Inoculation Procedure for Storage of Culture Isolates

   Steps to Preserve and Store MTB Cultures 

    From culture on solid media

1. Store Mycobacteria in the 7H9 Liquid medium.
2. Add 0.5 ml of double sterile distilled water in a Bijou bottle containing glass beads (3 mm. diameter).
3. Add bacterial growth from one slope of LJ medium with 3+ growth, and mix using vortex mixture for about 30 seconds. Allow the coarse particles to settle down.
4.  Pipette out 100 μl of bacterial suspension into McCartney bottles containing five ml of 7H9 medium and Incubate at 37° C for 10 days.
5. After 10 days, examine for any gross contamination (turbidity). If turbidity is observed, discard the subculture and attempt a fresh subculture.
6. Inoculate a loopful of media on a nutrient agar plate.
7. Incubate at 37°C for 24 hours and check for sterility.
8.  Record the observations are recorded in the Sterility Check Register.
9. Using a 1ml tuberculin syringe, prepare uniform suspension by aspirating and discharging the suspension for about 10 times within the vial.
10. Adjust the turbidity to McFarland No.1 standard.
11. Pipette out aseptically 750 μl of bacterial suspension into a cryovial. Add 750 μl of 40% sterile glycerol.
12. Label it with the lab number & date and arrange it in a cryovial rack.
13. Store Cryovials at –80 C freezer.
14.  Maintain a date-wise list of all cultures stored with particulars of the culture in the storage register.

    From liquid culture

• Label sterile screw-capped cryovials with laboratory number, study reference and date of preparation.
• If cultures are from quality control strains, label them with name, ATCC number, batch number and date of preparation.
• Distribute the suspension into sterile screw-capped cryovials.
• Put the cryovials into a numbered storage box. 
• Put the storage box into the -80C freezer.
• Record the location and strain identifiers in the -80°C Freezer Storage Logbook.

Resource

Mycobacteriology Laboratory Manual 

Assessment