152 DIFGO MANUAL 



and Chapman and Domingo^ staphylococci incriminated in food poisoning pro- 

 duce an orange pigment, coagulate plasma, ferment mannitol and give a 

 positive "Stone reaction" or gelatinase test on Staphylococcus Medium No. 110 

 when tested at the time of isolation. Velilla, Faber and Pelczar^ suggested the use 

 of both Mannitol Salt Agar and Staphylococcus Medium No. 110 to insure maxi- 

 mum recovery of coagulase producing staphylococci from suspected bovine 

 mastitis. 



Evans^° in his studies of coagulase positive staphylococci reported that 

 pigmentation was much stronger on Staphylococcus Medium No. 110 than on 

 Infusion Agar and some showed pigment on Chapman's medium but were white 

 on the Infusion Agar. Newman,ii in a study of the detection of food poisoning 

 attributable to dairy products, used Staphylococcus Medium No. 100 and Chap- 

 man Stone Medium for the isolation of staphylococci. The coagulase test was run 

 to assist in the detection of pathogenic staphylococci. The cultures for the per- 

 formance of coagulase test were grown in Brain Heart Infusion. 



Following streaking or smearing of the specimens on plates of Bacto-Staphylo- 

 coccus Medium No. 110 the medium is incubated at 30°C.i2 fQj. exactly 48 hours 

 or for exactly 43 hours at 37°C. The colonies are first observed for signs of the 

 production of any orange or yellow pigment. Such colonies are picked from the 

 surface of the medium and emulsified in 0.1-0.2 ml. of Brain Heart Infusion or 

 Bacto-Tryptose Phosphate Broth for the coagulase test. A drop of brom 

 cresol purple indicator is added to several of the areas from which typical pig- 

 mented colonies were removed. Any change in color of the indicator compared 

 with that of the uninoculated medium is indicative of fermentation of mannitol. 

 The plate is then flooded with 5 ml. saturated solution of ammonium sulfate kept 

 in the incubator and let stand 10 minutes. Any clear zone around areas from 

 which colonies have been picked, or around colonies, are gelatinase positive. 



Chapman^^ recently reported that a 20 per cent solution of sulfosalicylic 

 acid (Chapman-Stone Developer) is superior to saturated ammonium sulfate 

 solution for the test. The plate is flooded with the solution, and at the end of 

 10 minutes, a clear zone around the colonies denotes a positive "Stone reaction." 

 The lytic zones stand out as clear areas in an opaque white background. Chap- 

 man^* has also described a method of applying confirmation tests directly to 

 colonies on the medium. Chapman tubes (10 mm. length and diameter) are 

 sterilized and pressed slightly into the surface of the medium. In the cup thus 

 formed is added a drop of 0.04 per cent brom thymol blue. Replace cover on 

 plate and let stand 10 minutes before reading result. The formation of a yellow 

 color indicated a positive reaction, while a green color denotes a negative reac- 

 tion. In a similar manner the "Stone reaction" may be determined by using a 

 19 mm. tube, placing it around the colony to be tested. Add 1 ml. of Chapman 

 Stone Developer (20 per cent sulfosalicylic acid) or saturated Ammonium Sul- 

 fate solution. Examine after standing 10 minutes. A clear zone around the colony 

 denotes a positive "Stone reaction." 



To determine the coagulative power, use a 1 6-24 hour culture of the suspected 

 organism in Brain Heart Infusion or a Brain Heart Infusion suspension of a 

 16-24 hour culture on a slant of Heart Infusion Agar. Two drops of the culture 

 or suspension of the organism is added to 0.5 ml. of Bacto-Coagulase Plasma So- 

 lution. Incubate at 37 °C. Most coagulase positive staphylococci will clot Bacto- 

 Coagulase Plasma Solution within one hour. However, readings should be made 

 after two and three hours of incubation before discarding as negative. A positive 

 culture will show a definite clot often in 20-30 minutes. Chapman clearly states 

 that with few exceptions those colonies that produce pigment, coagulate plasma, 

 ferment mannitol and give a gelatinase reaction are food poisoning staphylococci. 

 According to Chapman^^ this type of staphylococcus may be isolated from only 



