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Technique for Isolation of Pure Culture

In this technique for isolation of pure culture post we have briefly explained about isolation of pure culture techniques, (i) spread plating on solid agar medium with a glass spreader (ii) streak plating with a loop, media, selective conditions, etc.

Isolation of Pure Culture Techniques

Most studies and tests of the physiological, serological and other characters of bacteria are valid only when made on a pure culture. The following methods may be employed for isolating pure cultures of bacteria from mixtures.

Streak Plate

Pure Culture

Isolation of Pure Culture Techniques: Streak Plate

The most common way of isolation of pure culture techniques cells on the agar surface to obtain isolated colonies is the streak plate. It provides a simple and rapid method of diluting the sample by mechanical means. 

As the loop is streaked across the agar surface, more and more bacteria are rubbed off until individual separated organisms are deposited on the agar. 

After incubation, the area at the beginning of the streak pattern will show confluent growth while the area near the end of the pattern should show discrete colonies.

Pour Plate

Another method of isolation of pure culture techniques is the pour plate method. With the pour plate method, the bacteria are mixed with melted agar until evenly distributed and separated throughout the liquid. 

The melted agar is then poured into an empty plate and allowed to solidify. After incubation, discrete bacterial colonies can then be found growing both on the agar and in the agar.

The spin plate method involves diluting the bacterial sample in tubes of sterile water, saline, or broth. Small samples of the diluted bacteria are then pipetted onto the surface of agar plates. A sterile, bent-glass rod is then used to spread the bacteria evenly over the entire agar surface in order to see isolated colonies.

Pure Culture

Isolation of Pure Culture Techniques: Pour Plate

Selective media

Enrichment, selective and indicator media are widely used for isolation of pure culture techniques from specimens such as feces, with varied flora.

i. Selective media

Selective media such as tellurite media for the diphtheria bacillus, have been devised so that, the majority of organisms likely to be associated with those for which the media are used will not grow, and the isolation of pure cultures is thus facilitated.

ii. Enrichment media

Enrichment media such as selenite broth for Salmonella sp, favor the multiplication of particular species as a step towards their isolation in pure culture.

iii. Indicator media

Indicator media, such as Willis and Hobbs medium for Clostridium sp, contain ingredients that change in appearance with particular organisms and so assist their isolation.

Selective treatment

Heating at 65°C for 30 minutes or at higher temperatures for shorter periods, Pre-treatment of specimens with appropriate bactericidal substances are two selective treatment for isolating pure culture.

i. Temperatures

Heating at 65°C for 30 minutes or at higher temperatures for shorter periods: can be used to separate spores from vegetative bacilli but there is no guarantee that spores will germinate under subsequent cultural conditions. 

For example, by heating a mixture containing vegetative and spore forming bacteria at 80°C the former can be eliminated. This method is useful for the isolation of tetanus bacilli from dust and similar sources.

ii. Substances

Pre-treatment of specimens with appropriate bactericidal substances: Pure cultures may be obtained by pre-treatment of specimens with appropriate bactericidal substances which destroy the unwanted bacteria. 

This method is the standard practice for the isolation of tubercle bacilli from sputum and other clinical specimens, by treatment with alkali, acid or other substances to which most commensals are susceptible but tubercle bacilli are resistant.

Growth Conditions

Isolation of pure culture techniques with different temperature optima and Cultivation under aerobic or anaerobic conditions.

i. Temperature

Separation of bacteria with different temperature optima: The temperature and atmosphere chosen for a culture automatically preclude the growth of many bacteria. Incubation at 37°C, used for most medically important bacteria, is too warm for some air contaminants, which subsequently appear as colonies when plates are kept at room temperature. 

Some pathogens are selectively favoured by growth at temperatures above 37°C. Only thermophilic bacteria grow at 60°C. A mixture containing Neisseria meningitidis and Neisseria catarrhalis can be purified by incubation at 22°C when only the latter grows.

ii. Aerobic or Anaerobic

Cultivation under aerobic or anaerobic conditions: Obligate aerobes and anaerobes may be separated by cultivation under aerobic or anaerobic conditions. Strict anaerobes will not grow in air and most facultative anaerobes grow less vigorously under anaerobic than under aerobic conditions. Shake cultures in Veillon tubes were in use formerly but are now obsolete.

Cragie’s tube

Pure Culture

Isolation of Pure Culture Techniques: Cragie’s tube

This consists of a tube of semisolid agar, with a narrow tube open at both ends placed in the center of the medium in such a way that it projects above the level of the agar. 

Inoculation of the mixture is made into the central tube. On incubation, subculture is taken from the surface of the medium in the outer tube because the motile bacteria alone traverse the agar and appear at the top of the medium outside the central tube. 

A U-tube also serves the same purpose, inoculation being performed in one limb and the subculture taken from the other. This method can also be used to obtain phase variants in Salmonella species.

Animal inoculation

Pathogenic bacteria may be isolated from mixtures by inoculation into appropriate animals due to the fact that laboratory animals are highly susceptible to certain organisms for example, the mouse to the pneumococcus. Advances in the development of culture media have now restricted the requirement for animal work to specialist centers.

Examples

Pneumococcus: If a mixture of organisms containing the pneumococcus, e.g. in sputum, is inoculated subcutaneously into a mouse, the animal dies of pneumococcal septicemia in 12 to 48 hours and the organism can be obtained in pure culture from the heart blood. 

Anthrax bacilli: Anthrax bacilli can be distinguished from other aerobic sporulating bacilli by inoculation into mice or guinea pigs. Anthrax bacilli produce a fatal septicemia and may be cultured pure from the heart blood. 

Tubercle bacillus: Similarly, the tubercle bacillus can be isolated from contaminating organisms by inoculation of an infected specimen into a guineapig. The tubercle bacillus is found in a pure state in the resulting lesions. 

Filters

Bacteria of differing sizes may be separated by the use of selective filters. Filters are widely used for separating viruses from bacteria.

Further Readings

Reference

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