Preparation of agar plates (LB and Light Glucose, see protocols below), with no colouring and with brilliant black ink (Pelican 4001).
Started at 11am, sterilisation 11.45-3.45, plating and spreading of bacteria 4-5pm.
Tests on 4 LB agar plates (see protocols below).
More details here.
Protocol To make 500mL of LB agar (makes about 25 LB agar plates):
Weigh out the following into a 500mL Schott Bottle:
- 5g NaCl
- 5g Tryptone
- 2.5g Yeast Extract
- 7.5g Agar
- add dH2O to 500mL
Light Glucose medium
In 500 mL Schott Bottle:
- 2 g Glucose
- 2 g Yeast Extract
- 1 g NaCl
- 8 g Agar
- add dH2O to 500mL
Petri agar plates
After weighting ingredients according to the recipes above, swirl to mix (the contents do not have to be completely in solution, but any powder left on the sides of the flask will caramelize on the glass during autoclaving).
Cover the top of the flask with aluminum foil or with a lid. Do not close tightly as it may explode during sterilisation!
Label with your name and the content. Add autoclave tape (if you have some).
Sterilise for 20 minutes using the Autoclave (be aware that the full autoclave's cycle takes about 3 hours to complete).
After removing the solution from the autoclave (at 75 °C), allow the agar solution to cool to 55 °C.
When pouring plates, keep your bench area sterile by working in a laminar flow hood (turn it on 30 minutes before and clean with ethanol before use).
Pour ~ 15-20mL of LB agar in each 96mm polystyrene Petri dish.
Note: Pour slowly from the flask into the center of the petri dish. When the agar has spread to cover about 2/3 of the dish stop pouring and the agar should spread to cover the entire plate. You may need to tilt the plate slightly to get the agar to spread out completely. If you pour in too much, the plate will be fine, but it will reduce the number of plates you can make per batch.
Do not place the lids on the plates before they are cold (allow them to cool for 30 minutes, until solidified), then invert the plates. Let sit for several more hours or overnight.
Label the bottom of plates with date and store in plastic bags or sealed with parafilm at 4°C.