Module 7
Reading cultures
Purpose
To provide you with the knowledge and skills to recognize
culture tubes that show the growth of colonies with the
appearance of M. tuberculosis and to identify and discard
contaminated cultures. To give you the appropriate time-frame
for reading cultures on solid and liquid media.
Prerequisite
modules
Modules 2 and 5
Module time
50 minutes, plus 1 hour of practical exercise in the laboratory.
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 suspect positive cultures.
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
Exercises
1. Reading solid cultures: Recognize contaminants and MTB
colonies
EXAMINATION SCHEDULE
All cultures should be examined 48 hours after inoculation in order to:



check absorption of liquid inoculated;
tighten caps to prevent drying out of media;
detect early contaminants.
Thereafter, cultures should be examined weekly or, if this is not feasible, at least three times during
the 8-week incubation period (see Figure 1).

7 day check :
 after 1 week, in order to detect rapidly growing mycobacteria, which may be mistaken for M.
tuberculosis.

3–4 week check
 to detect positive cultures of M. tuberculosis as well as other slow-growing mycobacteria,
which may be either harmless saprophytes or potential pathogens.

End of culture check
 after 8 weeks to detect very slow-growing mycobacteria, including M. tuberculosis, before
discarding and reporting the culture as negative.
Figure 1. Minimal examination schedule for solid cultures
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
evaporated, 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 slowgrowing
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
examination for negative report
Liquid cultures should be examined daily and reported negative after 6 weeks of incubation in the
absence of growth
PRESUMPTIVE IDENTIFICATION OF M. TUBERCULOSIS
With experience, a technologist can presumptively identify M. tuberculosis colonies, which are
typically rough, crumbly, waxy, non-pigmented (cream, buff-coloured) and slow-growing; a clear
morphology is apparent after 2-4 weeks of incubation.
M. bovis is a slow-grower – colonies appear white, small and round with a wrinkled surface and
irregular thin margins. This microorganism is infrequent and can be isolated from people in contact
with cows. In high-burden TB settings and if only solid media are in use, environmental
mycobacteria are also infrequent in relation to M. tuberculosis and generally represent
contamination or colonization. Suspicious colonies should be confirmed by Ziehl‒Neelsen (ZN)
staining.
Processing a positive culture entails increased biological risk, and biosafety measures must be
strictly enforced. To minimize cross-contamination risks positive cultures should be processed after
specimen processing is finished.
A very small quantity of bacteria is removed from the culture using a loop and gently rubbed into
one drop of sterile saline or water on a slide. The ease with which the organisms emulsify in the
liquid should be noted: unlike environmental mycobacteria, tubercle bacilli do not form smooth
suspensions. The smear is allowed to dry, fixed by heat and stained by the ZN method. Observed
by microscopy, TB bacilli are frequently arranged in serpentine cords of varying length or show
linear clumping. Individual cells are 3–4 µm in length.
If liquid media are used for culture, a positive TB culture may be recognized from the following
characteristics:
‒
‒
‒
signs of growth are observed at least 1 week after inoculation;
there is growth in the form of visible floccules in the medium;
floccules tend to remain isolated after shaking.
For ZN staining, deposit a drop of medium on a glass slide (albumin-coated smears are
recommended for liquid culture microscopy), let it dry in the BSC, fix, and proceed with the staining
protocol. M. tuberculosis bacteria grown in broth tend to form large side-to-side aggregates called
“cords” from the Latin corda meaning “rope”. Among MOTT the only species with a similar
characteristic is M. kansasii.
CULTURE REPORT
Positive cultures should be reported immediately – this allows prompt treatment of the TB patient
(if smear-negative).
Figure 2 shows a model TB culture report; more detailed explanations of the recording and
reporting system will be given in Module 9.
Figure 2. Model culture report
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+
CONTAMINATIONS
Even after the decontamination procedure, some bacteria and moulds can survive and
contaminate the culture. The most common early contaminants of solid media are moulds: any
culture showing the presence of mould contamination should be discarded.
Contaminated cultures are recognizable from various characteristics. The surface of contaminated
culture media may be completely covered by the growth of non-mycobacteria. Some bacteria
species can liquefy or discolour the solid medium: either the slant collapses to the bottom of the
tube or there is a very clear change of colour of Löwenstein–Jensen (LJ) medium (from very dark
blue-green to cream). In such cases, cultures should be sterilized and discarded. Certain
contaminating organisms produce acid from constituents of the medium and the lowering of pH
unbinds some of the malachite green from the egg that is the basis of the medium (indicated by the
medium changing to dark green). Tubercle bacilli will not grow under these conditions and cultures
should be discarded.
If the contamination is present only in part of the slant and the medium maintains its
characteristics, the cultures should be retained until the eighth week. The appearance of late
contamination does not exclude the presence of M. tuberculosis if colonies with suspicious
morphology are visible; a smear should therefore be prepared from these colonies or from the
surface of the medium. The smear should be stained by ZN staining: if microscopy indicates the
presence of acid-fast bacilli, an attempt could be made to collect bacteria from the slant’s surface,
followed by re-decontamination and re-inoculation of the culture.
If liquid media are used, contamination should be suspected if they show homogeneous turbidity.
ZN staining should be performed by depositing a drop of medium on a glass slide, letting it dry in
the BSC, fixing and proceeding with the staining protocol.
The different kinds of contaminants that should be considered are: mycobacteria other than
tuberculosis (MOTT), fungi, bacteria and yeasts.1
After the ZN staining, the culture should be handled according to the results:

Presence of only AFBs in the deposit, with no non-AFBs, indicates pure growth of
mycobacteria; the deposit should be processed for identification and DST (inoculation of a nonselective agar plate, such as blood agar, can be used for purity check).

Presence of AFBs with non-AFBs in the deposit indicates contamination of possible growth of
mycobacteria; the deposit should be processed for decontamination and culture on solid
media.

No AFBs and only non-AFBs in the deposit indicate growth of contaminants; the deposit should
be discarded.
1
Mycobacteria other than tuberculosis (MOTT):
‒ fast- or slow-growers
‒ acid-fast bacilli
‒ not usually arranged in cords
Fungi:
‒ usually slow-growers
‒ non-acid-fast
‒ hyphae are thicker than mycobacteria
Bacteria
‒ usually non-acid-fast except for some closely related genera (Gordonia, Tsukamurella, Nocardia, Rhodococcus,
Dietzia) and Legionella micdadei
Yeast
‒ usually non-acid-fast
Oocystis
-- usually non-acid-fast except for : Cryptosporidium, Isospora, Cyclospora,
Any presence of contaminants should be recorded in the laboratory register; if the culture is
discarded, it should be reported as “contaminated culture”.
Evaluation of the contamination rate should be performed every 6 months for quality assurance
purposes: as explained in Module 6, and extensively in Module 11, a contamination rate of 3–5% is
considered a good balance between the need to kill contaminant bacteria and the need to keep
alive the majority of tubercular mycobacteria present in the sample. A contamination rate of 0–1%
may indicate too strong a decontamination process. The contamination rate should be referred to
the number of contaminated tubes, not to the number of registered specimens.
KEY MESSAGES
 M. tuberculosis 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.
 M. tuberculosis 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 M. tuberculosis could be re-decontaminated.
 Culture results should be recorded regularly and reported promptly.
Module 7: Review
Find out how much you have learned by answering these questions.
List the principal characteristics for presumptive identification of TB-positive
cultures on solid or liquid media
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List some of the characteristics of contaminated cultures (liquid and solid media)
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What is the minimal reading schedule for TB cultures inoculated on solid media and
why?
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