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Culture Methods

Tuberculosis Research Centre


Indian Council of Medical Research
Chennai
MYCOBACTERIAL CULTURE
Advantages:

Increases number of cases found


Detects cases among smear negative patients
Establishes viability of organisms
Distinguishing between Mycobacterial species
Helps in performing DST
Helps in diagnosing cases of failure

Limitations:

Expensive
Require enriched media
Require considerable expertise
Time consuming
SELECTIVE USE OF CULTURE

DRS as a part of N T P evaluation


Diagnosis of TB patients with clinical /radiological
symptom where smears are neg.for AFB
Diagnosis of extra pulm.TB/ CH.TB
Follow-up of patients who fail on standard regimens to
check up for drug resistance
Investigation of high risk individuals who are
symptomatic
Decontamination Procedures
1915 Petroffs NaOH

1946 Trisodium Phosphate

1955 Pancreatin Desogen

1958 Pancreatin + 1% cetrimide

1962 Zephiran Trisodium PO4

1963 N-acetyl L-cysteine + 2%NaOH

1969 Swab culture technique + 1% cetrimide

1975 CPC + NaCl2


Culture Media : Solid

LJ
LJ with Na pyruvate
LJ with out asparagine
Middlebrooks 7H10 & 7H11
Selective 7H10 & 11
Ogawa
Tarshis Blood Agar
MODIFIED PETROFFS METHOD
Used widely in DEDCs since it is simple, reagents are
inexpensive and easy to obtain.
NaOH is toxic, both for contaminants and also for tubercle
bacilli; therefore, strict adherence to the indicated timings
is required

Reagents
4% sodium hydroxide (NaOH) solution
Sodium hydroxide pellets (analytical grade) 4g
Distilled water 100ml
Dissolve NaOH in the distilled water, distribute in conical
flasks and sterilize by autoclaving at 121o C for 20 minutes.
Sterile distilled water
Sterilize by autoclaving in 500ml flasks at 121o C
for 20 minutes
MODIFIED PETROFFS METHOD
PROCEDURE
To x ml of sputum add 2x ml of 4% NaOH
Tighten cap of container and shake to digest
Let stand for 15 minutes at RT with occasional
shaking
Centrifuge at 3000 g for 15 minutes
Carefully pour off the supernatant
Add approx. 20 ml sterile distilled water and re-
suspend the sediment
Centrifuge again at 3000 g for 15 minutes
Decant supernatant and inoculate deposit on to two
slopes of L-J and one slope of L-J with pyruvate
PETROFFS METHOD
Advantages:
Simple, inexpensive & control the growth of contaminants
Twenty samples can be processed in 2 Hrs, with centrifuge
capacity being the limiting factor
Sterilized NaOH can be kept for several weeks

Limitations:
The specimen exposure times must be strictly followed to
prevent over kill of tubercle bacilli. The initial kill is
independent of additional contributory factors such as heat
build-up in the centrifuge and centrifugal efficiency
Processing of sputum with CPC Method

If delay of more than 48 hours between


collection and processing is anticipated, the
sputum should be collected with 1%CPC and
2%NaCl2
CPC ( Cethyl Pyridium Chloride) acts as
homogenizing and decontaminating agent
It helps in retaining viability of Tubercle
bacilli up to 7 days
These specimens should not be treated with
NaOH ( Petroffs)
Processing of CPC containing samples
To the specimen with CPC, add 15-20 ml sterile
distilled water (to reduce the viscosity)
Tighten cap of container and mix well by inversion
Centrifuge at 3000 g for 15 minutes
Carefully pour off the supernatant
Add approximately 20 ml sterile distilled water
and re -suspend the sediment
Centrifuge again at 3000 g for 15 minutes
Decant supernatant and inoculate deposit on to
two slopes of L-J medium and one slope of L-J
with pyruvate
Egg based Medium
Advantages
Easy to make and inexpensive
Supports good growth of tubercle bacilli
Can be stored in refrigerator for several weeks
Other Factors:
Fresh eggs to be used
Bottle caps to be to tightly closed to minimize
drying
Inspissation and Malachite green suppresses the
growth of Non Mycobacterial organisms
Disadvantages
Prolonged delay 8 weeks!
Contamination covers usually entire surface -
recovery difficult
Colony Morphology of M.tuberculosis

