Autoclave Machine Lab Report
actococcus lactis ssp. lactis C2 and bacteriophages c2, ml3, sk1 were obtained from T. R. Klaenhammer (North Carolina State University, Raleigh, NC). Both bacterial Lactococcus lactis ssp. lactis C2 stains and bacteriophages c2, ml3, sk1 were stored in microcentrifuge tubes at -80℃ refrigerator to keep frozen until use.
3.3 Preparation of M17 medium, skim milk, Bottom agar, Top agar and CaCl2 solution M17 medium, skim milk, Bottom agar, Top agar and CaCl2 solution were all prepared in lab 201 in Garrugus building (University of Kentucky). All the materials and solutions involved in this research were autoclaved for 15 minutes at 121℃ in microbiology lab 202 (University of Kentucky). The complete cycle in the autoclave machine was about
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Top agar was dispensed into 50ml test tubes and then autoclaved for 15 minutes at 121℃ as well. Stored the top agar at -5℃ refrigerator when it cooled down. Preheated top agar for 20 minutes to liquefy before use. The optimal dispense temperature for top agar was 45℃.
3.3.4 Preparation of CaCl2 solution The molecular weight of CaCl2 was 111 g/mole. Weighed 11.1g CaCl2 and dissolved into 100ml distilled water. The final CaCl2 concentration would be 1M. CaCl2 solution was used to make bottom agar and initiate the infection cycle.
3.4 Selection of most sensitive strain to bacteriophages to make new stock culture Starting culture was prepared by inoculating 1ml (1×109 CFU/ml) stock Lactococcus lactis ssp. lactis C2 culture into 25ml M17 medium in a test tube. The mixture was incubated overnight for 16h in a 32℃ water bath to get fresh Lactococcus lactis ssp. lactis C2 culture. Inoculated the Lactococcus lactis ssp. lactis C2 culture onto the petri dish with bottom agar by using inoculation loop for streaking. The streaking was used to isolate the pure strain of Lactococcus lactis ssp. lactis C2 from the stock culture. The inoculation loop was sterilized in a fire for 5-7 seconds and then cooled to dip into the starting culture for microbes inoculation on the plate. Allowed the inoculation plate to grow in a constant 32℃ temperature incubator for another 16 hours. After 16 hours, the plate we streaked had shown colonies thinning with the streaking direction
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