Module 7

Reading cultures

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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.

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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

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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

Inoculation

0Time

2 days

• Check that liquid has completely absorbed, tighten caps in order to prevent drying out of media (solid)

• Detect contaminants (solid and liquid)

• On solid, detect rapidly growing mycobacteria

• On liquid, possible MTB or NTM

1 week

• Detect positive cultures of M. tuberculosis as well as other slow-growing mycobacteria

2–4 weeks

• Detect very slow-growing mycobacteria, including M. tuberculosis

• End of culture examination for negative report

8 weeks

• Liquid culture: detect slow growing mycobacteria

• Liquid culture: end of culture examination for negative report

6 weeks

(solid: weekly preferable)Liquid: daily preferable

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Preliminary identificationfrom 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 colonies7

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 colonies8

Preliminary identification of M. tuberculosis from liquid cultures

• Flocculation: granular, non-homogeneous suspension

• ZN: serpentine cords of varying length or district linear clumping

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Contamination – liquid media

• Homogeneous turbidity

• Perform a ZN staining: non-acid-fast bacteria

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Contaminants

• 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

Growth rate and microscopy aspects are considered.

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Contaminants

• Bacteria – Fast growers, usually non-

acid fast, with the exception of Rhodococcus equi (coccus-shaped) and of Nocardia spp (partially acid-fast and do not form cords)

• Yeasts– non-acid fast round in shape

and bigger than mycobacteria)

Growth rate and microscopy aspects are considered.

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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 re-inoculate 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 non-AFBs:

• Indicates growth of contaminants– discard the deposit.

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Record and report positive cultures

immediately!

Contaminated cultures should also be reported

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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 : cultureLaboratory register : culturePrimary culture serial

numberName and TB registration

identification number

New / Retrt. type

Diagnosis / Follow-up (month)

Centre of origin

Local lab ID number

Date collected / received Type

Local result

Culture lab

smear result

AFB-smearPatient Specimen

Media inoculated

Date inoculated Week 1 Week ../.. Week 8

ZN smear: morphology and % AFB

(Provisional) ID result

Date culture result

reported../..

Completed TB laboratory form

The completed form should be sent promptly to the treatment unit

Form should be adapted for reporting results of liquid cultures. 20

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

Culture result: previously reported on date _____/______/20_____ will follow

Mycobacterium tuberculosis complex ID # Contaminated

Neg Non-TB mycobacteria

(species) 1-9

colonies actual count

10 – 100 col 1+

>100 - 200 col 2+

>200 col 3+

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. 22

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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