LJ Inoculation, Reading, and Reporting TB Cultures

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LJ Inoculation, Reading, and Reporting TB Cultures

• Robert B. Ferguson, M.S.

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LJ Inoculation and Incubation of TB Cultures

• Inoculate 2-3 drops sediment per slant
• Leave caps loosened, for at least the first week,
  to allow for air exchange, then screw tight to
  prevent desiccation
• Lay slants out horizontally, inoculated surface
  facing upward, for at least 24-48 hours, to allow
  inoculum to absorb into media, after which they
  can be placed upright to save room in incubator

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Reading of L-J Cultures

• Inoculated L-J slants should be read weekly for 8
  weeks
• Read the same day each week
• Record each reading on worksheet

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Forms/Records- Documents

• Worksheet for each individual sample to record
  results of microscopy, culture readings, culture
  identification, and drug susceptibility
• Preliminary report form to issue at 4th week
• Log/register that lists final results for all
  patient samples
• Minimize duplication of forms to reduce
  transcription errors

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Forms and Records

• Keep all record documentation neat and legible
  (sloppy paperwork implies sloppy results)
• All documentation to be recorded in a ledger or
  paperwork secured in a 3-ring binderNO LOOSE
  PAPERWORK

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Preliminary Results

• Preliminary reports are important to keep
  physician informed of current status of culture,
  especially when delays are anticipated
• Examples
• No MTB at 4 weeks, final report to follow
• If AFB isolated, identification to follow
• If AFB identified as MTB, susceptibility test
  to follow

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Contamination Issues

• Contamination is the presence on
  non-mycobacterial growth (e.g. non acid fast
  bacteria and fungi)
• Can be determined by gross appearance of LJ slant
  (dark blue/green or bleached out/faded LJ slant)
• Can be determined microscopically by ZN stained
  smear

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Contamination Rate

• Contamination rates are determined by dividing
  the number of slants discarded due to overgrowth
  by the total number of slants inoculated
• Example 50 patient samples inoculated to 100
  L-J slants (2 slants/sample). If 6 LJ slants
  overgrew, 6/100 6 contamination rate

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Contamination Rate

• Target Contamination rate 5
• If much less than 5, probably over-decontaminatin
  g, and killing off too many mycobacteria as
  wellneed to reduce concentration of NaOH
• If much greater than 5, probably
  under-decontaminatingneed to increase
  concentration of NaOH (but no more than 4), or
  look into obtaining fresher samples

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L-J Culture Reports

• Reporting Scheme
• Colony Count Report
• 0 no growth
• 1-19 actual count
• 20-100 1
• 100-200 2
• 200-500 (almost confluent) 3
• gt500 (confluent) 4

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Identification of M. tuberculosis

• Colony morphology, rough dry, cauliflower-like
  appearance
• Growth Rate
• Biochemical Tests
• -Niacin positive
• - Nitrate positive
• - Catalase negative at 68 Centigrade pH 6.8

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Final Report of Cultures

• Report Mycobacterium tuberculosis, with colony
  count and sensitivity report status
• If non-MTB, report out as such (i.e.,
  Mycobacterium species other than MTB, with colony
  count)

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Post-Identification Considerations

• Storage of patient cultures for potential
  follow-up testing (e.g., secondary drug testing)
• Prepare stock cultures frozen in Middlebrook
  based broth with glycerol
• Store frozen broths in cryotubes at -70oC, and
  maintain storage log