If colonial characteristics of an organism are to be examined, the petri dish is an excellent container for the medium. The shallowness of the dish and the large surface area render macroscopic examination of colonies easy, and, if necessary, microscopic examination is possible. The dish should be flat-bottomed, and either of heat-resistant soda-free glass or plastic. The most commonly used petri dishes are 90 mm diameter disposable plastic. Glass petri dishes may be sterilized in copper tins which have a deep lid to prevent air penetration on cooling. They should be sterilized in a hot-air oven for 1 h at 160 °C and allowed to cool slowly in the oven.
Plate Inoculation Methods
To isolate single colonies, the medium in the petri dish should be inoculated as follows. Using a sterile loop, smear a loopful of the specimen over area 1. Sterilize the loop in the bunsen flame, and when cool streak over area 2. Repeat over area 3 and 4. Incubate the plates at 370C. The maximum available area should be used but care must be taken not to cross a previously inoculated area.
An alternative method is to use a sterile spreader. This is a glass rod, 3mm in diameter, bent at right angles and sterilized either by boiling, or by wrapping it in Kraft paper and placing in the hot-air oven at 160 °C for 1 hour. A small amount of the specimen is placed on the medium, and smeared over the whole surface using a sterile spreader. With the same spreader, another petri dish is inoculated. Any of the specimens remaining from the first inoculation will be transferred to the second petri dish, and single colonies should be obtained. In both methods, it’s important that the medium surface is dry so that discrete colonies are obtained. The drying of plates is performed by placing the flat surface of the lid onto an incubator shelf at 370C containing dish and angling the media containing dish either within or on the edge of the lid
Tube Cultural Methods
Slope cultures Many tests devised to differentiate organisms require solid cultures. It is not always necessary to grow an organism on a whole petri dish of medium, and slope cultures often suffice. 'Slopes' or 'slants' are tubes or bottles containing a small quantity of medium that has been allowed to solidify with the bottles slightly raised at one end. Such slopes are used only for maintenance or biochemical tests once the organism has been isolated in pure culture. Deep cultures `Anaerobic' organisms require an oxygen-free atmosphere. For cultivation of these organisms 'shake' or 'deep' cultures are sometimes made. The medium is distributed in 150 mm x 20 mm tubes to a depth of 6-7 cm and allowed to solidify. For use, the medium is melted, cooled to about 45 °C, inoculated with the organism, and mixed by rotation between the palms of the hands.
When it has solidified, the culture is incubated and the anaerobic organisms grow at the bottom of the tube. These shake, or deep, tubes can also be used for counts of viable organisms. In similar fashion, the medium is melted, cooled, inoculated with a known dilution of the organism and mixed. It is then poured into a sterile petri dish, and after incubation a count is made of colonies growing in and on the medium.
The 'roll rube' method is also useful for counting viable organisms. The medium is distributed into 6 x5/8 in tubes, 1-2 ml per tube, and stored. For use, the medium is melted, cooled to approximately 500C, and a known quantity of a known dilution of the test sample is added. The tube is then tilted and rolled between finger and thumb, allowing the medium to run all round the sides of the tube just below the half-way mark. This rolling is carried out under cold tap water. A thin film of agar solidifies around the sides of the tube, which is inverted for incubation. Colonies are counted on the following day. By varying the dilution of the bacterial inoculums and taking the mean of several readings, a fairly accurate count of viable organisms in a specimen can be obtained. Commercially made equipment is available for the rolling operation.