Plate count media preparation

Trypsinization is performed as described above. Cells in the suspension are counted and prepared in a suspension containing 4.2-6.7 x 104 cells/cm2. To each apical well of the 24-well plate 400 til of the cell suspension are added apically. The Feeder Tray contains 40 ml of complete medium (feeding cells from the basolateral side). 

Desoxycholate Agar is recommended by APHA for the counting of coliforms in dairy products. APHA recommend placing a sample (1 ml) in the petri dish and preparing a pour plate of medium. Once this has set, a further 5 ml of uninoculated medium is poured over the plate. Plates are incubated at 35 °G for 18-24 hours and coliform numbers can be estimated by counting all dark red colonies. Enteric bacteria which are non-lactose fermenters form colourless colonies, whilst non-enteric bacteria are inhibited by the desoxycholate. 

Prepare a cell suspension in fresh growth medium from a flask of cells in exponential growth and count cells. Dilute cells to density of 104 cells /mL (see Note 1) allowing 30 mL of cell suspension for 3 plates. Transfer the cell suspension to a 10 cm Petri dish and with a multichannel pipet add 100 pL to the central 10 columns of a round bottomed microtitre plate. Add 200 pL of growth medium to the wells in columns 1 and 12. Put plates in a plastic box and incubate in a humidified atmosphere at 37°C while drug solutions are prepared. 

The agar plate method was used to determine the minimum inhibition concentration (MIC) of CM, Q, and CMQ as follows the samples were prepared at a concentration of 1% (w/v) and then autoclaved at 121°C for 25 min. Duplicate twofold serial dilutions of each sample were added to nutrient broth (beef extract 5 g, peptone 10 g to 1000 mL distilled water, pH 7.0) for final concentration of 0.1%, 0.05%, 0.025%, 0.0125%, 0.00625%, and 0.00313%. Some samples were prepared and diluted by the same way except for a final concentration of 0.00065% and 0.00033%. The culture of each bacterium was diluted by sterile distilled water to 105-106 CPU mL. A loop of each suspension was inoculated on nutrient medium with sample or control added. After inoculation, the plates were incubated at 37°C for 72 h, the colonies were counted, and the MIC values were obtained. The MIC was considered to be the lowest concentration that completely inhibited against on agar plates comparing, disregarding a single colony or a faint haze caused by the inoculum (Speciale et al. 2002). 

Another procedure for dealing with samples insoluble in counting solution is to support them on a medium such as paper strips, filter discs, glass fibre or DEAE cellulose prior to adding them to a counting vial [235-237]. As indicated earlier, use of this has been in chromatography where the spot has been cut out of the paper or scraped from the plate. Although useful for materials insoluble in scintillator fluid, self-absorption for tritiated samples may constitute a major drawback in this technique, just as it does for the suspension methods. It should be evident why the recent developments in in combustion procedures are so important in the problem of sample preparation. 

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