Media formulations and explanations:

ACTINOMYCETE ISOLATION AGAR

Component	Amount (grams/L)
Sodium propionate	4.0
Soluble starch	10.0
Sodium caseinate	0.4
Potassium nitrate	2.0
Sodium chloride	2.0
Dipotassium phosphate	2.0
Magnesium sulfate (7H2O)	0.05
Calcium carbonate	0.02
Ferrous sulfate (7H2O)	0.01
Agar	18.0

pH 7.5-7.8

Use:

For the isolation of Streptomyces from soil. Streptomyces and molds are especially equipped with extracellular enzymes to hydrolyze the starch and casein to obtain carbon, nitrogen and energy. (Potassium nitrate serves as an supplemental nitrogen source.) Sodium propionate is added to discourage growth of molds. Streptomyces will be the predominant organism after several days of incubation at room temperature or 30°C, and the colonies are very distinctive.

AMINO ACID DECARBOXYLASE BROTH

Component	Amount (grams/L)
Peptone	5.0
Beef extract	5.0
Glucose	0.5
Brom-cresol purple	0.01
Cresol red	0.005
Pyridoxal	0.005

pH 6.0

Use:

For the determination of amino acid decarboxylase activities of the enteric bacteria. The desired amino acid (lysine, ornithine or arginine) is added to the above basal medium to a 1% final concentration prior to sterilization. The inoculated medium is overlaid with several ml of sterile mineral oil such that the organisms will respire all of the oxygen in the medium (now sealed against oxygen entry) and render it anaerobic. Fermentation of the glucose (to acidic products) will result in the bromcresol purple turning yellow unless decarboxylation of the amino acid also occurs, in which case a net alkaline reaction (purple color) will be seen. For each organism tested, a control tube of the basal medium (i.e., without the amino acid) is always inoculated. Failure to achieve an acid reaction in the basal medium means that the organism is probably a strict aerobe (therefore not an enteric) and that results in the test media are meaningless.

BRILLIANT GREEN LACTOSE BILE (BGLB) BROTH.

Component	Amount (grams/L)
Peptone	10.0
Lactose	10.0
Oxgall (a brand of bile salts)	20.0
Brilliant green	0.0133

pH 7.2

Use:

A highly-selective, lactose-containing broth medium which will support the growth of many gram-negative organisms such as coliforms and Pseudomonas. Lactose is a carbon source used by all coliforms and bile salts selects against gram-positive bacteria. With rare exceptions, coliforms are the only organisms which will grow in this medium and also produce gas from lactose fermentation at 35-37°C. Used in the confirmatory test for total coliforms.

EC BROTH

Component	Amount (grams/L)
Tryptose (a rich peptone)	20.0
Lactose	5.0
Bile salts	1.5
Dipotassium phosphate	4.0
Monopotassium phosphate	1.5
Sodium chloride	5.0

pH 6.9

Use:

A highly-selective, lactose-containing broth medium which will support the growth of many gram-negative organisms such as coliforms and Pseudomonas. The medium is especially selective because of the high temperature.. With rare exceptions, fecal coliforms are the only organisms which will not only grow in this medium but also produce gas from lactose fermentation at 44.5°C. Used in the confirmatory test for fecal coliforms.

EOSIN-METHYLENE BLUE (EMB) AGAR (LEVINE'S FORMULATION).

Component	Amount (grams/L)
Peptone	10.0
Lactose	10.0
Dipotassium phosphate	2.0
Eosin Y	0.4
Methylene blue	0.065
Agar	15.0

pH 7.1

Use:

For the detection and isolation of gram-negative bacteria. Used in the completed test for isolation of individual coliforms to be identified by IMViC and other tests. Eosin Y and methylene blue serve as selective agents, inhibiting gram-positive bacteria. These materials also serve as the pH indicator. Lactose-fermenting colonies will produce acid, resulting in a dark color. Non-lactose-fermenters produce pinkish or lavender colonies.

FLUID THIOGLYCOLLATE BROTH

Component	Amount (grams/L)
Yeast extract	5.0
Casitone	15.0
Glucose	5.0
Sodium chloride	2.5
Cystine	0.5
Sodium thioglycollate	0.5
Resazurin	0.001
Agar	0.75

pH 7.1

Use:

Mainly for the routine cultivation of anaerobes such as Clostridium. Occasionally as an all-purpose broth medium. Also used as a base for Thioglycollate Agar and Thioglycollate Medium .

Before inoculation, the medium is steamed (to melt the agar and drive out oxygen), cooled to 45-50°C, and then inoculated. Cystine and sodium thioglycollate are included in the medium to maintain a low oxidation-reduction potential. Agar decreases the diffusion of oxygen into the medium. Resazurin is used as an indicator of oxygen; this compound is pink or red in the oxi­dized state and colorless when reduced. (The color may not be seen if good growth is present.) Glucose is included as a fermentable energy source although some clostridia can ferment amino acids in the yeast extract and casitone; any of these organic compounds may be respired by various aerobes and facultative anaerobes.

GLUCOSE O/F MEDIUM.

