Making Milk Media

Many microbes grow better in milk than in nutrient broth. We will open this page with the results of an experiment comparing the growth rate of two common milk bacteria in those media. We will then show how you can prepare bacteriological media from milk. Remember, all measurements are grams per liter of final medium.

Comparison of Growth of Two Bacteria
in Milk and Nutrient Broth at 37oC

Bacterium              Medium      Cell doubling time (minutes)
Escherichia coli       broth                 17
Escherichia coli       milk                  12.5
Streptococcus lactis   broth + lactose       48
Streptococcus lactis   milk                  26
---------------------------------------------------------------

Discussion of the above table. Cell doubling time is the time required for a given number of bacteria to become twice as numerous. Such measurements should be made during the Logarithmic Growth Phase when the cells have plenty of food and are not too crowded (accumulated waste products are not slowing growth). A short doubling time means an organim grows fast. In this experiment, E. coli grew 2.08 times faster than S. lactis in milk. Escherichia coli is famous for being one of the fastest growing bacteria.

Streptococcus lactis is an important 'clean sour' bacterium commonly found in raw milk. S. lactis is often added to milk to cause the milk to sour for cheese making. Notice S. lactis grows slowly in nutrient broth even when lactose is added. S. lactis gets its energy for growth by fermenting lactose (milk sugar) to lactic acid.

Escherichia coli is a common fecal organism which on balance is beneficial in human and animal intestines, but some rare strains cause diarrhea or toxins which can be fatal. Since E. coli grows rapidly, is a common contaminant in milk, causes off flavors, and some strains have serious health effects, we try to exclude it from milk supplies by good sanitation practices.

Skim Milk is a Good Medium

The results of the above experiment show that milk is an excellent medium for many common bacteria and sudents should try it. Since most bacteria do not digest fats or do so very slowly, bacteriologists usually use skim milk in media. Since dry skim milk lasts for years and works just as well as fresh, you may prefer to buy dry skim milk and keep it in a tightly closed jar. Save jars that have tight fitting plastic lids such as mustard jars for such uses.

Composition of Skim Milk

Skim milk contains a single carbohydrate. It is the disaccharide lactose which consists of one molecule of glucose bound to one molecule of galactose. When milk is autoclaved a tiny amount of lactose is hydrolyzed to glucose and galactose. Since only a few bacteria can use galactose, beginners can ignore the trace of galactose. You may consider autoclaved milk to contain a large amount of lactose and a trace of glucose.

Milk contains two proteins, a trace of lactalbumen and a large amount of caseinogen. Lactalbumen forms the thin skin you have seen on boiled milk. Caseinogen is the main protein in milk and it occurs as fine suspended particles which cause the opacity and white color of milk.

Pure Skim Milk Medium

Use fresh skim milk or add 100 grams of dry skim milk to water to make a final volume of 1 liter. Fill tubes 1/3 full and autoclave 15 minutes at 15 pounds. Experienced bacteriologists sometimes use 10 minutes at 10 pounds pressure or Tyndallization to reduce the hydrolysis of lactose. Tyndallization is simple, place your tubes in a steamer or pressure cooker with a lid but leave the steam ports open. Let the steam flow out of the ports for 30 minutes on 3 successive days. Be sure to begin with enough water because considerable water will be boiled away. If you wish, leave the medium in the cooker for 2 day period--you save effort and avoid re-contamination.

Autoclaved skim milk is the one of the simplest media to prepare but it is very useful. If you have no litmus you can still learn much with pure milk medium. See the discussion under Litmus Milk for ideas. Incubation should be continued for a week or two and the appearance recorded each day. You may see a sequence of reactions. Consider the following uses and experiments for milk medium.

Litmus Milk

This an old standby of the early bacteriologist, but students may not have litmus powder. The colors produced are pale red and pale blue about the color of litmus papers. red = acid; blue = alkaline. Autoclaved litmus milk is a lavender color (not blue or red).

100 g skim milk powder
    5 g Litmus powder - this comes from a lichen
    1 liter of distilled water. Tap water should work.

If a bacterial species, which ferments glucose but not lactose, is inoculated in litmus milk, the litmus may turn pink due to the acid formed from the trace of glucose. The color change will not be great and may only be detectable by comparison to an uninoculated tube of the same lot of litmus milk incubated with the inoculated tubes. If the bacterium is able to produce alkaline products from either protein, ususally the lactalbumen, the color will change back to lavender or even blue.

If the bcterium is able to ferment lactose, a deep, permanent, red color is produced due to the large amount of lactose in milk. Rarely, does the bacterium produce enough alkaline substances to reverse the deep red color to pink, lavender, or blue.

Reactions in litmus milk after inoculaton and during incubation

See b028b for more useful details about the curdling and peptonization of milk, stormy fermentations, and additional details of Litmus and and Ulrich milk.

Milk with Thioglycollate

100 g Skim milk powder
  10 g Peptone
 0.5 g Sodium thioglycollate
    5 g Litmus Powder
    1 Liter distilled water

This medium permits growth of Clostridium species and allows observation of their action on litmus milk. Autoclave at 10 pounds pressure for 10 minutes; then push rubber stoppers in tightly. Screw cap tubes are more convenient.

Many Clostridum species will 'storm' milk without use of thioglycolate

Methylene Blue Milk

100 g Skim milk powder
    1 g Methylene blue (see notes below)
    1 liter of distilled water

This is a professional medium used in identification of enterococci. Students may use 5 mg of methylene blue as in Ulrich milk.

Ulrich Milk

100 g Skim milk powder
    5 mg Methylene blue
  15 mg Phenol red
    1 liter tap water

This medium works exactly like Litmus Milk but is more dependable and probably cheaper. Colors differ from Litmus Milk. Phenol red is red at alkaline pH. Phenol red is yellow at acid pH. Oxidized methylene blue is blue. Reduced methylene blue is colorless. Recall red plus blue is purple. Results are shown in the table:

Reaction Color
Uninoculated medium Medium bluish-gray
Slight acid Pale yellow-green
Acid with reduction Pale yellow-orange
Alkaline Purple clot or milky
Alkaline with reduction Red clot or milky
Alkaline with peptonization Clear, transparent red
or clear purplish

General Comments

37oC ... SO4


First installed Feb 1998      Revision #2 1998 Feb 5       indbio@disknet.com
Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164
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