# Presentation: 023016

## LJ Inoculation, Reading, and Reporting TB Cultures

- Robert B. Ferguson, M.S.

## 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

## 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

**Notes:**
C02 enhances growth on L-J, but not required to obtain growth

## 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

## 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 binder...NO LOOSE PAPERWORK

## 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

## 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

## 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

## Contamination Rate

- Target Contamination rate= 5%
- If much less than 5%, probably over-decontaminating, and killing off too many mycobacteria as well...need to reduce concentration of NaOH
- If much greater than 5%, probably under-decontaminating...need to increase concentration of NaOH (but no more than 4%), or look into obtaining fresher samples

## 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+
- 		>500 (confluent)			4+
- _  _

## 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

## 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)

## 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