533559183
Document type: SOP
Document code: TB 05-05
MYCOBACTERIAL SUSPENSIONS AND
VIABLE COUNT PROCEDURE
Confidentiality: none
TABLE OF CONTENTS
1.
PURPOSE ...................................................................................................................... 2
2.
SCOPE ........................................................................................................................... 2
3.
RESPONSIBILITIES ...................................................................................................... 2
4.
CROSS-REFERENCES ................................................................................................. 2
5.
PROCEDURES .............................................................................................................. 2
5.1. Reagents and equipment required .............................................................................................. 2
5.2. Preparation of suspensions from solid cultures ........................................................................... 3
5.3. Determining the viable count of mycobacterial suspensions ...................................................... 3
6.
REFERENCES ............................................................................................................... 3
7.
CHANGE HISTORY ....................................................................................................... 4
This SOP template has been developed by FIND for adaption and use in TB laboratories
Release date: ddMMMyy
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533559183
1.
PURPOSE
This SOP describes the techniques of preparing mycobacterial suspensions and performing viable
counts. Standardized suspensions of mycobacteria, particularly M. tuberculosis, are routinely
prepared for experimental purposes and for preparation of inoculated sputum, where it is important to
estimate the approximate viable count of the input suspension so that performance of diagnostic tests
can be evaluated in advance of viable count data. Viable counts are routinely performed to determine
the number of viable organisms in a suspension.
2.
SCOPE
This SOP relates to all procedures involving preparation of mycobacterial suspensions for
experimental purposes in the ________________TB Laboratory. Viable counts must be performed on
all mycobacterial suspensions prepared for experimental purposes, unless stated otherwise in specific
SOPs.
3.
RESPONSIBILITIES
All staff members working in the ____________TB Laboratory are responsible for the implementation
of this operating procedure. All users of this procedure who do not understand it or are unable to carry
it out as described are responsible for seeking advice from their supervisor.
4.
CROSS-REFERENCES
See:
Document Matrix_TB 01-01_V1.0.doc
Location:
5.
PROCEDURES
5.1. Reagents and equipment required
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1µL disposable loop
Middlebrook 7H9 medium or phosphate buffer pH 6.8
Sterile glass ‘bijou’ bottle
≥3 mm diameter glass beads
Vortex mixer
Timer
0.5 McFarland Standard
Middlebrook 7H11 agar plates
Plastic bags
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533559183
5.2. Preparation of suspensions from solid cultures
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Take a 1µL (one microlitre) loopful of mycobacterial culture from Löwenstein Jensen (LJ) or
Middlebrook 7H11 media (no antibiotic present) using a sterile loop. Fresh cultures (cultures
aged up to 2 weeks from positive confirmation of M. tuberculosis (that have been stored at
room temperature or 37°C) should be used.
Add the fresh growth to 5ml of Middlebrook 7H9 medium in a sterile screw cap bottle
containing 6-8 glass beads.
Vortex for 15-20 seconds to homogenize the suspension. Allow any large clumps to settle by
standing for 10-15 minutes. The supernatant will be used as the test suspension.
Adjust the concentration of the supernatant to 0.5 McFarland Standard (0.5 mL 1% Barium
Sulfate in 99.5 mL 1% Sulphuric acid) by visualization. Use 7H9 medium to dilute if the
supernatant is too concentrated, or add more growth if the supernatant turbidity is too low.
A 0.5 McFarland standard suspension corresponds to approximately 107 cfu/ml for M.
tuberculosis and M. kansasii and 108 cfu/ml for other mycobacteria (and other bacteria).
Dilute the suspension according the requirements of the particular experiment.
5.3. Determining the viable count of mycobacterial suspensions
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Dilute the suspension by a series of 1:10 dilutions (0.1 mL of the suspension into 0.9ml 7H9
medium) until an expected concentration of 102 cfu/mL is obtained.
Mix each dilution by several inversions.
Prepare 7H11 agar plates.
Use:
Media Preparation_TB 02-09_V1.0.doc
Location:
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Inoculate two 7H11 plates each with 0.5 ml of each appropriate dilution.
Calculate the dilutions expected to give between 20 and 300 colonies on the plate, and include
at least one dilution either side to ensure that a countable number of colonies will be obtained
Allow the plates to dry in the biological safety cabinet.
Seal the plates in a plastic bag, and incubate inverted at 37°C for up to 8 weeks. Check for
the presence of growth on a weekly basis.
After incubation, count the number of colonies on each plate. Plates should yield 20-300
colonies.
Add the number of colonies from each dilution with between 20 and 300 colonies together
(mean colony count).
Determine the viable count (cfu/ml) by using the following formula:
Viable count (cfu/ml) =
6.
mean colony count x 100
dilution factor
REFERENCES
Kent P.T., and G.P. Kubica. 1985. Public Health Mycobacteriology. A Guide for the Level III
Laboratory. U.S. Department of Health and Human Services, Centers for Disease Control, Altlanta,
GA.
Isenberg H.D. (Ed.). Clinical Microbiology Procedures Handbook. Second edition. NY
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7.
CHANGE HISTORY
New version #
/ date
Old version #
/ date
No. of
changes
Description of changes
Source of
change request
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