Introduction to Enrichment and
Selective Media for Bacteria

You select or enrich the desired bacterium by providing the food and conditions it needs.

When you isolated bacteria by streaking or pour plate methods, you found you could only isolate the majority bacteria from a sample. If a bacterium is present in a population at a low level such as one percent or fewer, then it is not likely to be found by streaking or some other method for isolating a colony that grew from a single bacterium. For example if you are looking at creek water for bacteria from septic tanks, such bacteria may be rare and seemingly absent.

Each bacterium has the enzymes to grow in certain environments. By reading about a bacterium in books or on the Internet, you should be able to make a medium that is favorable for it and hostile to many other bacteria. Some bacteria grow very slowly, but after a couple days to a few weeks, you should have an enriched culture.

Sucessive enrichments. Once you have suitable medium and conditions for a bacterium, 1 or more successive passages may be useful.

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The items below are being used as references for writing this page.

This page will have some simple introductory enrichment expts.

Pond mud set in the light. perhaps milk, pH affects enrichment. desulfo-bacteria, rocks-sulfur-water,

see oxy relations expt. methods for handing anaerobes to be written under advanced methods.

meat, brain, egg, liver, can add to TGY and other bases. add agar o gel or use as broth.

see page 022 E-broth for methods.

Some Links to Indiana Biolab Enrichments

203 - Vibrio phosphoreum - bioluminescence, 4o C, seawater
204 - Desulfo-bacteria - provide sulfate and anaerobic conditions
mm - Bacillus - heat kills veg cells but not spores, the air for growth
mm - Clostridium - heat kills veg, then anaerobic conditions.
mm - Janthinobacterium - rice grain on soil in refrigerator; nice purple bacterium

Some Links to Enrichments on the Web

Composition of meat extract

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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

First installed February      Revision #2 1999 Sep 29       H2SO4  1022m  90oC
Bacteria Index Page | INDIANA BIOLAB Web Sites |
Written by Harold Eddleman, Ph. D., President, Indiana Biolab, 14045 Huff St., Palmyra IN 47164