Methods in Microbiology
In order to get a pure culture. It is necessary to first achieve an increase in the relative number of species, preferably to the point where the species becomes the numerically dominant component of the population. This can be achieved by using the growth of desired species, while killing or inhibiting others. There are following three categories of methods in order to obtain particular kind of organism:
1. Physical methods
2. Requirement of Gases
3. Chemical methods
4. Biological methods
1. Physical conditions for growth
i. Heat Treatment
This is applied in case of bacteria which form spores for example Bacillus spp. Lactobacillus spp. Etc. to select the endospore-forming bacteria, a mixed culture can be heated at 80⁰ C for 10 min. before being used to inoculate culture medium. In such case spore forming bacteria will survive and all the bacteria will be killed.
ii. Incubation temperature
Most of the microorganisms require a particular temperature to grow efficiently. Such a temperature is called optimum temperature. To select psychrophilic or psychrotrophic bacteria, cultures are incubated at extreme low temperature varying zero to 5⁰ C. the mesophilic (20-40⁰C) and thermophilic (45⁰C and above) microbes will be killed, and only psychrophiles will survive.
It is important factor which influence the growth of microorganisms. Most of the lactobacilli spp. grow at acidic range of pH (below 7) while some bacteria grow at alkaline medium e.g. Vibrio cholera.
iv. Cell Size and Motility
Most of the microorganisms has a definite size and are motile in nature. Due to 0.15 mm size Treponema can be isolated by placing gingival scrapping on the surface of an agar plate. It will penetrate from the filter and will swim through solid agar medium. The hazy appearance in the agar indicates the presence of Treponema.
2. Requirement of Gases
Oxygen and carbon dioxide are the gases which affect bacterial growth. Aerobic bacteria require about 21% oxygen while anaerobic bacteria do not require oxygen as it is toxic to them. Facultative anaerobic bacteria may use it for growth. They are not inhibited by oxygen. Microaerophilic bacteria require low levels of oxygen for growth but cannot tolerate the level of oxygen present in an air atmosphere. This is due to inactivation of enzymes, reduced thiol (-SH) group. On the other hand, various cellular enzymes catalyze chemical reactions, involving molecular oxygen. Sometimes addition of a single electron to an oxygen molecule forms super oxide radical (O2).
The inactivation of cell component is also observed due to production of even more toxic chemical substances such as hydrogen peroxide () and hydroxyl radicals of the reaction given below:
Aerobic and facultative microbes have protective mechanism against oxygen due to super oxide dismutase. The () produced can in form the dissipated by catalase and peroxidase enzyme.
- Chemical Methods
The chemicals select a particular microorganism. If it is supplemented in a medium only those microorganisms will grow well which can utilize the supplemented chemicals. This particular kind of selection is based on addition of either carbon or nitrogen source. Such method is often called enrichment culture and medium is called enrichment medium.
In addition certain dyes such a s rose Bengal are often used for the isolation of fast growing fungi, while certain antibiotics such as nystatin is added to avoid bacterial contaminants in the medium. Similarly, yeast mannitol agar (YEM) medium supplemented with congo red allows to differentiate rhizobia with that of Agrobacterium. Rhizobia do not take colour while Agrobacterium colonies become ping. Many Gram – positive bacteria do not grow in the presence of dyes due to their inhibitory action while Gram-negative bacteria grow well in the presence of dyes.