Agar plate dilution method

You will determine the number of cells in each bacterial suspension by use of the agar plating method. In this method, you spread a small sample of known volume (or a known dilution of such a sample) over the surface of a sterile Petri plate containing an agar-stabilized growth medium (Fig. 20-1). The sample dif-... 

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. 

The agar diffusion method (Kirby—Bauer) is also sometimes used for the evaluation of antibacterial activity of textiles. This is a relatively quick and easily executed semiquantitative method to determine antibacterial activity of diffusible antimicrobial agents on treated textile material. The bacteria are grown in nutrient broth medium and after appropriate dilution (e.g., lOOx) from the culture, test organisms are swabbed over the surface of agar plates. Ten-millimetre-diameter disks of the test fabric and control fabric are then gently pressed onto the surface of the plate. The plates are then incubated at 37 °C for 18—24 h. The antibacterial activity of the fabrics is demonstrated by the diameter of the zone of inhibition in comparison to the control textile sample. 

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). 

Savings in time and materials can be made by removing the need for the dilution series in the standard plate colony count. Various methods based on the Thompson plate loop method have been described. In the technique, two loops that retain 0.01 and 0.001 ml are dipped into the sample. A Petri dish is positioned under each loop and the loops are flushed with diluent, agar is then added, and the contents of the Petri dish are mixed. These machines can plate 10 and 10 dilutions from 300 samples per hour. The technique is suitable for enumerating bacteria in the range 3000-300 000 ml Manufacturers of one commercially available automated plate loop machine (PetriFoss) claim that the relationship with the reference method has a correlation coefficient of 0.99. 

Spread plate method A technique used to prepare pure cultures by placing a diluted sample of cells on the surface of an agar plate and then spreading the sample evenly over the surface. 

Fuel emulsification. Emulsify 1 ml of fuel in 9 ml of Ringer s solution with Tween 80. Prepare serial dilutions of the emulsion in sterile phosphate buffer and pour or streak plate the dilutions. If the fuel is viscous, 0.1ml of fuel can be added to 0.2 ml of the emulsifying agent. Then add 9.7 ml sterile deionized water to make a 1 100 dilution. Plate this solution directly or prepare dilutions for plating on or with agar media. This method has a lower level of detection compared to the filtration method. 

Standard plate count method. Various diluted volumes of the sample are added to a solid agar culture medium and incubated at 35°C for 72 h. The colonies are counted and recorded as the number per miUiUter. 

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