Use of a plating method to estimate extracellular protein production by staphylococci

Employing 4% NZ-Amine type NAK medium fortified with thiamine and niacin and solidified with 0.75% agar, staphylococci were streaked onto this medium and incubated for 24 hr at 37 C. After the gentle removal of growth with cotton swabs, the plates were flooded with 25% trichloroacetic acid and allowed to stand for about 15 min, after which various degrees of protein precipitate were noted under the areas of growth. Organisms that produced the most intense precipitate or extracellular protein (ECP) were designated 4+, followed by 3+, 2+, and 1+ for those that produced progressively less ECP to 0 for those that produced none. Of 585 strains of coagulase-positive staphylococci tested, 91% produced 3 to 4+ ECP, whereas only 8.9% of 112 coagulase-negative strains produced these amounts. The NZ-Amine media including type A were far superior to five other media in permitting ECP production. Of other organisms examined, only the three salmonellae species tested produced ECP. Maximum visual quantities of ECP were produced between 22 and 24 hr at pH values between 6.6 and 7.0 and in the presence of <3% NaCl. The visual estimation of ECP production correlated well with the production of certain other specific extracellular products of staphylococci. This plate method provides a simple means of characterizing staphylococci on the basis of extracellular protein production.