleic acid and
polyoxyethylated sorbitol
• M. kansasii - positive result in 3 to 6 hours.
• Two scotochromogens with similar-appearing colonies, M.
gordonae (positive) and M. scrofulaceum (negative), can
be differentiated.

74. Catalase Activity
• Most of the mycobacteria produce catalase except
INH resistant strains (heat-stable catalase)
• Catalase activity is assessed semi-quantitatively
• By measuring the height achieved by the column of
bubbles produced when 30% hydrogen peroxide is
added to growing colonies in a tube culture
• Tubes of LJ medium must be poured in an upright
position to produce a flat rather than a slanted
surface

75. • This surface is heavily inoculated with the test organism
and incubated for 14 days before adding the hydrogen
peroxide reagent
• A column higher than 45 mm is considered a positive test
• Quick method- few drops of hydrogen peroxide on
colonies growing on the surface of Middlebrook 7H10 agar
rapid effervescence of bubbles

76. Growth on MacConkey Agar
• MacConkey agar, from which the crystal violet
has been removed, will support the growth of
the rapidly growing mycobacteria
• However, most other Mycobacterium species
cannot grow on this medium.

77. Arylsulfatase Activity
• Differentiate rapidly growing mycobacteria from group III
nonphotochromogenic mycobacteria
• This enzyme can also be produced by M. marinum, M.
kansasii, M. szulgai, and M. xenopi. ( in small quantity)
• The development of a red color in the test medium,
indicating a release of free phenolphthalein, is a positive
result

78. Pyrazinamidase
• Useful in distinguishing weakly niacin-positive M.
bovis from M. tuberculosis.
• Pyrazinamidase is an enzyme that deaminates
pyrazinamide to form pyrazinoic acid, which produces
a red band in the culture medium.

79. Growth Inhibition by Thiophene-2-Carboxylic
Acid Hydrazide(T2H)
• (T2H) selectively inhibits the growth of M.
bovis, whereas most other mycobacteria,
including M. tuberculosis, can grow in a
medium containing this compound.
• This feature is particularly helpful for
differentiating certain strains of M. bovis.

80. Drug Susceptibility Testing
Several methods which are grouped as:
• Phenotypic methods
MGIT
Proportion method
VersaTREK
BACTEC 460
Resistant ratio and absolute concentration
methods-obsolete now
• Genotypic methods
GeneXpert
Line Probe Assay

81. • DST is essential due to the emergence of multidrug-resistant
strains.
• Susceptibility testing should be performed on the first isolate of
MTBC recovered from every patient and should be repeated after
3 months of appropriate therapy if cultures remain positive.
• Initial testing should include all primary drugs: INH(Isoniazid) at
the critical concentration, rifampin, ethambutol,
PZA(pyrazinamide) and streptomycin
• The CDC suggests that mycobacteriology laboratories strive to
report results of first-line testing within 15 to 30 days of receipt
of the specimen in the laboratory
• Ideally, susceptibility results for the primary drugs should be
available within 7 to 14 days after isolation of MTBC.

82. Proportion Method
• The reference method for susceptibility testing of
MTBC to INH, rifampin, and ethambutol and
streptomycin
• Recommended concentrations in LJ agar media are
as follows: INH, 0.2 µg/mL; rifampin, 40 µg/mL; and
ethambutol, 2.0 µg/mL, streptomycin- 4.0 µg/mL
• Reference strain of M. tuberculosis, H37Rv (ATCC
27294) is used
• Results are reported after incubation for 3 weeks

83. • This method calculates the ratio of number of colonies on drug
containing media to the number of colonies on drug free media
proportion of resistant bacilli present in the strain.
• Two appropriate dilutions of the bacilli 10-2 and 10-4 are
inoculated on drug containing and drug free media
• Below a certain proportion (critical proportion=1%), the strain is
classified as sensitive and above that is resistant.
• If 1st reading on 28th day is “resistant”, give report as resistant.
• If reading made on 28th day is “sensitive”, a second reading is
made on 42nd day only for sensitive strains; and then final
definitive results for all 4 drugs should be reported.

84. MGIT 960
• The fully automated, nonradiometric system
• Approved by the FDA for susceptibility testing of MTBC to
INH, rifampin, ethambutol, PZA, and the secondary drug
streptomycin.
• The MGIT system uses a two-tier testing protocol: INH,
rifampin, and ethambutol are in the first-tier, and PZA is
tested separately.
• An MGIT drug susceptibility testing set includes a drug-
free growth control tube and drug containing tubes
• When the GU(growth unit) of the growth control reaches
400 within 4 to 13 days, the system flags the completion
of the testing and interprets results as follows:
 Susceptible-if GU of tube <100 Resistant- if GU of
tube >100

85. • The test is invalid if the GU of the growth control reaches
400 in 13 days; this indicates that the inoculum is either
too heavy or contains contaminants, or it is too light.
• The broth in the MGIT PZA tube has a reduced pH, the
tube has a separate supplement, and the PZA growth
control tube is inoculated with a 1:10 dilution of the
inoculum to PZA-containing tube
• MGIT performance for PZA is inferior to that of other
primary drugs
• There appears to be a problem with false resistance,
which, based on data from one study, might be corrected
by reducing the inoculum from 0.5 to 0.25 mL.

86. VersaTREK
• Fully automated
• FDA-approved
• For testing susceptibility of MTBC to all primary drugs.
• The inoculum can be prepared from either broth
(VersaTREK Myco or 7H9) or a solid medium (LJ or
7H10/7H11).
• Inoculated drug-containing and drug-free control bottles
are placed in the instrument, which automatically
monitors growth every 24 hours
• Test is valid when growth in a control bottle is detected
3 to 10 days after inoculation.
• An isolate is resistant- if growth detected in <3 days
• An isolate is sensitive- if growth detected in >3 days
after that of control bottle

87. TREK Sensititre MYCOTB MIC
• It’s a 96-well plate, which includes both first-line (except
PZA) and second-line drugs.
• The inoculum is prepared from colonies grown on a solid
medium.
• Results are available 10 to 21 days after the plate is
inoculated.
• Performance of this method compared to agar proportion
is satisfactory to excellent.
• Advantage of testing both first- and second-line agents
simultaneously.
• Does not require an instrument for interpretation of
results.
• It does not include PZA, which is a major drawback, and
the turnaround time is longer than automated broth
systems.