Molten sterile medium

In this method, 25 ml of a molten sterile medium is poured into presterilised Petri dishes and allowed to solidify at room temperature (RT). The agar plates are seeded with 0.1 ml of (1 X 10 spores/ml) of a fungal/bacterial culture using the spread plate method. Subsequently, 10 mm wide wells are bored into these agar plates using a sterile cork borer. 250 pi of a stock solution of an antimicrobial compound is filled into the wells and the plates are incubated at 25 2 °C. The antimicrobial activity is interpreted from the size of the zone of inhibition(s) (ZOI) measured to the nearest millimetre, i.e., the clear zones surrounding the wells, as shown in Figure 11.1 [20-23]. 

Pour plate method. An aliquot of the neat or diluted sample is added to a sterile Petri dish and mixed with the selected molten agar medium. When the agar has solidified, the plates are incubated for a predetermined time at the specified temperature. Surface or subsurface colonies will develop in some of the agar plates, which can be counted to provide a quantitative value for the bacterial density of the original sample. They can also be picked for further qualitative study. 

Dispense 120 ml. volumes into 250 mL conical flasks and add 2 g. of agar to each. Plug the flarits and autoclave them at 120°C. for 15 minutes. The sterile medium is stored at 4°C. and has a storage life of 4 to 6 weeks. When required for use, the flasks are steamed for 30 to 40 minutes to melt the agar, and then 10 ml. of glucose solution is added to each. The glucose is sterilized separately as a 13% w/v solution for 15 minutes at 120°C. The molten medium is allowed to stand in a water bath at a temperature of 46-48°C. 

This medium was prepared by adding sterile defribrinated sheep blood (10%) to sterile molten nutrient agar (Oxoid) at 55 °C. 

The fuel water bottom can be plated directly onto the surface of solid agar media or pour plated with molten (cooled to 50°C) liquid agar. If the microbial counts are suspected to be high, the water bottom should be diluted to 10 to 10 in an appropriate medium such as sterile phosphate buffer prior to plating. Sediment (0.01 to 0.1 g wet weight) can be dissolved in 100 ml of a suitable dilution broth prior to agar plating (Swift, 1988). 

Strains were inoculated into liquid YPD medium and grown overnight at 37°C. The cells were then pelleted and washed three times with distilled water. Approximately lO cells/ ml were inoculated in molten agar media at 40°C and poured into 100-mm-diameter petri plates. The test materials initially dissolved in 1% DMSO were further diluted in distilled water to concentration ranges of 10-fold of their respective MICs. 4-mm-diameter sterile filter discs were impregnated with the test compounds as reported previously [61]. 1% DMSO (solvent) and 100 pg/ml of fluconazole were also applied on the discs to serve as negative and positive controls, respectively. The diameter of zone of inhibition was recorded after 48 h and was compared with that of control. The type of halo produced depicts fungicidal/static characteristic of test entity. 

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