Commonly Used Solid Media

Commonly Used Solid Media

These media will be prepared and used for a variety of experiments during the quarter in Microbiology. Note the similarities and differences between them. You should become familiar with the purposes of each. Identify which are selective media, and which are differential media, and why. Note that all but the first are formulated as a complete powder, and therefore you weigh out the amount indicated in bold below the title. For specific directions on how to handle these powders, dissolve them, autoclave and pour plates, refer to Solid Media Prep: 4% Glucose Nutrient Agar. Note that most of these require only the single weighing out the premixed powder. (The number of 600 mL bottles needed for a class of 20 students is indicated.)
NUTRIENT AGAR WITH 4%GLUCOSE (Mark the side of plates with four lines)
(For cultivating yeast and non-fastidious bacteria)
(5 bottles = 3000 mL for 20 students)

Dissolve the following in 600 mL dH2O:

weigh out four items in bold:
Nutrient Broth Powder            6.0 g
(Beef Extract        2.25 g)
(Peptone              3.75 g)
NaCl                                      3.0 g
Glucose                                24.0 g
Agar                                       9.0 g
PLATE COUNT AGAR (For examining milk and dairy products.)
NOTE: Aliquot out 15 mL in 16×150 test tubes, cap and then autoclave.
(make 1500 mL for 20 students)

weigh out 14.1 g/600 mL

Ingredients per liter:
Tryptone 5.0 g
Yeast Extract 2.5 g
Glucose 1.0 g
Agar 15.0 g

LEVINE EMB AGAR (Selection and differentiation of E. coli and other Gm- bacteria)
(5 bottles = 3000 mL for 20 students)

weigh out 22.5 g/600 mL

Ingredients per liter:
Peptone 10.0 g
Lactose 10.0 g
Dipotassium phosphate 2.0 g
Agar 15.0 g
Eosin Y 0.4 g
Methylene blue 0.065 g

MacCONKEY AGAR (Selection and differentiation of E coli and other Gm- bacteria)
(3 bottles = 1800 mL for 20 students)

weigh out 30 g/600 mL

Ingredients per liter:
Peptone 17.0 g
Proteose peptone 3.0 g
Lactose 10.0 g
Bile Salts No. 3 1.5 g
Sodium Chloride 5.0 g
Agar 13.5 g
Neutral Red 0.03 g
Crystal Violet 0.001 g
OTHER COMMON MICROBIOLOGICAL MEDIA

The following media have been used in past years, and may be used under some circumstances:

TRYPTONE GLUCOSE EXTRACT AGAR

14.4 g/600 mL

(a Standard Plate Count Agar, for enumeration of bacteria in milk, meat, etc.)

Ingredients per liter:
Tryptone 5.0 g
Beef Extract 3.0 g
Dextrose 1.0 g
agar 15.0 g

TRYPTIC SOY AGAR:
(Enriched for fastidious organisms)

24.0 g/600 mL

Ingredients per liter:
Tryptone (pancreatic digest of casein) 15.0 g
Soytone (Papiac soybean meal digest) 5.0 g
Sodium Chloride 5.0 g
Agar 15.0 g
BRAIN HEART INFUSIONAGAR:
(Enriched for fastidious organisms)

31.0 g/600 mL water

Ingredients per liter:
Calf Brains, Infusion from 200.0 g
Beef hearts, infusion from 250.0 g
Proteose Peptone 10.0 g
Glucose 2.0 g
Sodium Chloride 5.0 g
Disodium phosphate 2.5 g
Agar 15.0 g

Commonly Used Media for Phage Growth

Commonly Used Media for Phage Growth

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