Saburo OHKAWA, Director of Ohkawa Microorganism & Medium Laboratory
Biography of Mr. Ohkawa
Bacillus cereus is an environmental bacterium which is widely distributed in natural surroundings (soil, air, river, etc.) and food/feedstuff of agricultural, fishery, and livestock products. It is a relatively large facultative anaerobic bacillus that forms Gram-positive spore. As it is widespread in nature, it has an increased likelihood of contaminating foods and can cause food poisoning without hygienic handling of food materials and products. Therefore, food hygiene efforts put importance on countermeasures against this bacterium.
The bacterial name, Bacillus cereus, comes from its property of forming wax-like colonies on a medium (the Latin word cereus meanswax in English).
Commercially-available selective media for Bacillus cereus are; NGKG (Nacl-Glycine-Kim-Goepfect) agar, MYP (Mannitol Yolk-Polymixin) agar, PEMBA (Polymixin pyruvate Egg-Yolk mannitol Bromthymol blue Agar), CHROMagar, B.cereus agar, CHROMagar C-BC, etc. Standard Methods of Analysis in Food Safety Regulation of Japan adopts NGKG agar, MYP agar, and PEMBA agar. NGKG is commonly used in Japan, whereas MYP is dominant in other countries. This piece of column will deal with NGKG agar.
NGKG agar (Nacl-Glycine-Kim-Goepfect agar)
NGKG agar 30°C, 24hours
Mossel, et al. first developed a selective medium for Bacillus cereus called MYP agar (1967) , and then H.U. Kim and J.M. Goepfert of University of Wisconsin developed KG medium (Egg yolk-Polymixin medium) which is superior in spore forming of Bacillus cereus (1971). The basal medium of NGKG agar is composed of peptone added with yeast extract. Its selective agents are polymyxin B and glycine which respectively inhibit the growth of Gram-negative bacteria and environmental B.subtilis. Egg yolk is contained to differentiate Bacillus cereus (LV reaction (+)) from other bacteria(LV reaction (-)). Bacillus cereus forms matte, wax-like, rude, wet, and gray to dark-gray colonies with irregular circumference in this medium. White halo is formed due to LV reaction around colonies. Also, their surroundings turn pink.
|Peptic Digest of Animal Tissue||1g|
|Egg yolk (20%)||100ml|
Peptic Digest of Animal Tissue
Essential nutrients for bacterial growth are ①nitrogen source and ②carbon source. Bacteria cannot break down protein unless it is digested or decomposed to be the form of polypeptide or peptide. A substance generated from digestion or decomposition of protein is called peptone.
There are several types of peptones such as casein peptone, soy peptone, meat peptone, myocardial peptone, and gelatin peptone. Peptones are selected according to the agar composition. In NGKG agar, meat peptone (pepsin digestion of meat) is used. Though meat peptone is poor in tryptophan, it is rich in sulfur amino acid, vitamins, and other growth factors.
Yeast extract is not an essential nutritional element for general bacteria to grow. It is usually used as a supplementary nutrition of peptone. Adding extracts will make up for the loss of nutrients and promote bacterial growth. Also, vitamins in extracts are rich in coenzymatic function (increasing enzymatic activity of bacteria).
Sodium chloride is mainly used to maintain uniform osmotic pressure between inside and outside of bacteria. In cell division, rise in cytoplasmic volume and cell wall synthesis are important. During the early stage of culture, the two factors sometimes become unbalanced and cell division progresses with incomplete cell wall synthesis. Protoplast formed in this process easily lyses in hypotonic solution, which can be prevented by adding sodium chloride.
Glycine is added in order to prevent the growth of B.subtilis, which is present in the natural environment with the greatest numbers among Bacillus.
Polymyxin B is an antibiotic with an antimicrobial effect on Gram negative bacilli. It inhibits the growth of glucose fermenters such as intestinal bacteria and non-fermenters of glucose such asPseudomonas aeruginosa. (Polymyxin B has no antimicrobial effect on Proteus and some types of serratia bacteria.)
Agar is used to solidify media. 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 (for its reasonable price). 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. It can maintain water and store nutrition in it. For that reason, 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.
Egg yolk contains lecithin (phospholipid) and triglyceride (lipid). Although Bacillus cereus can break down these two components, many other bacilli cannot. Harnessing this property, egg yolk is added as a selective agent for Bacillus cereus. If lecithin is degraded, circumference of colonies will become white-turbid by lecithin’s crystallization (halo phenomenon). Also, breakdown of triglyceride will make surface of colonies pearlescent, which is called lipovitellin lipase reaction.
① Dilute 10% food emulsion tenfold in phases.
② Deliver 0.1ml of each diluting phase by drops onto NGKG agar and spread it with a bacteria spreader.
③ Culture them for 18-24hours at 30°C.
④ Count colonies with positive egg-yolk reaction and figure out the bacterial count per gram.
1. Bacteria other than Bacillus cereus may grow in this medium and have a positive reaction to egg yolk.
Bacteria other than Bacillus cereus, such as Bacillus anthracis, Bacillus amyloliqufaiens, Bacillus circulans, Bacillus thuringiensis, Bacillus mycoides, S.aureus, Micrococcus luteus, S.saprophyticus, and S.schleiferi, can grow and generate turbidity around colonies (halo) similar to that of Bacillus cereus. Even when typical colonies are formed in this culture medium, a textbook-based test for identification of Bacillus cereus is needed. (See Reference 6)
2. Bacteria other than Bacillus cereus may grow with a negative reaction to egg yolk.
Enterococcus, Listeria spp., S.epidermidis, Serratia marcescens, Proteus spp., and P.aeruginosa grow in this medium and form colonies. However, they have a negative reaction to egg yolk. (See Reference 6)
3. Some types of Bacillus cereus have a negative reaction to egg yolk.
Some types of Bacillus cereus are slow-reacting to egg yolk. They show negative reaction with 24-hour incubation, whereas positive after 48 hours. There are other types of Bacillus cereus that do not react to egg yolk at all. (See Reference 5)
1) Standard Methods of Analysis in Food Safety Regulation ---for Microorganisms---, Japan Food Hygiene Association, 2004.
2) Mossel,D.A, A.,M.J Koopman and E.Jongerius 1967. Appl.Microbiol.18;650-653
3) H.U.Kim.,and J.M.Goepfert,1971 J.Milk Food Technol.34:12-15
4) Sakazaki, R.: New Medium Science, Kindai Shuppan, 1988.
5) H.U.Kim. and J.M.Goepfert,1971.Appl.Microbiol.22:581-587
6) Sandra M.Tallent et.al, 2012 95:446-451
7) Ueda, S.: The Chemical Times, 228:11-18, 2013