Dry wrinkled warty


growth.
Eugonic
Reading and Reporting

Characteristics of Tubercle bacilli


Growth of Primary culture takes 2 4 weeks to
obtain visible colonies
Colonies are buff colored and rough, having the
appearance of bread crumbs or cauliflower
Not easily emulsified but give a granular
suspension
Microscopically frequently arranged in serpentine
cords of varying length or show linear clumping
Reporting of culture results

Reading Report
No Growth Negative

1 19 colonies Positive ( No.of colonies)

20-100 colonies Positive (1+)

>100 colonies Positive (2+)

Confluent growth Positive (3+)

Contaminated Contaminated
Culture: Extra-Pulmonary Samples

Aseptically collected samples


Body fluids:
Spinal ,Pleural, Pericardial, Syanovial, ascitic, Blood,
Pus & Bone marrow

Tissues:
Lymph node, Needle biopsies or Tissue biopsies

Specimens known to contain contaminating flora:


Gastric lavage, Bronchial washings & Urine
Extra-Pulmonary samples

Pauci-bacillary
Direct inoculation
Milder decontamination
5% H2SO4treatment for most
specimens
Heavily contaminated
Pus, Gasric aspirate - Petroffs method

Media:
LJ, LJP & SKLM (PACT or PANTA )
Culture

Conventional:
LJ
LJP
7H10
7H11
7H9
Kirchners
Other Culture Methods

Septi-check AFB : Non radiometric, Non


automated
MGIT 960 : Automated. Monitors
continuously every 60min. O2 utilization,
Intensification of O2 quenching fluorescent dye
MB/Bact : Non radiometric,
colorimetric detection of CO2
ESP Culture ii : Reliable non radiometric
alternative system
Microscopic Observation of Broth Culture & MODS
Micro Colony Detection System
BACTEC 460 TB System
Developed in 1969 by Deland and Wagner.

Principle
When the BACTEC 12B vial is inoculated, mycobacteria, if
present, utilize the 14C labelled substrate (fatty acid) and
release 14CO2.
The BACTEC instrument measures quantitatively the
radioactivity on a scale ranging from 0-999, as GI.
The daily increase in GI is directly proportional to the rate
and amount of growth in the medium.
When an inhibitory agent is introduced in the medium,
inhibition of metabolism is indicated by reduced
production of 14CO2 and decrease in GI.
This basic principle is utilized in drug susceptibility and
identification tests in this method.
Recovery rate (%) of Mycobacteria from
positive specimens by different systems
Mycobact. MB/BacT B 460 LJ
species

MTB 148 125 (84.5) 135 (91.2) 105 (70.9)


NTM (31) 16 (51.6) 21 (67.7) 10 (47.1)
Total 179 141 (78.8) 156 (87.1) 115 (64.2)
All clin.sig.
135 (83.3) 144 (88.9) 113 (69.7)
Myco(162)

Piercimoni C. et al. J.clin.microbiol.2001,89,651-659


Various culture methods for recovery
of Mycobacteria from clinical samples
No. (%) Isolates detected
organisms
MGIT/LJ MB/BacT+LJ B 460 +LJ
ALL (120) 106(88.3) 106 (88.3) 103 (85.8)
M.TB complex (96) 88 (91.7) 88 (91.7) 83 (86.5)

All NTM sp.(24) 18 (75.0) 18 (75.0) 20 (83.3)


M.kansasii 12 (85.7) 14 (100.0) 14 (100.0)
genotype I (14)
M.xenopi (5) 4 (80) 2 (40.0) 3 (60.0)
Detection time(days) 12.6 15.9 11.8

