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since 11 July 2001.
These media will be prepared and may be
used for a variety of experiments related to the propagation, titering
and genetic crossing of bacterial viruses (bacteriophage). See the protocol
Microbiological
Media Preparation for principles and practices of media prep. You
are to weigh out each reagent listed in bold, add the appropriate
amount of distilled water, and dissolve it by heating to 95°C (except
for Tryptic Soy Broth which does not need high temp to dissolve because
it does not have agar). All media should then be sterilized immediately.
The top agar will be melted as needed, aliquoted out in 2 mL portions and
kept melted in a 45°C hot block for titering phage.
TRYPTIC SOY BROTH [TSB] 18 g/600 mL
(Soybean-Casein Digest Medium, USP)
For the cultivation of fastidious and non-fastidious organisms, culturing of coliphage.
Ingredients per liter:
Bacto-Tryptone 17.0
g
Bacto-Soytone 3.0 g
Bacto-Dextrose 2.5
g
Sodium Chloride 5.0
g
Dipotassium Phosphate
2.5 g
TRYPTONE AGAR (For growth of phage)* grams/600 mL:
(Make 3 bottles for a class of 20)
tryptone
6.0 g
NaCl
0.5 g
1 M CaCl2
(2 mL/L medium) 1.2 mL
agar
6.6 g
TOP AGAR:*
For use in titering phage, and preparing a poured a lawn of bacteria in top agar.
grams/200 mL:
tryptone (1%)
2.0 g
KCl (0.5%)
1.0 g
agar (0.6%)
1.2 g
Mix, distribute 2 mL/13 x 100 mm test tube, cap and sterilize.
Before use, melt in 100°C water, cool
to 45°C. (Alternately, sterilize in bottle, melt just before use, aliquot
out into 45°C capped sterile Wasserman tubes.)
LB AGAR, MILLER (Luria-Bertani) 24 g/600 mL
For the cultivation Escherichia coli, titering of coliphage, especially the fastidious lambda bacteriophage.
Ingredients per liter:
Bacto-Tryptone
10.0 g
Bacto-Yeast Extract
5.0 g
Sodium Chloride
10.0 g
Bacto-Agar
15.0 g
* From Bob Kirchen at Carolina Biological, 2/7/95