VMC 311: Systematic Veterinary Bacteriology and Mycology (2+1)

CULTURAL, BIOCHEMICAL CHARACTERISTICS AND RESISTANCE

• Mycoplasmas are highly fastidious organisms and most require specific growth factors, an isotonic medium and the absence of inhibitory substances for growth.
• Mycoplasma and Ureaplasma require reduced sterol or cholestrol for their growth.
• Because the Mycoplasma are unable to synthesise purines and pyrimidines, they require complex media.
• The agar which contains bovine heart infusion, 20% horse serum, (Pooled serum from several animals), 10% yeast extract, 20ml of adenine dinucleotide, 50 units of penicillin and 0.25 mg of thallous acetate (inhibitory to Gram –ve and fungi) and the optimal pH is 7.5.
• Most grow aerobically but some require N2 with 5% to 10% Co2 after 2 to 6 days of aerobic incubation at 370C, colonies on solid media are 0.1 to 0.6 mm in d.m.
• under low power magnification, the colonies appear transparent, flat and often resemble a fried egg.
• Colonies grow into the medium and are difficult to remove from the agar surface.

• The colonies are best studied by stained with Diene's method
• A block of agar containing microcolonies is placed, colony side upwards, on a microscopic slide.
• A light film of Diene’s stain (alcoholic solution of methylelne blue and azure) is placed on a coverslip and allowed to dry.

• This is then put, stain-slide downwards on the microcolonies on the agar block then examined under low power of microscope.
• The dense center of the microcolonies, which grow down into the agar, stain dark blue, the less dense peripheral zone, resembling surface growth, stains light blue.
• Most mycoplasmas are haemolytic in swine blood agar. Mycoplasmas may grow in chicken embryos (yolk sac route) and cell cultures.
• Ureaplasma produce tiny colonies (T-mycoplasma) than the conventional mycoplasmas

Biochemical characters

• Mycoplasmas are chemoorganotrophs, the metabolism being mainly fermentative.
• Most species utilize glucose or arginine as the major source of energy.
• Urea is not hydrolysed except by ureaplasmas.
• They are generally not proteolytic.

Resistance

• Mycoplasmas are more fragile because of the absence of cell wall.
• Drying, sunlight and the usual disinfectants readily kill them.
• They are resistant to penicillin and sulfonamides.