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Microbiological Quality Control Procedure for Selective FastMedia™ LB Liquid

Test the sterility, selectivity and ability to support growth of E.coli strain of a particular lot of selective FastMedia™ LB Liquid.

FastMedia™ being tested	Positive control strain	Negative control strain
LB Liquid Amp, #M0011	JM109 pUC57 Ampr	JM109

• One unautoclaved but clean 500 ml glass bottle or flask
• 3 autoclaved 100 ml flasks with "cotton" plugs.
• 200 ml of deionized or distilled water
• 1 autoclaved LB Agar plate as control media
• Control strains: appropriate glycerol stock

1. Pour the pouch contents into a 500 ml glass bottle or flask.
2. Add 200 ml deionized or distilled water.
3. Heat in a microwave oven on medium power until bubbles start to appear (2-3 minutes). Swirl gently to mix the preparation.
4. Reheat the media for 30 seconds and swirl gently again. Do not over boil!
5. Repeat step 4 if necessary until the media is completely dissolved.
6. Allow the media to cool.
7. Pour 25 ml of the media into 3 sterilized flasks. Label these flasks "I", "II", "III".
8. Inoculate flask "II" with the negative control strain.
9. Inoculate flask "III" with the appropriate positive control strain.
10. Incubate all the flasks overnight in a rotative incubator at 37°C.
11. Streak autoclaved conventional LB Agar plate with positive and negative control strains. Label this plate "IV".
12. Incubate at 37°C.

Steps 7-12 have to be performed in duplicate.

Flasks & Plates	Conditions	Purpose	Expected Result
Flask "I"	Uninoculated	Control for sterility of equipment and procedure	No growth
Flask "II"	Inoculated with negative control strain	Control for antibiotic presence in the media and no growth of negative control strain	No growth
Flask "III"	Inoculated with positive control strain	Control for growth support of positive control strain	Growth
Plate "IV"	Autoclaved conventional LB media streaked with positive and negative control strains	Control for viability of positive and negative control strains	Growth

After 16, 24 and 48h incubation, check each group of flask for growth and record in chart below:
(+) growth,
(-) no growth.

Flasks & Plate	Result
	after 16 hours	after 24 hours	after 48 hours
Flask "I"
Flask "II"
Flask "III"
Plate "IV"

Results should indicate flask "III" as having growth,  flask "I" and flask "II" as having no growth. The plate "IV" should have growth. Results are measured by visual inspection. If results agree with these indications, the lot passes. If there is growth in flask "I" or flask "II", the lot fails.

If lot fails "QC", repeat the QC test in it's entirety.

If lot fails "QC" again, it's identified, segregated and destroyed.

Updated gegužės 09, 2007 16:19