Hayflick Agar Base

Principle And Interpretation

Mycoplasma, represents a group of minute bacteria devoid of cellwalls belonging to class Mollicutes (1). These are responsible for causing serious contamination in cell and or tissue cultures used to generate compendial articles. They may also cause contamination of filtered sterilized Soyabean Casein Digest Broth. Infection of cells in a culture can affect nearly every pathway of cell metabolism including alteration of the cells phenotypical characterization and normal growth. The presence of mycoplasma species does not always result in turbid growth in cultures or visible alteration of the cells. Hayflick et al have reported complex medias for growth of Mycoplasmas (2,3). Testing of mycoplasmas is necessary to assure reliably pure biotech products and allied materials used to generate these products. Hayflick Agar media (solid) is recommended for general detection of Mycoplasmas in Pharmacopoeias (4,5) for testing of products for Mycoplasma. When testing for Mycoplasmas, at least two known Mycoplasma species or strains as positive controls, one of which should be dextrose fermenter (i.e. M. pneumoniae or equivalent species and strain) and one of which should be an arginine hydrolyzer (i.e. M. orale or equivalent species and strain) should be included in each test. Only when testing insect cell lines should one include a Spiroplasma control strain (e.g., S.citri ATCC 29747, S. melliferum ATCC 29416, or equivalent species and strains.) Additionally these strains may be a little more fastidious in their nutritional requirements. They require lower incubation temperatures (as do insect cell lines).

This medium contains BI Broth containing HM infusion powder and peptone which provides nitrogen, vitamins, aminoacids and carbon sources. Sodium chloride maintains the osmotic balance. Many Mycoplasmas require serum for their good growth. Addition of Penicillin supress growth of unwanted flora. Phenol red in the medium indicates the growth of Mycoplasma on change of colour of medium from red to yellow or purple. Added Horse serum provides growth factors including lipid components to Mycoplasma. DNA provides additional nutrients to Mycoplasma.Yeast extract serves rich source of Nicotinamide-Adenine Dinucleotide (NAD) required by M.synoviae. Agar acts as a solidifying agent. Mycoplasma species are either aerobic or facultative anaerobic but some are microaerophilic. Few are anaerobic saprophytic Mycoplasma which grow best at 22-35°C while pathogenic strains grow at 35°C. Tubes should be incubated in an atmosphere containing 5-10% carbon dioxide and examined after incubation of 48 hours but they should not be discarded as negative until after incubation for three weeks. M. synoviae is not able to grow on Hayflick broth medium because growth depends on NAD. M. hyorhinis ATCC 29052 which is recommended as a fastidious strain for use in indicator cell method, is not able to grow on this medium.