In the pour plate method, you need to add a small quantity of sample into a sterile Petri plate using a pipette. The next step is pouring sterile molten agar medium into the plate so that it covers this sample. Rotating the plate a few times in both directions ensures mixing of the agar with the sample. When the agar completely solidifies, the plates are inverted and incubated at the prescribed temperature.
Some of the colony forming units present in the sample migrates to the top of the agar layer on inverting the plate. As the agar solidifies further, other colonies cannot make this transition and remain trapped within the agar gel. These colonies appear as being dispersed within the agar medium. As a result, when you observe the agar plate following incubation, you will observe both surface and subsurface colonies.
Obligate aerobes such as Micrococcus luteus that require oxygen for their growth do not grow within the agar medium. Facultative anaerobes such as E. coli are capable of growing in both aerobic and anaerobic conditions. Microaerophiles like Campylobacter grow with as little as 2 to 10 percent of oxygen. The pour plate method is conducive for the growth of microaerophiles and facultative anaerobes, but not obligate aerobes because colony forming units trapped within the agar gel have no access to oxygen.
The subsurface colonies in a pour plate are difficult to recover and characterize by counting. These colonies are smaller than surface colonies and it is important to observe the plate carefully to ensure you don't miss them while counting. Besides, certain types of bacterial cells are unable to withstand the temperature of the molten agar and may die. If you keep the Petri plate open for a long time when pouring the agar, there is a possibility of environmental contamination that results in surface colonies unrelated to the sample.