Bacteria Control Strain Source

An integral part of quality control testing include quality control organisms. Microorganisms should be obtained from reputable sources, for example, the American Type Culture Collection (ATCC) or other commercial sources.

Maintenance / Frozen Stock Cultures

If using commercial stock cultures, follow the manufacturer's recommendations for growth and maintenance.

To prepare frozen stock cultures of Staphylococcus species, Streptococcus species, Enterobacteriaceae and Pseudomonas aeruginosa :

1.Reconstitute the stock culture, if necessary.

2.Inoculate multiple plates of a general purpose medium (e.g., TSA or blood agar).

3.Incubate plates for 18-24 hours in an appropriate atmosphere and at the recommended temperature.

4.Check for purity and correct colony morphology.

5.If necessary, verify biochemical tests.

6.Remove sufficient growth from a confluent area to prepare a 0.5 McFarland standard (1-2 x 10 CFU/ml). For fastidious organisms, adjust to a 1.0 McFarland.

7.Suspend the growth in 50-100 ml of cryoprotective medium, e.g., Tryptic Soy Broth with 10-15% Glycerol, Skim Milk or sterile defibrinated sheep blood.

8.Dispense 0.5-1.0 ml into sterile glass plastic freezing  vials, Prepare enough vials for one year of storage. Assume only one freeze/thaw cycle per vial. Assume  at least one fresh culture every four weeks.

9.Store vials at or below -50C (freezer) for one year. Organisms will keep longer (indefinitely) if stored in ultra low temperature freezer or in a liquid nitrogen tank.

To use a frozen culutre :
1.Thaw the vial quickly.
2.Use the culture directly subcutlure.
3.Discard any unused cell suspension.

Working Cultures

Prepare no more than three serial subcultures from a frozen stock culture.

1.Inoculate an agar slant or plate with the frozen stock culture and incubate overnight.
2.Store the working culture at 2-8C or at room temperature for up to four weeks.
3.Check for purity and appropriate colony morphology.

Or

1.Use the frozen stock culture directly as a working culture.

Maintain anaerobic cultures in Cooked Meat Medium or another suitable anaerobic medium. Alternatively, use frozen anaerobic cultures.

TEST PROCEDURE
1.Inoculate an agar plate from the "working culture".
2.Incubate overnight.
3.Suspend 3-5 isolated colonies with typical appearance in a small volume (0.5-1.0 ml) of TSB. Incubate 4-5 hours in an appropriate atmosphere and temperature.
4.Adjust the turbidity to 0.5 McFarland and 0.08-0.1 absorbance units at 625 nm.

Or

1.Adjust an overnight culture to a 0.5 McFarland.
2.Plate 0.01 ml of the specimen to confirm a colony count of 1-2 x 10 CFU/ml. If using a frozen culture, confirm the appropriate density.

To Test Cultural Response

Non-Selective Media
Dilute the cell suspension 1:100 in normal saline or purified water. Inoculate each plate with 0.01 ml to give 1-2 x 10 CFU/plate. Reduce the inoculum ten fold, if necessary, to obtain isolated colonies.

Selective Media And Tubed Media
Dilute the cell suspension 1:10 in normal saline or purified water. Streak each plate with 10.01 ml of the suspension to provide 1-2 x 10 CFU/plate. Reduce the inoculum ten fold, if necessary, to avoid overwhelming some selective media.

Results

For general-purpose media, sufficient, characteristic growth and typical colony morphology should be obtained with all test strains. For selective media, growth of designated organisms is inhibited and adequate growth of desired organisms is obtained. Color and hemolytic reaction criteria must be met.

Reference

National Committee for Clinical Laboratory Standards. 1996. Quality assurance for commercially prepared microbiological culture media, 2nd ed. Approved standard. M22-A-2, vol. 16, no. 16. National Committeefor Clinical Laboratory Standards, Wayne, PA.