Anaerobic media

 Anaerobes require special media where all O₂ must be excluded. These media contain reducing substances or has other mechanisms to ensure absence of oxygen. Special anaerobic media contain reducing agents such as thioglycollate or cysteine which will eliminate any dissolved O₂ remaining in the medium so that anaerobes can grow.

 

Thioglycollate medium

Thioglycolate broth is a multipurpose, enriched, differential medium used primarily to determine the oxygen requirements of microorganisms. It acts as an enrichment broth in diagnostic bacteriology. This broth supports the growth of all organisms with varied oxygen requirements: anaerobes, aerobes and facultative anaerobes.

Obligate aerobes grow only at the top of the tube of medium, microaerophiles in the middle, while anaerobes grow only at the bottom. The medium contains yeast extract, casein, sodium chloride, L-cystine, thioglycollic acid, agar, methylene blue and deionized water at a final pH of 7.2. Dextrose, pancreatic digest of casein, yeast extract, L-cystine provide the growth factors necessary for bacterial multiplication. Thioglycollate and L-cystine in the medium act as reducing agents and create an anaerobic atmosphere. L-cystine and sodium thioglycollate allows Clostridium and other strict anaerobes to grow in this medium even under aerobic conditions. An oxygen indicator turns the medium pink or blue at the top of the tube. This medium is boiled before use to eliminate oxygen, which is less soluble at hot temperatures.

Uses

• Thioglycollate broth is recommended for the cultivation of aerobic, microaerophilic, and anaerobic microorganisms. It allows the differentiation of obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, and aerotolerant organisms. Thioglycollate broth is used to find out the growth characteristics of various bacteria based on their oxygen requirements.
• It is recommended for sterility testing of antibiotics, biologicals, and foods and for determining the phenol coefficient and sporicidal effect of disinfectants.
• When anaerobic infection is suspected, thioglycollate medium is recommended to isolate strict anaerobes from the blood.

Limitations of Thioglycollate Broth

• It is essential that the medium should be freshly prepared or boiled and cooled within four hours of use.
• Storage at lower temperatures increases oxygen absorption.
• Thioglycollate media should not be re-heated more than once because toxic oxygen radicles are formed on reheating.

  

Robertson’s cooked meat media (RCM)

Robertson’s cooked meat medium was originally developed by Robertson for the cultivation of certain anaerobes isolated from wounds. It contains beef heart, the muscle protein, which provides amino acids and other nutrients. Beef heart also contains glutathione, a reducing substance that permits the growth of obligate anaerobes. Cooked meat is added in the medium since the sulfhydryl groups, which give the reducing effect, are more available in denatured protein. Dextrose allows rapid and heavy growth of anaerobic bacteria in a short time and leads to more rapid identification of important anaerobes.

For best results, medium should be used on the day it is prepared, otherwise it should be boiled or steamed for a few minutes and allowed to cool without agitation and then inoculated. Inoculation should be made near the bottom of the tube in the meat particles for anaerobic cultures. Aerobes grow at the top while more anaerobic species grow deeper in the medium.

Robertson’s Cooked Meat (RCM) medium is used to cultivate anaerobic microorganisms, especially Clostridium species.  It is also known as cooked meat broth (CMB) as it contains pieces of fat-free minced cooked meat of ox heart and nutrient broth. It supports the growth of both spore-forming and non-spore-forming obligate anaerobes and also differentiates between putrefactive and saccharolytic species.

Before inoculation, RCM/CMB medium is boiled to make it oxygen-free. After inoculation, it is covered with a layer of sterile liquid paraffin oil to prevent the entry of oxygen into the medium. The medium’s ingredients help maintain the anaerobic (reduced) environment.

1. Unsaturated fatty acids in meat utilize oxygen for auto-oxidation.
2. Glutathione and cysteine (both reducing agents) present in meat also utilize oxygen.
3. Sulfhydryl compounds (present in cysteine) also contribute to a reduced oxidation-reduction potential

The cooked meat medium is inoculated using a swab or wire loop inserted to the bottom of the container.

A saccharolytic reaction is shown by reddening of the meat with a rancid smell due to carbohydrate decomposition. A proteolytic reaction is shown by blacking of the meat with very unpleasant smells due to protein decomposition.

1. Clostridium perfringens: Saccharolytic anaerobes (turn the color of meat pieces into red)
2. Clostridium tetani: Proteolytic anaerobes (blacking of the meat)

Uses of Robertson’s cooked meat medium

1. Cultivation of aerobic, microaerophilic, and anaerobic microorganisms, especially Clostridium species. It supports the growth of both spore-forming and non-spore-forming obligate anaerobes.
2. It is useful as an enrichment broth for cultivating organisms from a very small inoculum.
3. The cooked meat medium preserves the viability of organisms over a long period of time and is useful in maintaining anaerobic stock organisms.
4. The Food and Drug Administration recommends its use in the enumeration and identification of Clostridium perfringens from food.