Component	Amount (grams/L)
Peptone	2.0
Glucose	10.0
Sodium chloride	5.0
Dipotassium phosphate	0.3
Brom-thymol blue (don't make up in ethanol)	0.08
Agar	2.0

pH 6.8

Use:

For the detection of acid not only from fermentation of glucose but also from respiration. Thus, many strictly aerobic bacteria (such as most strains of Pseudomonas) will grow at the top of the medium and produce acid from glucose respiration. Fermenting bacteria will produce more acid that will cause the entire tube to turn yellow. Glucose O/F usually inoculated in duplicate (by single-stab inoculation) with one of the tubes receiving an overlay of several ml of mineral oil. Used only for gram-negative bacteria; inhibition of others is probably due to the concentration of brom-thymol blue.

KLIGLER IRON AGAR (KIA).

Component	Amount (grams/L)
Beef extract	3.0
Yeast extract	3.0
Peptone	15.0
Proteose Peptone	5.0
Lactose	10.0
Glucose	1.0
Ferrous sulfate	0.2
Sodium chloride	5.0
Sodium thiosulfate	0.3
Phenol red	0.024
Agar	12.0

pH 7.4

Use:

Used only for gram-negative bacteria, mainly enterics and Pseudomonas. Aids in the primary differentiation of enterics. Differentiation can be made between organisms which ferment both lactose and glucose or only glucose or neither sugar. Organisms which produce hydrogen sulfide from the reduction of thiosulfate are easily detected; the H₂S reacts with the iron in the medium to produce ferrous sulfide, a black precipitate. Medium is inoculated with the needle, first stabbing down the center to the bottom of the tube and then streaking up the slant. The various combinations of reactions are explained and illustrated.

LACTOSE LAURYL TRYPTOSE BROTH (LLTB; sometimes called "LST Broth").

Component	Amount (grams/L)
Tryptose	20.0
Lactose	5.0
Dipotassium phosphate	2.75
Monopotassium phosphate	2.75
Sodium chloride	5.0
Sodium lauryl sulfate	0.1

pH 6.8

Use:

A moderately-selective, lactose-containing broth medium which will support the growth of most common gram-negative organisms including coliforms and Pseudomonas. The selective agent in this broth is sodium lauryl sulfate, which will inhibit many organisms. Coliforms will grow in this medium, fermenting the lactose and producing visible gas at 35-37°C. Medium is used in the presumptive test for total coliforms; a tube showing growth and gas leads to the presumption that at least one coliform was initially inoculated into the medium. As medium contains no strong inhibitory agents - as it is formulated to be friendly to organisms possibly damaged in the environment (from inhibitory chemicals or osmotic problems caused by suspension in low-solute-containing water) - some gram-positive organisms may also grow, including occasional (i.e., relatively rare) strains of Bacillus and Clostridium which ferment lactose to acid and gas, giving rise to a false-coliform-positive reaction in this medium.

MacConkey Agar

Component	Amount (grams/L)
Peptone	17.0
Proteose Peptone	3.0
Lactose	10.0
Bile salts	1.5
Sodium chloride	5.0
Neutral red	0.03
Crystal violet	0.001
Agar	13.5

pH 7.1

Use:

For the detection and isolation of gram-negative organisms, especially the enterics and Pseudomonas. This medium is used as the primary example of a selective-differential medium; Other sugars may be used in place of lactose, depending on specific experimental requirements. Medium may be modified to allow detection of hydrogen sulfide; see next item.

MacConkey Agar, MODIFIED

Formula: After autoclaving the ingredients of the above medium in 900 ml distilled water, 100 ml of the following filter-sterilized solution are added: 6.8% sodium thiosulfate+0.8% ferric ammonium citrate.

Use: The primary purpose of this medium is to illustrate how some media used in hospital, food and industrial labs not only isolate enterics (and similar organisms) and differentiate them according to fermentation reactions, but also differentiate according to production of hydrogen sulfide. H₂S produced by the organism from the thiosulfate will react with the iron, resulting in ferrous sulfide, a black precipitate which is seen in the centers of the colonies.

MINERAL SALTS SOLUTION FOR PHOTOSYNTHETIC MICROORGANISMS

Component	Amount (grams/L)
Magnesium sulfate (7H2O)	0.2
Dipotassium phosphate	1.0
Sodium chloride 0.5
Ferrous sulfate (7H2O)	0.01
Calcium chloride	0.02
Manganese chloride (4H2O)	0.002
Sodium molybdate (2H2O)	0.001

pH not to be adjusted

Use:

Used as a basal medium for the enrichment and cultivation of photosynthetic microorganisms. The medium may be supplemented and/or incubated in various ways depending on the particular group of organisms being studied. As used in Experiment 11.1 for purple non-sulfur photosynthetic bacteria: succinate, yeast extract and ammonium chloride are added as nutritional supplements; agar is also added for use in plates. The succinate is a non-fermentable carbon source, thus preventing many microbes from using it under anaerobic conditions.

MOTILITY INDOLE ORNITHINE (MIO) MEDIUM.

