MICLAB-140 Maintenance of Culture Collection

5.1. Materials and Reagents Required

5.1.1. Tryptone Soya Agar (TSA) slopes

5.1.2. Sabouraud Dextrose Agar (SDA) slopes

5.1.3. White adhesive labels

5.1.4. White, green, blue and yellow adhesive dots

5.1.5. Diagnostic media plates

5.1.6. Storage baskets

5.1.7. TSA & SDA plates

5.1.8. Metal file/ diamond pen

5.1.9. 10ml Tryptone Soya Broth (TSB) in test tubes / MacCartney bottles

SAFETY NOTE: All microbiological cultures used in the microbiology laboratory should be

classified as either Risk Group 1 (low individual & community risk) or Risk Group 2 (moderate individual risk, limited community risk) micro-organisms.

5.2. Opening of Freeze Dried Ampoules

NOTE: All work related to opening of freeze- dried ampoules and subculturing of cultures must be performed in the biohazard safety cabinet.

All cultures used in the laboratory must be obtained from an approved source.

5.2.1. Decontaminate ampoules by swabbing with 70% alcohol.

5.2.2. Place the ampoule on a bench previously decontaminated with 70% alcohol and score the glass above the cotton plug with metal file.  Cut the glass all the way around the ampoule starting from the score mark.

5.2.3. Break the ampoule, applying gentle pressure away from the body and keeping fingers away from file marks.

5.2.4. Using a sterile 1 ml pipette remove a few drops of TSB from a MacCartney bottle/test tube and add to the ampoule. Use SDA for bacterial and fungal cultures.

5.2.5. Dissolve the pellet by gently washing with TSB/SDA.

5.2.6. Aseptically transfer all the TSB/SDA from the ampoule into the MacCartney bottle/test tube from step

5.2.4.  Label the bottle with culture name, number and date.

5.2.7. Incubate broths for 24-48 hours at a temperature specified for each organism.

5.3. Subculturing                         

Culture collection is subcultured on a bi-monthly basis onto a fresh set of agar slopes. Subcultures can be streaked from master cultures up to five times after which a new freeze-dried ampoule must be opened.

5.3.1. Establish viability and purity of all master cultures by streaking onto Tryptone Soya Agar (TSA) plates for bacteria and Sabouraud Dextrose Agar (SDA) plates for fungi.  Incubate bacterial plates for 24-48 hours and fungal plates for 5-7 days at a temperature specified for each organism.

5.3.2. If culture is impure proceed as in 5.4.

5.3.3. If culture is pure, streak onto 2 slopes of TSA or SDA (as applicable) and inoculate 1 MacCartney bottle of Tryptone Soya Broth (TSB) if required. Ensure the new white Master slope is inoculated first, followed by the new routine slope and broth.

5.3.4. Label all slopes and broths as in section 5.5.

5.3.5. Incubate slopes and broth as in 5.3.1.

5.3.6. When cultures have grown, check purity of white Master slope by streaking onto TSA or SDA plates.  Incubate plates at a temperature recommended for each organism.  Check culture performance by streaking (inoculating) diagnostic media.

5.3.7. If culture is impure, proceed as in 5.4.

5.3.8. If culture is pure, document and store as described in the standard operating procedure.

5.3.9. For the first and fifth subculture obtain a biochemical profile for bacterial cultures via an API identification system (if possible).

5.4. Isolation of Pure Culture

5.4.1. Pick a single isolated colony and streak onto TSA/SDA plate.

5.4.2. Incubate for 24-48 hours at a required temperature.

5.4.3. Remove plate and check for purity and colony morphology.

5.4.4. Check Gram reaction and other appropriate biochemical tests.

5.4.5. Set up an API system.

5.4.6. If results obtained are satisfactory, return culture into culture collection.

5.4.7. If results are not satisfactory, open new freeze- dried ampoule (see section 5.2).

5.5. Documentation and Labelling of Culture Collection

5.5.1. Each culture must be labelled with the bacteria/fungus name, reference number, subculture number and date of subculture.

5.5.2. Each culture is given a laboratory number written in black on a self-adhesive white, yellow and blue dot which is placed on the top of the lid.

5.5.3. Slopes with white dots are called “master cultures” and must not be used for routine work.

5.5.4. Slopes with yellow dots are called “working cultures” and are used for routine work.

5.5.5. Broth cultures receive a blue dot, are also called “working cultures” and are used for routine work.

5.6. Environmental Culture Collection

5.6.1. A collection is kept of cultures isolated from environmental samples.

5.6.2. Due to their short life span, each environmental isolate should be replaced with the most recently isolated sample of the same species.

5.6.3. Environmental cultures receive a green dot with culture number written in black.