Chang Lab:Notebook/CBE/08/148/2008/12/04

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Preparation of LB Agar:

Weight 14g of Agar and dissolved in 400ml of distill water
Autoclaved the flask at 121 degree celsius for 15 minutes
- (An autoclave is a pressurized device designed to heat aqueous solutions above their boiling point at normal atmospheric pressure to achieve sterilization)
As time is needed for the pressure to built up for the temperature to rise, the whole process took about 2 hours to complete
The temperature was let to cool below 75 degree celsuis before we could open the autoclave machine to take out our flask
The LB Agar was then pour into about 40 petri disks in the ventilation hood.
The LB Agar in the disk was cooled down before storing them in the fridge
The remaining LB Agar was stored in the 70 degree incubator

Preparation of LB Broth:

Ecoli Culturing:

Before we start, the UV light in the Biosafety hood was switched on for about 20 minutes
After 20 minutes, turn off the UV and wait for about 5 minutes before using the safety hood.
- (This is to allow all the O3 to clear)
Ecoli Strand (K12 W3110) was mixed with LB Broth in the ratio 1:9 in the 15ml test tube
The test tube is left in the incubator at 30 degree, 30rpm for 24hour

Plate Streaking:

After 24 hour, the Ecoli culture was taken out of the incubator into the biosafety hood
One loopful of E coli streaked (quadrant streaking method) onto LB(A) plate (as immediate culture)
A slant stock culture was also prepared with the same method
Two E coli plate and slant was incubated at 37 C overnight (time start: 1020 hrs)

Improtent Notes

Always Parafilm petri dishes when storing (this is to avoid contaimination)
Overturn dishes upon storing(this is to avoid the water droplets from falling onto the agar)
For streaking, we must always ensure that the agar is free from moisture before we streak)