Component	Amount (grams/L)
Yeast extract	3.0
Peptone	10.0
Tryptone	10.0
L-ornithine hydrochloride	5.0
Glucose	1.0
Brom-cresol purple	0.02
Agar	2.0

pH 6.5

Use:

For differentiation of enteric bacteria. Medium is carefully tab-inoculated through the center of the medium to the bottom of the tube. Three reactions are assessed using this medium. The medium contains a glucose as a carbon source that is fermented to acid by enteric bacteria, turning the bottom of the tube yellow. If the microbe is capable of ornithine decarboxylation, a net alkaline reaction will occur in the bottom of the tube and it will appear grey, blue, or purple. Indole is tested by adding Kovac's reagent, with a red ring indicating a positive reaction. Finally the medium has a low concentration of agar and a motility test is preformed by determining the cloudiness of the medium. A turbid tube indicates motility, while a clear tube is a non-motile organism.

MR-VP BROTH

Component	Amount (grams/L)
Buffered Peptone	7.0
Glucose	5.0
Dipotassium phosphate	5.0

pH 6.9

Use:

For the performance of the Methyl Red and Voges-Proskauer tests with the addition of appropriate reagents.Tests must be read after two days of incubation at 37°C. A half-dropperful of methyl red reagent is added to the tube. A red color is a positive reaction, indicative of mixed-acid fermentation; pH has dropped and remained at or below 4.4.A yellow color is a negative reaction, indicative of butanediol fermentation; pH has dropped and then risen to 6.2 or above. An Orange color is a equivocal reaction.

NITRATE BROTH

Component	Amount (grams/L)
Beef extract	3.0
Peptone	5.0
Potassium nitrate	2.0

pH 7.0

Use:

With the use of a Durham tube in the medium and appropriate reagents, reduction of nitrate to nitrite or nitrogen gas can be detected. A bubble in the Durham tube indications the reduction of nitrate in the Nitrate Broth all the way to nitrogen gas (i.e., denitrification). To test for the reduction of nitrate only to nitrite, add a dropperful of each of the two nitrite reagents - sulfanilamide and N-(1-naphthyl)-ethylenediamine - to each of the tubes. Any appearance of a red color (which may or may not persist) indicates the presence of nitrite. If no red color was seen, add a few more drops of each reagent, mix well, and observe again. If no indication of gas or nitrite is seen, the nitrate may not have been reduced, or it was reduced to a product for which we are not testing. One can check for unreduced nitrate by adding a small amount of granulated zinc to any tube not showing a positive reaction for nitrite or gas. Mix the tube well. If nitrate is present, it will be reduced by the zinc to nitrite which will react with the reagents already added, forming a pink or red color. (Disregard any gas generated with the addition of the zinc.) Do not record for the organism any reaction you see only upon addition of the zinc.

NITROGEN-FREE BROTH AND AGAR

Component	Amount (grams/L)
Sucrose or Mannitol	5.0
Dipotassium phosphate	0.8
Monopotassium phosphate	0.2
Magnesium sulfate	0.2
Calcium sulfate	0.1
Ferrous sulfate	0.003
Molybdic anhydride	0.001
Calcium carbonate	2.0
Purified agar (optional addition)	12.0

pH 6.8-7.2

Use:

For the cultivation and isolation of Azotobacter and other non-symbiotic nitrogen-fixing bacteria. As there are no fixed nitrogen compounds in the medium, the only organisms which should grow as pure cultures are those which can obtain and utilize N₂ from the atmosphere. Calcium carbonate is included to buffer acid produced from fermentation of the sugar by many facultatively anaerobic and strictly anaerobic nitrogen-fixers - such as many strains of Klebsiella and Clostridium. (Acid-producing colonies can be detected by a clearing of the white CaCO₃ precipitate.) In Appendix D, this medium is given as an example of one made selective by the deletion of an important nutrient. Note its use in Experiment 11.3.

PHENYLALANINE AGAR

Component	Amount (grams/L)
DL-phenylalanine	2.0
Yeast extract	3.0
Sodium chloride	5.0
Sodium phosphate	1.0
Agar	12.0

pH 7.3

Use:

For differentiation of enteric bacteria based on ability to produce phenylpyruvic acid from phenylalanine. With rare exceptions, only members of the Proteus Group of enterics (Proteus, Providencia, and Morganella) will give a positive result. A half-dropperful of FeCl₃ reagent is added to the tube and the immediate presence of a green color is a positive test. This test is much more definitive in helping to identify members of this group than is the traditional test for urea hydrolysis. Must be read (with the addition of the appropriate reagent) after just 1 day of incubation at 37°C.

SIMMONS CITRATE AGAR

Component	Amount (grams/L)
Magnesium sulfate	0.2
Monoammonium phosphate	1.0
Dipotassium phosphate	1.0
Sodium citrate	2.0
Sodium chloride	5.0
Brom-thymol blue	0.08
Agar	15.0

pH 6.8

Use:

For the differentiation of gram-negative bacteria (especially enterics) on the basis of citrate utilization. If citrate, the sole source of carbon and energy, is utilized, an alkaline reaction results, and the brom-thymol blue indicator turns a bright metallic blue.

References:

1. http://inst.bact.wisc.edu/inst/index.php?module=Book&func=toc&book_id=3