The following points highlight the top four techniques used for the isolation of microbial mutants. The techniques are: 1. Direct Observation 2. Enrichment Technique 3. Replica-Plating Technique 4. The Ames Test.
1. Direct Observation Technique:
In some cases, a colony growing on an agar plate can easily be seen to be different from the normal parental type (wild-type). For example, if the parental strain is pigmented, the observation of non-pigmented colonies may indicate the presence of mutants. Indicators can also be incorporated into the medium to detect microorganisms with and without particular metabolic capabilities.
For instance, pH indicators can be used into the medium to detect the production of acidic products. The indication of acid production by one microbial strain and not by other of the same microorganisms growing under identical conditions would show the presence of a mutant.
2. Enrichment Technique:
Enrichment technique is employed especially in isolating mutants resistant against phages, antibiotics, or toxic chemicals. Phage-resistant mutants can be isolated simply by plating the mutagenized, phenotypically expressed microbial population on plates containing phage particles.
Cells expressing the parental wild-type phenotype are killed; only phage-resistant mutants develop into colonies. Such colonies are isolated. Similarly, mutants resistant to an antibiotic or a toxic chemical can be isolated by plating the microbial population with the antibiotic or the chemical.
3. Replica-Plating Technique:
Replica-plating technique is often used to isolate nutritional mutants (auxotrophs) as well as various other type of mutants, e.g., antibiotic resistant mutants.
For convenience, if one wants to isolate nutritional mutants employing replica-plating techniques (Fig. 29.13), he is required to follow the following steps:
(i) Bacterial cultures are diluted, and the cells are spread on the surface of semisolid nutrient agar medium in a Petri dish (called “master plate”). The medium in the master plate is a complete medium i.e., containing all the nutritional components required by the bacterial population. After a sufficient incubation period, each bacterium produces a visible colony on the surface of the agar in the master plate.
(ii) A piece of sterile velvet cloth is stretched over a cylindrical block of wood or metal that is slightly smaller in diameter than the Petri dishes used in the process.
(iii) The master plate is now inverted and gently pressed onto sterile velvet. Since the fibres of the velvet act as fine inoculating needle, some cells from each colony of the master plate stick to the velvet.
(iv) Other Petri dish (called “replica plate”) is taken containing a minimal medium i.e., a medium deficient with specific nutritional component,
(v) The replica-plate is now inverted and gently pressed onto the velvet thus stamping the bacterial cells onto the surface of its minimal medium. The replica plate is identically oriented at the application on the velvet with respect to mark placed on its rim so that the colonies that appear on the replica plate after incubation occupy positions congruent with those of their siblings on the master plate.
(vi) After sufficient incubation, it is observed that a colony that develops on the complete medium of the master plate fails to develop on the minimal medium of the master plate that lacks a specific nutritional component. Such colony is marked on the master plate and is isolated; it represents mutant for that specific nutritional component not used in the minimal medium of the replica plate.
The replica-plating technique was developed by Joshua and Esther Lederberg in 1952 in order to provide direct evidence for the existence of pre-existing mutations originated spontaneously in microorganisms.
4. The Ames Test:
This test was developed by Ames and coworkers and is based on histidine-requiring (his–) auxotrophic mutants of Salmonella typhimurium. Different his– mutants carry different types of mutations, i.e., transitions, transversions and frame- shifts.
In the Ames test, the frequency of reversion to his+ (prototrophy) is scored in the especially constructed his– mutants. This is done by placing a known number of mutant cells on medium lacking histidine and scoring the number of colonies formed. The frequency of cells forming colonies gives the frequency of reversion. The frequency of spontaneous reversion to his+ is quite rare, i.e., 10-8.
Ames test is routinely used to investigate the mutagenicity of various chemicals. Some of the chemicals may become mutagenic only when they are acted upon by liver enzymes.
For example, nitrates themselves are neither mutagenic nor carcinogenic. But in eukaryotic cells, nitrates are converted to introsamines, which are highly mutagenic and carcinogenic. In addition; some chemicals may be mutagenic only to replicating DNA.
The routine Ames test addresses to both these needs as follows:
1. The his– cells are plated onto a medium that contains traces of histidine, which is enough to allow a few cell divisions, but inadequate for visible colony formation.
2. The test chemical is incubated with rat liver extract containing the liver enzymes, i.e., the microsomal fraction. This allows modification of the chemical in the same way as it would be in the liver of animals.
The procedure of Ames test is as follows. The his– bacterial cells are incubated with the liver extract, and then plated onto a medium containing traces of histidine; this serves as the control plate.
The test plates contain the same medium but the his– cells are not treated with liver extract. The test chemical is treated with the rat liver extract and a filter paper disc is soaked in this solution. The filter paper disc is placed onto the medium of test plate (Fig. 29.14).
The chemical present in the filter paper acts on the his– cells growing in the test plate. The frequency of colonies formed in the control plate and the test plate are compared. An increase in the frequency in the case of test plate will indicate the test chemical to be mutagenic.
In order to increase the efficiency of the test, the his– strains used in the test are defective in DNA repair, and have increased permeability to chemicals. It has been observed that more than 90% of the chemicals that are mutagenic are also carcinogenic.