All procedures should be carried out on a Class 2 safety bench or cabinet using gloves.

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1 Use one non-nutrient agar plate for each test including a control. Allow the plates to warm up to RT.

2 Pipette about 0.5 ml Page's saline onto the blood agar plate with the E. coli culture. Emulsify some of the growth with a 10^l bacteriological loop. Place three to four drops of bacterial suspension onto each non-nutrient plate. Spread out the bacterial suspension with a 10^l loop and allow the fluid to absorb into the agar.

3 Inoculate each specimen in the centre of a separate E. coli - seeded plate. Fluid specimens are first centrifuged at 500g, the supernatant removed and the deposit inoculated onto the plate. Avoid flooding the plate with liquid inoculum. Incubate the inoculated plates inverted at 30°C in a moist chamber, for example, a plastic bag containing some moistened absorbent tissue.

4 Before setting up the control strain, swab the working surfaces with 70% alcohol.

5 Culture the Acanthamoeba reference strain by cutting a 1cm2 piece of agar from the plate with a sterile scalpel. Place it upside down in the centre of a seeded non-nutrient agar plate. Incubate as for the clinical specimens but in a separate moist chamber.

All plates are incubated for 1 week and examined daily.

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