Granada Agar Base

The production of an orange carotenoid pigment is a unique characteristic of Streptococcus agalactiae and serves as the basis for several media used for its detection and identification.

Proteose peptone No. 3 provides nitrogen, vitamins, minerals and amino acids essential for growth. Soluble starch in the medium acts as a growth factor, probably functioning like a colloid protector, and neutralizes toxic products that form during the development of the organisms. It also enhances the pigment formation. Dextrose is the fermentable carbohydrate providing carbon and energy. Magnesium sulphate is a magnesium ion required in a big variety of enzymatic reactions, including DNA replication. MOPS hemisodium salt and disodium phosphate anhydrous act as a buffer system. Crystal violet inhibits Gram-positive bacteria. Bacteriological agar is the solidifying agent. Methotrexate, added to the mixture, acts as a pigment enhancer. Colistin sulphate and metronidazole inhibit undesired flora.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 78.2 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 100 °C for 7 minutes. Cool to 50-55 °C and add 10 mL of a sterile solution (containing 6 mg of methotrexate, 5 mg of colistin sulfate and 10 mg of metronidazole in sterile water) and 50 mL of sterile horse serum. Mix well and pour into Petri dishes or tubes.

Instructions for use

Streak plate method:

1. In a Petri dish, add 12-15 mL of molten agar and let it solidify.
2. Inoculate 10 µL of the initial suspension and/or diluted sample.
3. Spread the inoculum with a sterile loop on the agar surface.
4. Incubate the plates in an inverted position at a temperature of 35 ± 2 °C for 18-24 hours.

• De la Rosa M., Perez M., Carazo C., Pareja L., Peis J.I., Hernandez F., 1991. New Granada Medium Detection and Identification of Group B Streptococci. Journal of Clinical Microbiology. Apr 1992, p 1019-1021
• Schuchat, A. 1998. Epidemiology of group B streptococcal disease in the United States. Clin. Microbiol. Rev. 11: 497-513
• Islam, A.K.M. 1977. Rapid recognition of group B streptococci. Lancet I: 256-257.
• Pritzlaff, C.A., et al. 2001. Genetic basis for the beta-haemolytic/cytolytic activity of group B Streptococcus. Molec. Microbiol. 39: 236-247.