Preparation and Maintenance of QC STOCK Cultures

Improper storage and repeated subculturing of a microorganism can produce phenotypic alterations. A phenotypic alteration is observed when a microorganism fails to produce known and predictable growth, biochemical, physiological, or serological characteristics for which it was selected. Establishing a routine maintenance procedure can prevent alterations from occuring.

CLSI has modified its recommendations for the maintenance of stock cultures for routine quality control procedures. However, maintenance of stock cultures for antimicrobial susceptibility procedures still requires weekly subculturing and monthly regeneration of organisms.(2)

MATERIALS REQUIRED BUT NOT PROVIDED

Each laboratory must provide the selected LYFO-DISK® or KWIK-STIK™ Microorganisms; non-selective, primary, growth agar media; non-selective, storage agar media; inoculating devices; 35 degree C. incubator; and refrigerator (2-8 degrees C.) to properly process the LYFO-DISK® or KWIK-STIK™ Lyophilized Microorganisms.

1. Primary Growth Media
A non-selective agar medium must be used to grow the microorganisms following hydration. Refer to the LYFO-DISK® or KWIK-STIK™ Microorganism instructions or Product Insert for detailed information on media selection.

2. Storage Media
Following growth, store working control cultures at 2-8 degrees C. on Tryptic Soy agar for non-fastidious organisms or on Chocolate agar slants for more fastidious strains. See the HUGO™ document "Stock Organism List and Maintenance Program at Hardy Diagnostics".

PROCEDURE FOR ORGANISMS USED FOR ROUTINE qc PROCEDURES

Once per year a cycle will begin by processing a LYFO-DISK® or KWIK-STIK™ Microorganism.

1. Hydrate the LYFO-DISK® or KWIK-STIK™ Microorganism as outlined in the directions.

2. Immediately saturate the swab with the hydrated material and transfer the material to the primary non-selective agar medium. On primary agar media, with pressure, rotate the swab, and inoculate a circular area (i.e. one inch or 25mm in diameter) of the medium. Using a sterile loop or the swab, repeatedly, (about 10 to 20 times) streak through the inoculated area and then continue to streak the remainder of the agar surface in order to obtain isolated colonies.

3. Immediately incubate the inoculated media at temperature and environmental conditions favorable for growth of the organism.

4. Following incubation, select representative well-isolated colonies for subsequent testing, indicated subcultures or QC Stock Culture Maintenance. Prepare both a 'Stock Control' and 'Working Control' as follows:

1. Stock Control
Using the culture growth from the processed LYFO-DISK® or KWIK-STIK™ microorganism (Step 4 above), a subculture is performed by transferring growth from the original 'growth medium' to 2 slants of appropriate non-selective agar medium. Label the cultures with the date, challenge strain identification, and designate one slant as the 'Stock Control' and the other as the 'Working Control'. Following incubation, the cultures are stored at 2-8 degrees C. The 'Stock Control' may be stored for less than or equal to 12 months. The growth on the 'Working Control' is used as a source for quality control challenges for a period of one month.

Note: Molds and certain fastidious bacteria such as N. gonorrhoeae may require 25 to 35 degrees C storage. Additionally, fastidious organisms have a limited survival on stored slants and may require more frequent subculture.

2. Working Control
One month after preparing the 'Working Control', transfer growth from the 'Stock Control' to a new non-selective agar slant. If plates are used, seal them tightly. Label the new slant as the 'Working Control', the name of the challenge strain and the date of inoculation. Following incubation, the culture is stored at 2-8 degrees C. (see above information for fastidious organisms). Discard the 'Working Control' that was prepared in the previous month and use the newly prepared 'Working Control' for daily or weekly routine quality control challenges. For less fastidious bacteria, prepare a new 'Working Control' each month from the 'Stock Control'. Avoid multiple serial subcultures of quality control organisms over extended periods of time.

PROCEDURE FOR ORGANISMS USED FOR ANTIMICROBIAL SUSCEPTIBILITY PROCEDURES

Once per month a cycle will begin by processing a LYFO-DISK® or KWIK-STIK™ Microorganism.

1. Hydrate the LYFO-DISK® or KWIK-STIK™ Microorganism as outlined in the directions.

2. Immediately saturate the swab with the hydrated material and transfer the material to the primary non-selective agar medium. On primary agar media, with pressure, rotate the swab, and inoculate a circular area (i.e. one inch or 25mm in diameter) of the medium. Using a sterile loop or the swab, repeatedly, (about 10 to 20 times) streak through the inoculated area and then continue to streak the remainder of the agar surface.

3. Immediately incubate the inoculated media at temperature and environmental conditions favorable for growth of the organism.

4. Following incubation, select representative well-isolated colonies for subsequent testing. A total of two subcultures must be done prior to use in testing procedures and/or preparation of 'Stock Control'.

1. Stock Control
Using the culture growth from the processed LYFO-DISK® or KWIK-STIK™ microorganism (step 4 above), a subculture is performed by transferring growth from the original 'growth medium' to another non-selective medium. A total of two subcultures must be done prior to use in testing procedures. Label the cultures with the date, challenge strain identification, and label as the 'Stock Control' and the others as 'Working Control'. Following incubation both cultures are stored at 2-8 degrees C. The 'Stock Control' may be stored for less than or equal to 1 month.

Note: Certain fastidious bacteria such as N. gonorrhoeae may require 25 to 35 degrees C storage. Additionally, fastidious organisms have a limited survival on stored slants and may require more frequent subculture.

2. Working Control
Working cultures are subcultured from the 'Stock Control' each week for no more than three successive weeks. Following incubation, the 'Working Control' is stored at 2-8 degrees C. (see above information for fastidious organisms). Discard the 'Working Control' that was prepared in the previous week. Use the newly prepared 'Working Control' for quality control challenges.

TECHNICAL NOTES

During stock culture storage at 2-8 degrees C., moisture loss in the agar medium must be avoided. Store plated agar medium subcultures in a sealed 'zip-lock' bag and store tubed agar medium with the caps sealed.

REFERENCES

1. Technical Bulletin, MicroBioLogics, Inc., St. Cloud, MN.

2. Quality Control of Commercially Prepared Microbiological Culture Media; Approved Standard-Third Edition. M22-A3, Vol. 24, No. 19, Clinical and Laboratory Standards Institute (CLSI).

3. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard--Ninth Edition M2-A9, Vol. 26, No. 1, Clinical and Laboratory Standards Institute (CLSI).


LYFO-DISK is a registered trademark of MicroBioLogics, Inc., St. Cloud, MN.
KWIK-STIK is a trademark of MicroBioLogics, Inc., St. Cloud, MN.

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