Alcaide F J.Clin. Microbiol. 2000. 398-401 Vol38.No.1


MODS versus other culture methods*
Pos.each Pos. by Sens. Median
Method Method(%) atleast one % detection
cult.(%) days
Auramine 0 76 98 78
MODS 89 97 92 9 (4-31)
MGIT 88 95 93 10 (3-39)
LJ 73 96 76 24 ( 6-59)
Micro COL 7H11 75 96 78 14.5(4-28)
PCR 81 90 90
* Based on 172 samples
Caviedes.L. et al J..Clin.Microbiol. 2000, 38, 1203
Cost: MODS versus other methods
Detection Susceptibility cost ($)
Method
cost ($) Two drugs Four drugs
MODS 0.77 1.72 1.80
MGIT 7.00 35.02 63.03
BACTEC 2.55 12.75 23.00
LJ 0.14 1.60 1.57
Micro 7H11 0.29 1.60 2.92
MABA 1.23 - 2.43 5.62 6.87
PCR 2.90 - -
AFB smear 0.10
Caviedes.L. et al. J.Clin. Microbiol. 2000, 38, 1203
Identification of M.tuberculosis

No single test can differentiate M.tb from


other mycobacteria.
The following tests, along with the colony
morphology may enable an identification
>95% of the M.tb strains:
Susceptibility to p-nitrobenzoic acid (PNB)
Niacin production test
Catalase activity at 68oC/pH 7
Nitrate reduction test (optional)
Susceptibility to p-nitrobenzoic acid
(PNB)
Procedure:
Weigh out 0.5g PNB and dissolve in the minimum
amount of dimethyl formamide (~ 15ml).
Add to 1 litre of L-J fluid, distribute in 6 ml amounts
and inspissate. Store in the cold.
Inoculate with the neat bacterial suspension two
slopes of LJ medium without drugs and one slope of
LJ medium containing PNB at a conc. of 500mg/ l and
incubate at 370C. Read after 28 days.
Results and interpretation:
M.tb does not grow on PNB medium. All other
mycobacteria are resistant.
NIACIN PRODUCTION TEST

Although all mycobacteria produce niacin,


comparative studies show M.tb accumulates the
largest amount of nicotinic acid and its detection is
useful for diagnosis.
Niacin negative M.tb strains are rare, while very few
other mycobacterial species yield positive niacin tests.
Cultures grown on LJ with asparagine yield consistent
results in the niacin test and hence LJ is preferred.
A culture must be at least three to four weeks old and
must have sufficient growth of at least 100 colonies.
NIACIN PRODUCTION TEST

