# Microbiology Protocols

(under construction)

• LB only broth
• LB/Agar only plates
• LB/Ampicillin broth
• LB/Agar/Ampicillin plates
• Overlay plates

Ampicillin is a beta-lactam class antibiotic, derived from penicillin, and is used to select for cells with plasmids containing a gene producing beta-lactamase.  It also helps to prevent unwanted growth of other cells on the plates.  If it is warmed too much it is destroyed, so media has to cool to 55 C or below before adding ampicillin.

The final concentration of ampicillin should be 50 ug/ml or 0.05 mg/ml (divide by 1,000 to convert ug to mg).  This 0.005 g per 100 ml solution.  A 100X stock can be made up with 0.5 g / 100 ml (of 50% EtOH to promote dissolving the AMP powder) and 1 ml stock added to 99 ml solution.  (Aliquot the 100X AMP solution and keep frozen so you only have to take out and thaw a few milliliters at a time.)

Luria broth is made up of

• Casein Peptone 10 g/l
• Yeast Extract 5 g/l
• Sodium Chloride 10 g/l

Luria Agar mix also has 15 g/l agar added.

Liquid LB mix is 2.5 g per 100 ml solution.  Ready made powder can be ordered (USB/Affymetrix stock #75854).  Freshly made mix without antibiotics should be autoclaved or placed in boiling water for 30 minutes to sterilize.

The LB/agar is 4 g per 100 ml solution.  Ready made powder can be ordered (USB/Affymetrix stock #75853).  Microwaving the mix (with an unsealed bottle) does not work well; it sputters and makes a mess.  It is better to melt in a boiling water bath in a sealed autoclavable bottle, which will also  sterilize the solution if antibiotics are not used later.

Ampicillin can only be added to the agar solution when it has cooled to 55C.  Bubbles tend to form in the media when poured at this or lower temperatures.  They can be removed by lightly passing a flame over the surface (don't hold it in place or the ampicillin will be destroyed).

Overlay plates can be used to get a nice even lawn of bacterial growth.  The top LB/agar is made at half concentration, 2 g per 100 ml solution.  The bacteria are added only when the mixture has cooled to 45 C.  They must then be immediately vortexed and poured on a pre-warmed (37C) agar plate.

(to be continued...)

Ordering: (not an endorsement, links to company products are put here for convenience; this list is incomplete and we have not tried and compared many competitive products)

Ampicillin (Sodium Salt) from Affymetrix

Calcium Chloride (Dihydrate) from Affymetrix

Glycerol from Affymetrix

Luria Agar (Powder) from Affymetrix

Luria Broth (Powder) from Affymetrix