User:Sim Huay Ping/Notebook/CBE/08/150/2008/12/10
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Validating bacteria concentration
1. Extract 0.5 ml of the bacteria inoculums left overnight into 5ml of broth.
2. Incubate for 3-4 hours
3. Bring the samples to centrifuge at 1000rpm for 5 minutes.
4. Remove the supernatant and wash with PBS 3 times.
5. Extract the sample into the spectrometer vials and keep the concentration at a standard of 0.05.
Performed kinetics experiment on E.Coli, using 6.2ul of Zhou’s AMP
1. Label the petri dishes and the microtiter plate. For identification sake, the wells are identified as (row, column). For example, (D, 6) indicates row D and column 6.
2. Obtain 2ml of bacteria from the inoculums and transfer it into the vials. Bring the vial to centrifuge to discard the supernatant. The pellet is then washed three times with physiological buffered saline (PBS) by centrifugation for 7 minutes at 1000rpm and vortex. After washing, the bacteria is resuspended in 1ml of the PBS.
3. Measure the bacteria sample to 0.05 on the spectrometer by trial and error.
4. Pipette 180ul of PBS into all the wells except for wells (A,1), (A,2) and (A,3).
5. Now, dilutions on the bacteria without AMP is carried out. From the vial of bacteria in PBS, pipette out 20ul and transfer it into (A,4). Next, obtain 20ul of the mixture from (A,4) and transfer it into (A,5). Do subsequent dilutions until (A,8) which is the 10-5 sample. Pipette 20ul from (A,8) and drop it in the center of the petri dish. Obtain 2 samples for each dilution. Do the next subsequent dilution in (A,9) which is the 10-6 sample. Pipette 20ul from (A,9) and drop it in the center of the petri dish. Obtain 2 samples for the dilution.
6. Add 200ul of bacterial solution in each of the 3 wells, (A,1), (A,2) and (A,3). Add the desired amount of AMP (6.2ul for E.Coli, 6.2ul for Pseudomonas, 50ul for Serratia, 6.2ul for Staphylococcus and 25ul for Candida). This time, there is only one AMP to perform experiment on.
7. After 5 minutes of interaction, pipette 20ul from (A,1) and add it into (B,1). Pipette up and down to ensure good mixing, and then transfer 20ul from (B,1) into (B,4). Do subsequent dilutions until 10-5 and 10-6 (Note that there will not be enough wells down the row, hence we place these dilutions in wells (H,1) and (H,2)). Before pipetting solution out from (H,1), samples are to be extracted and placed on petri dishes. Obtain 2 samples for each dilution.
8. Continue the steps and collect samples at time intervals of 10, 15, 30, 45 and 60 minutes.
9. While waiting for the time, Nutrient Agar (NA) can be added to the petri dishes one at time. Yeast Medium (YM) is used for Candida. Pour agar to cover half of the petri dish, swirl the plate to allow equal distribution of the samples within the agar.
10. Place the petri dishes into the incubator or water bath and leave for overnight.
Left the bacteria inoculums in the incubator overnight