Module 7
Reading cultures
1
Learning objectives
At the end of this module you will be able to:
 examine cultures at appropriate times;
 identify presumptive M. tuberculosis colonies;
 recognize contaminations;
 report the suspected positive cultures.
2
Content outline
• Examination schedule for cultures on solid
media
• Examination schedule for cultures on liquid
media
• Appearance of positive solid cultures
• Appearance of positive liquid cultures
• Criteria for presumptive identification of M.
tuberculosis
• Appearance of contaminations
• Preliminary report
3
Examination schedule
• Once a week
• On four major occasions:
• For detection of contamination/ rapidly
growing NTM detection:
after 2 days
at week 1
•For assessment of culture negativity:
at week 6 (for liquid cultures)
at week 8 (for solid cultures)
4
Minimal examination schedule
Liquid: daily preferable
0
2 days
(solid: weekly preferable)
1 week
2–4 weeks 6 weeks
8 weeks
Time
Inoculation
• Check that liquid
has completely
absorbed, tighten
caps in order to
prevent drying out
of media (solid)
• Detect
contaminants
(solid and liquid)
• Detect positive
cultures of M.
tuberculosis as
well as other
slow-growing
mycobacteria
• On solid, detect
rapidly growing
mycobacteria
• On liquid, possible
MTB or NTM
• Detect very
slow-growing
mycobacteria,
including M.
tuberculosis
• End of culture
examination for
negative report
• Liquid culture: detect slow
growing mycobacteria
• Liquid culture: end of culture
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examination for negative report
Preliminary identification
from solid media
• Rate of growth: visible isolated
colonies in 2–4 weeks.
• Colony morphology:
– buff-coloured (never pigmented)
– rough
– waxy
– appearance of bread crumbs or
cauliflower.
From colonies , ZN
staining should be
performed
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Preliminary identification
M. tuberculosis colonies
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Preliminary identification
M. bovis
•Visible growth in 3–6weeks
•Colonies:
– white
– small
– round
– wrinkled surface
– irregular, thin margins
Pictures of M.bovis
colonies
M. bovis colonies
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Preliminary identification of
M. tuberculosis from liquid cultures
• Flocculation: granular,
non-homogeneous
suspension
• ZN: serpentine cords of
varying length or district
linear clumping
9
Contamination – liquid media
• Homogeneous turbidity
• Perform a ZN staining: nonacid-fast bacteria
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Contaminants
Growth rate and microscopy aspects are considered.
• Mycobacteria other than
tuberculosis (MOTT/NTM)
– fast- or slow-growers
– acid-fast bacilli
– microscopy: usually not
arranged in cords
• Fungi
– usually slow-growers
– non-acid fast
– microscopy: hyphae are
thicker than mycobacteria
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Contaminants
Growth rate and microscopy aspects are considered.
• Bacteria
– Fast growers, usually nonacid fast, with the exception
of Rhodococcus equi
(coccus-shaped) and of
Nocardia spp (partially acidfast and do not form cords)
• Yeasts
– non-acid fast round in shape
and bigger than
mycobacteria)
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Contamination –
solid media
• If partial contamination: retain until
the eighth week.
• Late contamination does not
exclude the presence of M.
tuberculosis
• Prepare a smear from the surface
of the medium.
• Re-decontaminate and reinoculate the culture.
• Additional samples are necessary
if both LJ tubes are heavily
contaminated
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Appearance of contaminated
solid media
• Media colour
change
• Liquefaction
• Growth of
moulds/bacteria
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Contamination – solid media
• Surface has been
completely contaminated
• Medium has liquefied
• Medium is discoloured
• Medium is changing to
dark green
Autoclave and discard
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If contaminants are detected by
microscopy on solid or liquid cultures
Presence of AFBs with non-AFBs in the deposit:
•
Contamination of possible growth of mycobacteria
– process the deposit (or pellet from liquid cultures)
for decontamination and culture on solid media.
Absence ofAFBs in the deposit (or pellet) – only nonAFBs:
•
Indicates growth of contaminants
– discard the deposit.
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Record and report
positive cultures
immediately!
Contaminated cultures
should also be reported
17
WHO scoring
No growth reported
0
Fewer than 10 colonies
Report number of
colonies
+
10–100 colonies
More than 100
colonies
Innumerable colonies
or confluent growth
++
+++
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Laboratory register : culture
Patient
Primary
culture
serial
number
../..
Name and TB registration
identification number
Specimen
New /
Retrt.
type
Media
Date
inoculated inoculated Week 1
Diagnosis /
Follow-up
(month)
Week ../..
Date
Centre of Local lab ID collected /
origin
number
received
Week 8
AFB-smear
Type
Local
result
ZN smear:
Date culture
morphology (Provisional) ID
result
and % AFB
result
reported
Culture
lab
smear
result
Completed TB laboratory form
Reference laboratory results:
Date received in the Reference Laboratory _____/______/20_____ Reference Laboratory specimen
ID:__________
Microscopic examination: previously reported on date _____/______/20_____
ID #
Neg
1-9
1+
2+
3+
 hot Ziehl-Neelsen
 cold staining

fluorescence
 direct smear
 concentrated smear
 will follow
Culture result: previously reported on date _____/______/20_____
ID #
Contami
nated
Neg
Non-TB
mycobacteria
(species)
Mycobacterium tuberculosis complex
1-9
colonies
actual
count
10 – 100 col
1+
>100 - 200 col
2+
>200 col
3+
The completed form should be sent promptly to the treatment unit
Form should be adapted for reporting results of liquid cultures.
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True and false exercise
1. The morphology of M. tuberculosis is used
for preliminary identification.
2. M. tuberculosis cultures on LJ medium
should be examined daily to rapidly detect
TB bacilli.
3. Positive liquid cultures should always be
tested for presence of AFB bacilli by ZN
staining.
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Module review: take-home messages
 MTB is a slow-growing microorganism: solid media
cultures should be read at day 2 for contamination
and then weekly for 8 weeks before being reported
as negative.
 MTB colonies on solid media show a characteristic
morphology used for presumptive identification.
 Presumptive identification from positive liquid culture
can be performed by ZN staining (presence of
“cords”).
 Contaminated cultures showing presence of MTB
could be re-decontaminated.
 Culture results should be recorded regularly and
reported promptly.
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Self-assessment
• List the principal characteristics for
presumptive identification of TB-positive
cultures on solid or liquid media.
• List some of the characteristics of
contaminated cultures (liquid and solid
media).
• What is the minimal reading schedule for TB
cultures inoculated on solid media, and why?
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