Reagents
o-tolidine, 1.5%
o-tolidine 1.5 g
Ethanol 100ml
Mix in an amber bottle and store in the dark
in the refrigerator, prepare fresh weekly.
Cyanogen bromide solution, approx. 10%.
A saturated aqueous solution of cyanogen
bromide is approx 10%. Store at 4oC in the
refrigerator.
NIACIN PRODUCTION TEST
Procedure:
Check for water of condensation in the culture tube. If
needed add 1 ml sterile distilled water to the tube.
Place the bottles horizontally in the autoclave at 121oC
Allow 30 minutes for the extraction of niacin
Place the bottles upright for 5 minutes to allow the fluid to
drain to the bottom
Remove 0.25 ml of the extract to a clean screw-capped
tube
Sequentially add 0.25 ml of o-tolidine and 0.25 ml of 10%
cyanogens bromide. Mix well
Close the tube and observe the solution for the formation
of a pink color (positive) within 5 minutes
Add 2-3 ml of 4% NaOH to each tube and discard.
CATALASE TEST at 68oC/pH 7.0
Catalase is an intracellular, soluble enzyme capable of
splitting hydrogen peroxide into water and O2.
Mycobacteria posses several kinds of catalase that vary in
heat stability.
The O2 bubbles into the reaction mixture to indicate
catalase activity.
All mycobacteria possess catalase enzymes, except for
certain INH res. mutants of M.tb and M.bovis
Quantitative differences in catalase activity can be
demonstrated by 680C test at pH 7 (indicates loss of
catalase activity due to heat).
Drug susceptible strains of M.tb lose catalase activity when
heated to 680C for 20 minutes.
For these tests cultures on LJ should be used.
CATALASE TEST at 68oC/pH 7.0
Reagents
Phosphate buffer solution, 0.067M (M/15)pH 7.0
Dissolve 9.47 g disodium phosphate in 1 litre water to
provide 0.067 M solution (Solution 1)
Dissolve 9.07 g potasium dihidrogen phosphate in 1
litre water to give 0.067 M KH2PO4 solution (Solution 2)
Mix 61.1 ml of solution 1 with 38.9 ml of solution 2. Check pH.
Hydrogen peroxide 30% solution. Store in the refrigerator.
Tween-80, 10% Mix Tween-80 with 90ml distilled water
and autoclave at 121oC for 10 minutes. Allow to cool. Store
in the refrigerator.
Complete catalase reagent (Tween-peroxide mixture):
Immediately before use, mix equal parts of 10% Tween-80
and 30% hydrogen peroxide. 0.5 ml reagent for each test.
CATALASE TEST at 68oC/pH 7.0
Procedure
With a sterile pipette, aseptically add 0.5 ml of 0.067 M
buffer into 16 x 125 mm screw-capped test tubes
Suspend a loopful of the test culture in the buffer solution,
using a sterile loop
Place the tubes with the emulsified culture in a previously
heated water bath at 68oC for 20 minutes. Time and
temperature are critical
Remove the tubes from heat and cool to RT. Add 0.5 ml of
freshly prepared Tween-peroxide mixture to each tube
Observe the formation of bubbles appearing on the surface
of the liquid. Do not shake the tubes as it will result in
false positives.
Hold negative tubes for 20 minutes before discarding
NITRATE REDUCTION TEST
Reagents
(1) Substrate: 0.1M sodium nitrate in buffer
Sodium nitrate 0.85 g
M/45 phosphate buffer, pH 7.0 100 ml
Prepare M/45 buffer by diluting M/15 buffer 1:2 with d.
water. Dissolve the sodium nitrate in buffer and distribute
in 2-ml amounts in screw-capped test tubes. Sterilize at 15
lbs pressure for 15 min. and store in the cold.
(2) Hydrochloric acid (50 %) in d. water
(3) Sulphanilamide, 0.2% : 0.2 g in 100 ml d. water
(4) Coupling agent
N- (1-naphthyl)-ethylene diamine di-HCl 0.1 g
Distilled water 100 ml
(5) Zinc dust
NITRATE REDUCTION TEST
Procedure
Emulsify a loopful of growth from an actively growing
culture in one tube of the substrate Incubate in a
water bath at 37oC for 2 hr. Remove from the water
bath and add in the following order:
I drop of 1:1 HCl
2 drops of sulphanilamide solution
2 drops of the coupling agent

Development of a pink to deep red color indicates a positive


reaction. If no color develops, report as negative.
NITRATE REDUCTION TEST

Confirm all negative reaction by adding a pinch


of zinc dust. Development of a pink color at this
stage indicates that the initial negative reaction
was genuine. If no color change occurs after
adding the zinc dust, the reaction has proceeded
beyond nitrite into other components. Repeat
the entire test.
Controls
Use strain M.tuberculosis, H37Rv as positive
control and a reagent control without organisms
as negative control.
Identification of M. tuberculosis

SUMMARY
Growth temperature 35o-37oC only
No pigmentation
Niacin positive
Catalase negative at 68oC
No growth on LJ medium containing PNB
Positive reaction for nitrate reduction
Differentiation of Mycobacteria
M.tuberculosis NTM
Colony Rough, eugonic Mostly smooth
morphology
Growth at 37oC + +/ -
Growth at 25oC - +
Pigmentation - +/ -
Niacin + -
PNB - +
Nitrate reduction + -
Catalase at 68oC - +