Projet Sylvan

Protocols

LB Agar

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

Note: If your lab has pre-mixed LB agar powder, use the suggested amount instead of the other dry ingredients 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 and label with autoclave tape.
Autoclave on the liquid setting for 20 minutes or according to your autoclave's specifications.
After removing the solution from the autoclave, allow the agar solution to cool to 55°C.
Note: This can be done by placing the flask in a 55°C oven or water bath, as this will hold the temperature and it can be left unattended for some time.
When pouring plates, keep your bench area sterile by working near a flame or bunsen burner.
Add the appropriate amount of desired antibiotic to the solution (500µL if you are using a 1,000x antibiotic stock) and swirl to mix.
Pour ~20mL of LB agar per 10cm 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.
Note: If bubbles are introduced during the pouring, these can be removed by quickly passing the flame of an inverted bunsen burner over the surface of the plate. Be careful, if you leave the flame too long it will melt the petri dish. Also be careful not to burn yourself.
Place the lids on the plates and allow them to cool for 30-60 minutes (until solidified), then invert the plates. Let sit for several more hours or overnight.
Label the bottom of plates with antibiotic and date and store in plastic bags or sealed with parafilm at 4°C.

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