Pure Culture: Best No. 1 Microbiology Concept

Introduction of Pure Culture-Microbiology

The microbial population in our environment is both large and complex. Many different microbial species are normally present in air, water, soil as well as various parts of human or animal body. To study a specific role played by a specific microorganism in the environment, one must isolate the organism in pure. No reliable physiological determination can be made on mixed cultures of bacteria.

The pure cultures, once obtained must be preserved and maintained. These cultures are necessary for further studies, for which the pure cultures must be prevented from contamination by other microorganisms. Whether the isolated organism is pathogenic to humans, animals or plants, or what specific ability the organism possess? Such questions can be studied only after identification of the organism. Thus isolation of pure culture, maintainance and preservation, and systematic study of pure cultures are the important steps in microbiology.

A subject can only develop according to the techniques available. It must be evident from the history of microbiology how important is the suitable methodology in the development of a discipline. In general, three techniques are to be mastered before the science of microbiology could evolve beyound
a primitive visionary state; which include Microscopy, sterilization methods and Pure Culture methods. Once it is possible to obtain suitable sterile growth medium, it becomes particable to introduce methods to separate different microbes from each other and to maintain them in pure culture. Then only it will be possible to study their individual characteristics.

Because microbiology is a new field to most persons in a survey course of this type, the students must be introduced to the new terms and its meanings before we discuss this chapter in detail.When bacteria are transferred from one medium to another the material being transferred to another the material being transferred is called the INOCULUM and the resultant growth, whether it be in a liquid or a solid medium, is termed a CULTURE or SUBCULTURE.

If the transfer is made to a solid medium, the visibel growth which appears after a suitable incubation period is a COLONY. A PURE CULTURE consists of a population of only one species of microorganism, all derived from a single parent microorganism. CALTIVATION of microorganisms in the laboratory is accomplished by providing necessary nutrients in a medium and a favourable environment for their growth. The separation of one kind of microorganism from a mixture of many different kinds is called the ISOLATION or PURE CULTURE TECHNIQUE.

A pure culture, sometimes called an axenic culture, is the growth of a single type of microorganism in environment free of any other kind of living thing. Many microbiologists prefer to use the term “axenic” in order to avoid the misunderstanding that pure cultures are genetically pure.

A pure or axenic culture is not genetically pure; rather ,it is a population of microorganisms of the same species that may contain some individual with mutation.Such genetically different individuals occur in populations of all kinds. But a small degree of genetic variation does not usually interfere with the identification or understanding of a species because the mutants are such a small portion of the group being tested.


Till 1860, no good method of isolation of organisms in pure culture was known, Joseph Lister (1827 – 1912) physicist, physician and pioneer in antiseptic surgery, described a method of isolating bacteria from aqueous suspensions by successive dilutions to the point that the highest dilutions would contain only one of the desired organism. To determine this point, however, was extremely difficult.

In 1872, Schroeter, a microbiologist, had observed the growth of different bacteria in isolated colonies of various colours on the slices of decaying potato with his microscope, he observed that all the micro-organisms in any one colony were always exactly the same. It was, thus confirmed that a solid nutrient surface is necessary to obtain isolated colonies of any single kind of organism. In this case, each isolated colony represents a pure culture.

Use of solid nutrient surface Media for Pure Culture


Extending this principle, the search was undertaken for the substances that will help to prepare a moist, sticky nutrient surface on glass. In 1880, Koch used 5-10% gelatin, various nutrients and test substances to prepare a transparent, solid jelly on flat pieces of glass. In summer, however, the gelatin melted. Being a protein it was often digested and liquefied by many microorganisms, especially molds. However, modified in technical details, it is currently used in pure culture study of viruses, cells of cancer and of human, plants, and insects.


In 1881 Mrs. W. Hesse, suggested agar agar (commonly called agar) as a substitute for gelatin. This gum is a polysaccharide derived form the seaweed-Gelidium spp., which was used at that time in making jellies. For most bacteria, it is wholly indigestible and has no nutritive value. Agar is transparent and colorless, melts in water only at boiling temperature and once melted, does not set again until approximately body temperature (40°C). It is now commonly used in culture media in 1.5 to 3.0 percent concentrations to form fairly stiff gels.

Soon after the first uses of agar as solidifying agent, it was discovered that several important species of chemolithotrophic microorganisms of the soil are inhibited by the presence of an organic substance such as commercial agar or impurities present in it. A number of heterotrophic species of micro-organisms of the sea and soil readily digest and liquefy agar. Hence there was a need to search for an inorganic solidigying agent. Silica (SiO2) was found to be an acceptable substitute for agar to prepare silica gel. Preparation of silica gel is not difficult but requires attention to details such as pH, temperature, concentration silica and various ions.


Several substances other than agar, gelatin or silica gel are widely used to prepare solid microbiological media. Serum, blood and whole mixed eggs, which coagulate readily on heating at 80°C or above, are often used either alone of as the basis for mixtures. In addition, slices of potato, bread, carrot, as well as pieces of meat can serve as natural solid nutrient media for cultivating numerous species of microorganisms.


In order to prevent contamination of the pure culture by dust, R.J.Petri, Koch’s student, suggested the simple method of pouring the melted nutrient medium into circular, shallow dishes and immediately covering them with a glass cover. This permitted prolonged examination of the cultures and excluded contamination. Such dishes are widely used today and are called Petri plates.

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