Triphasic mycoplasmatales detection method

Claim 1

A method for detecting Mycoplasmatales in a sample with a triphasic culture device comprising a container having first and second, opposed, transparent sidewalls, an opening and a closure means for sealing the opening, the inner wall surface of the first sidewall being covered with an adherent layer of Mycoplasmatales nutrient agar medium, the container also containing a Mycoplasmatales liquid nutrient medium and a gas phase, the nutrient agar medium being spaced apart from the liquid nutrient medium with the gas phase in between the mediums when the first sidewall is positioned in a horizontal plane above the second sidewall, the distance between the outer surfaces of the first and second sidewalls and the surface quality of the first sidewall permitting microscopic examination of the nutrient medium through the first sidewall, said method comprising (a) introducing inhibitory quantities of a thallium salt and a cell-wall inhibiting antibiotic through the opening in the container, inoculating the Mycoplasmatales nutrient agar medium and the Mycoplasmatales liquid nutrient medium with a sample, and closing the opening to seal the container; (b) incubating the container at a temperature of from 35° to 37° C. for at least 48 hours with the container oriented to maintain the nutrient agar medium spaced apart from the liquid nutrient medium with the gas phase between the mediums; (c) inoculating the Mycoplasmatales nutrient agar medium with the Mycoplasmatales liquid nutrient medium in the container without unsealing the container; (d) reincubating the container at a temperature of from 35° to 37° C. for at least 48 hours with the container oriented to maintain the nutrient agar medium spaced apart from the liquid nutrient medium with the gas phase between the mediums; and (e) examining the Mycoplasmatales nutrient agar medium through the first sidewall with a microscope without unsealing the container to detect Mycoplasmatales colonies therein.

Claim 2

A method for detecting Mycoplasmatales in a sample with a triphasic culture device comprising a container having first and second, opposed, transparent sidewalls, an opening and a closure means for sealing the opening, the inner wall surface of the first sidewall being covered with an adherent layer of Mycoplasmatales nutrient agar medium, the container also containing a Mycoplasmatales liquid nutrient medium and a gas phase, the nutrient agar medium being spaced apart from the liquid nutrient medium with the gas phase in between the mediums when the first sidewall is positioned in a horizontal plane above the second sidewall, the distance between the outer surfaces of the first and second sidewalls and the surface quality of the first sidewall permitting microscopic examination of the nutrient medium through the first sidewall, said method comprising (a) introducing inhibitory quantities of a thallium salt and a cell-wall inhibiting antibiotic through the opening in the container, inoculating the Mycoplasmatales nutrient agar medium and the Mycoplasmatales liquid nutrient medium with a sample, and closing the opening to seal the container; (b) incubating the container at a temperature of from 35° to 37° C. for at least 48 hours with the container oriented to maintain the nutrient agar medium spaced apart from the liquid nutrient medium with the gas phase between the mediums; (c) inoculating the Mycoplasmatales nutrient agar medium with the Mycoplasmatales liquid nutrient medium in the container without unsealing the container; (d) reincubating the container at a temperature of from 35° to 37° C. for at least 48 hours with the container oriented to maintain the nutrient agar medium spaced apart from the liquid nutrient medium with the gas phase between the mediums; and (e) examining the Mycoplasmatales nutrient agar medium through the first sidewall with a microscope without unsealing the container to detect Mycoplasmatales colonies therein.