User:Sim Huay Ping/Notebook/CBE/08/150/2008/09/18
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Streaking microbial cultures
Streaking microbial cultures on plates allows bacteria and fungi to grow into discrete colonies. The entire surface of the plate is to be used, and only one loopful of broth culture is to be streaked. The plates are to be streaked lightly so that the agar is not gouged.
1. A BD calibrated disposable inoculating loop was dipped into the broth culture.
2. The inoculating loop was then gently streaked over half of the plate using a back and forth motion (area 1). Area 1 yields heavy growth of the microbial cultures.
3. Going back to the edge of area 1, the streaks are then extended into the adjacent quadrant of the plate (area 2). Area 2 yields weak growth of the microbial cultures.
4. Going back to the edge of area 2, the streaks are then extended into the remaining quadrant of the plate (area 3). Area 3 yields single colonies.
5. The plate is then placed into the incubator.
6. After the colonies have grown, all colonies should have the same general appearance. If there is more than one type of colony, each type should be streaked again on a separate plate. The colony of strain of interest would have to be determined.
To quantify the amount of protein in the bacteria by sonification
1. Wash the bacteria innoculum with three times PBS at normal temperature in a vial
2. Wash the bacteria innculum with three times ice cold PBS
3. Add in 200ul of SDS to break the membrane of the cells
4. Add in 200ul of Triethylammonium bicarbonate buffer
5. Bring the sample to vortex
6. Sonicate the sample
7. The turbid sample will become transparent
8. Let the sample rest for 10 seconds and then sonicate for a second time