Saburo OHKAWA, Director of Ohkawa Microorganism & Medium Laboratory
For food producing companies, quality control tests (microbial tests) are essential. They have to adopt proper and accurate testing methods. To deal with problems that occur during quality tests, the testing staff members are required to have an extensive knowledge and experience in, such as, product property, bacteriology, and limitation of testing methods. However, medium science is still in a black box for many people. In this column series titled “Medium Science Series”, I will offer detailed descriptions of bacteriological testing methods of food from a medium-science perspective.
In this piece of column, let us look at Standard methods agar popularly used to determine viable bacterial count. Standard methods agar was developed in 1953 as a viable count medium for food by Buchbinder and his colleagues at the request of APHA (American Public Health Association). APHA and FDA recommend Standard methods agar as a medium for total viable count.
Standard methods agar
(Apporoximate formula for per Liter)
|Pancreatic digest of casein||
Pancreatic digest of casein
Peptone means “digestion” in Greek. Bacteria require minimum nutrients of nitrogen and carbon sources to grow. As they cannot break down protein on their own, they cannot draw nutrient directly from meat, milk, etc. If protein is broken down into polypeptide by digestive process, however, bacteria can break the peptide form down into nutrients and use them. A substance made through digestion or breakdown of protein is called peptone. There are several types of peptone such as casein peptone, soy peptone, meat peptone, myocardial peptone, and gelatin peptone. In Standard methods agar, casein peptone (pancreatin digestion of the pancreas) is used. Among various types of peptones, we should choose the most appropriate one from a nutritional standpoint (content of amino acid, vitamin, carbohydrate, etc.) and the kind of targeted bacterium. Casein peptone is most frequently used for bacterial medium due to its nutritional and economical superiorities.
There are two purposes for adding Dextrose (carbohydrate) to a culture medium: ①energy obtainment and utilization as a carbon source, and ② identification of bacterial species according to the breakdown process of carbohydrate. As for Standard methods agar, Dextrose is added for the purpose ① (casein peptone contains only a small amount of carbohydrate). Adding glucose will enhance growth of bacteria.
Yeast extract are not necessarily needed for the growth of general bacteria. In Standard methods agar, Yeast extract are generally used as supplementary nutrient to promote growth of bacteria. Since Yeast extract are rich in vitamins, they have an effect to increase enzymatic activity of bacteria (coenzymatic function). Extracts of yeast, beef, potato, etc. are used for media.
Yeast extract ---rich in vitamins, amino acids and minerals--- are applied to Standard methods agar in order to accelerate the growth of bacteria and to supplement the nutrient deficient in casein peptone.
Agar is used to solidify media. As grown bacteria can form isolated colonies in agar, bacteria can be classified and distinguished. Also, total viable cells can be counted in pour cultures.Raw material of agar is seaweed such as Gelidiaceae and Gracilaria vermiculophylla. The latter one is generally used to make agar for a culture medium. A principal component of agar is agarose with linear sugar, which makes bacterial degradation difficult. Agar can contain relatively high weight of water molecules and has a spongy structure. As it can maintain water and nutrition, agar is suitable for a culture medium of bacteria. The temperature at which agar medium begins to melt by heating is called “melting point”, and that melted agar medium begins to solidify is called “congealing point”. The melting point of agar is 85-93 °C and the congealing point is 33-45 °C. These figures vary according to the ingredients mixed into agar. Quality of agar determines quality of the medium. Fine agar has superior transparency, jelly strength, viscosity, and water retentivity.
pH is an important factor to a medium. Optimum pH for many bacteria is 6.8-7.4. At a pH out of this range, bacteria show little or no growth. pH of this medium is 7.0±0.2
Standards methods agar(Dilution plate method)
Although many of the bateria living in food grow well in Standard methods agar, growth insufficiency / inability can occur in the following cases.
1. <Damaged bacteria in food grow insufficiently>
If a cell membrane/wall of bacterium in food is damaged by heating, drying, freezing and/or other production processes, the “damaged bacterium” cannot grow sufficiently in this medium.
Sodium pyruvate is generally used to repair damaged bacteria. Adding magnesium is said to be effective for the repair of S.aureus and iron is for L.monocytogenes. Conditions required for these repair (renewal) processes are different depending on bacteria. There remains much to be learned in the causes of and measures against the damaged bacteria.
2. <Bacteria that cannot grow due to the medium Formulation>
① Bacteria whose growth require specific nutrients cannot grow in the Standard methods agar (ex. bacteria that cannot grow without blood component).
② Some bacteria (such as Bordetella ) cannot grow inhibited by the unsaturated fatty acid in agar. To let them grow, use liquid culture media or add unsaturated fatty acid adsorbent (such as activated carbon) to Standard methods agar.
③ Some bacteria do not grow sufficiently due to the poverty of sodium chloride contained in the medium.Halophiles (vibrio) do not grow without a certain amount of sodium chloride.
④ Some bacteria do not grow sufficiently due to nutritional excess of the medium Formulation (heterotrophic bacteria).
3. <Bacteria that cannot grow due to the medium condition>
① Anaerobes (ex. Clostridium perfringens) and microaerophil (ex. campylobacter) cannot grow in this medium.
② High temperature growth bacteria and psychrophilic bacteria does not grow well.
4. <Insufficient growth caused by the way of testing (for technical reasons) >
Deterioration of powder medium or poor temperature control of incubator or medium during pour might cause insufficient growth.
Buchbinder, L., et al, Am J. Public Health 43: 869-972, 1951.
Barak，E. Ricca and S. M. Cutting: Mol. Microbiol. 55，33 0-338 2005.
Tomochika, K.: “VNC Bacteria and Their Hygienic Problems”, Journal of Antibacterial and Antifungal Agents, 30: 85-90, 2002.
Bloomfield, S.F.: Microbiology. 144, 1-2. 1998.
Sakazaki, R.: New Medium Science, Kindai Shuppan, 1988.