Giolitti-Cantoni Broth ISO

Casein peptone and beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group essential for bacterial growth. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Mannitol is the fermentable carbohydrate providing carbon and energy. Lithium chloride inhibits the growth of Gram-negative bacteria. Polysorbate 80 is incorporated to neutralize phenols, hexachlorophene and formalin. The growth of staphylococci is encouraged by sodium pyruvate and glycine. Gram-negative contaminants are inhibited by potassium tellurite.

This method is recommended for products where staphylococci are expected to be stressed and in low numbers such as dried products. Coagulase-positive staphylococci will mostly be Staphylococcus aureus, but Staphylococcus intermedius and some strains of Staphylococcus hyicus are also coagulase-positive.

The confirmation of staphylococci which produce coagulase is based on a strongly positive coagulase reaction, but it is also known that some strains of coagulase-positive staphylococci give weak positive coagulase reactions. These latter strains can be confused with other bacteria but can be differentiated by the use of additional tests such as one for the production of thermonuclease.

Preparation

Suspend 55.2 grams of the medium in one liter of distilled water for the preparation of single strength broth. Suspend 110.4 grams of the medium in one liter of distilled water for the preparation of double strength broth. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Distribute 9 mL portions into tubes for the preparation of single strength broth and 10 mL for the preparation of double strength broth. Sterilize in an autoclave at 121 °C for 15 minutes. Cool to 45-50 °C and aseptically add 0.1 and 0.2 mL per tube of 1% potassium tellurite solution, for single and double concentration respectively.

According to ISO 6888-3

For the enumeration of coagulase-positive staphylococci according to ISO 6888-3:

1. Inoculate a specified quantity of the test portion or the initial suspension for the detection method, or serial dilutions for the enumeration method, in a selective culture medium (Giolitti-Cantoni Broth).
2. Add 1 mL of the initial suspension to 9 mL of single-strength Giolitti-Cantoni Broth.
3. Add 10 mL of the initial suspension to 10 mL of double-strength Giolitti-Cantoni Broth.
4. For larger volumes of test portions, prepare the initial suspension by adding x mL or x g of test portion to 9x mL of the diluent. Then add the entire initial suspension to 90x mL of single-strength Giolitti-Cantoni Broth, previously deaerated and with potassium tellurite added.
5. Incubate the tubes at 37 °C anaerobically for 24-48 hours (carefully pour a plug of agar or paraffin, cooled to between 44 °C and 47 °C, onto the top of the medium and allow it to solidify to form a seal).
6. The presence of presumptive coagulase-positive staphylococci is indicated by the reduction of potassium tellurite (blackening or black precipitate).
7. Subculture the presumptive positive tubes on plates of Baird Parker Agar and incubate at 37 °C for 24-48 hours.
8. The presence of presumptive coagulase-positive staphylococci is indicated by the reduction of potassium tellurite and egg emulsion.
9. Confirm the typical and/or atypical colonies by a coagulase reaction.

• International Standard ISO 6888-3 Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) Part 3: Detection and MPN technique for low numbers.