Module 7 Reading cultures

1. Module 7Reading cultures

2. 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.

3. 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

4. Examinationschedule • 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)

5. Minimal examination schedule Liquid: daily preferable (solid: weekly preferable) 0 2 days 1 week 2–4 weeks 6 weeks 8 weeks Time Inoculation • Detect very slow-growing mycobacteria, including M. tuberculosis • End of culture examination for negative report • 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 • Liquid culture: detect slow growing mycobacteria • Liquid culture: end of culture examination for negative report

6. 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

7. Preliminary identification M. tuberculosis colonies

8. 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

9. Preliminary identification of M. tuberculosis from liquid cultures • Flocculation: granular, non-homogeneous suspension • ZN: serpentine cords of varying length or district linear clumping

10. Contamination – liquid media • Homogeneous turbidity • Perform a ZN staining: non-acid-fast bacteria

11. 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

12. Contaminants Growth rate and microscopy aspects are considered. • 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)

13. 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

14. Appearance of contaminated solid media • Media colour change • Liquefaction • Growth of moulds/bacteria

15. Contamination – solid media • Surface has been completely contaminated • Medium has liquefied • Medium is discoloured • Medium is changing to dark green Autoclave and discard

16. 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.

17. Record and report positive cultures immediately! Contaminated cultures should also be reported

18. WHO scoring

19. Laboratory register : culture ../..

20. 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.

21. True and false exercise • The morphology of M. tuberculosis is used for preliminary identification. • M. tuberculosis cultures on LJ medium should be examined daily to rapidly detect TB bacilli. • Positive liquid cultures should always be tested for presence of AFB bacilli by ZN staining.

22. 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 showa 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.

23. 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?