Methods of isolating Pure Culture
A number of techniques have been employed for the isolation of microorganisms from natural environments. Culture methods employed depend on the purpose for which they are intended. The indications for culture are mainly to
-isolate bacteria in pure culture.
-demonstrate their cultural characteristics.
-Obtain sufficient growth for tests.
-determine sensitivity and/or resistance to test substances,
-estimate viable counts and
-maintain stock cultures.
The basic principle of all is the provision of a type of physical environment and a medium where the desired organism will grow ideally,
The methods of isolationg pure culture are as follows –
1st Method of isolating pure culture
1 THE STREAK PLATE :-
Plating is the most widely used method for isolating the cultures. Streak plates are prepared by streaking a small amount of the diluted sample over the surface of the solid medium in a petri dish with a platinum or nichrome wire loop. The sample is streaked in such a way as to provide successive dilution of the sample.
Streaking may be done in any one of the ways shown in FIGURE 1 and when properly performed, the bacterial cells will be sufficiently far apart insome areas of the plate to ensure that the colony developing from one cell will not merge with that growing from another. The plate is incubated at proper temperature for overnight so that the transferred organism grow and multiply to give isolated colonies. Each isolated colony is the progeny of a single cell and hence a PURE CULTURE.
Figure 1. Different ways of streaking on agar plates. The arrows indicate the direction of streaking needle.
2nd Method of isolating pure culture
2. SPREAD PLATE PURE CULTURE TECHNIQUE:-
In the spread plate technique, a serially diluted inoculum is transferred over the surface of the solid medium in petridish. It is then spread uniformly with a sterile bent glass rod in order to separate aggregates of cells in the sample which on incubation give isolated colonies. A portion of a colony transferred to a tube of medium becomes a pure culture. Growth from each culture should be checked microscopically and culturally to verify that it is a pure culture.
Spread plate technique is shown in Figure 2
Figure 2. Streak Plate Method
The streak-plate and spread plate techniques can be performed easily and with a minimum amount of sample. These are now routine procedures for isolation of bacteria in pure culture. However, one of the limitations is that only a small amount of the specimen can be spread over the surface of the medium.
3rd Method of isolating pure culture
3. POUR PLATE PURE CULTURE TECHNIQUE:-
The principle of the pour plate technique is dilution of the specimen in the tubes of liquified or melted agar medium. Since the number of microorganisms in the original specimen is not known, it is necessary to dilute the culture to obtain well-isolated Colonies. An aliquot of diluted inoculum is added in the tubes of melted and cooled (45 to 50 C) agar medium, mixed well and the contents of the tubes are then poured into sterile petriplates, and allowed to solidify.
The plates are then incubated. Alternatively, an aliquot of the diluted sample may be first added to sterile petriplate followed by addition of melted and cooled nutrient agar medium and mixing the sample with medium by slowly rotating the plate. After solidification, the plates are incubated.
A series of agar plates showing decreasing number of colonics resulting from the dilution procedure in the pour-plate technique is shown in Figure 3.In this technique, some of the organisms are traped beneath the surface of the medium when it solidifies. Thus, pour plates exhibit both surface and subsurface (within the agar) colonics. The subsurface colonies (also known submerged) are smaller than the surface colonies of the same species and their shape is often different. This may have difficulty in cultural characteristics; the undesirable feature of this technique.
Figure 3.Steps of pour plate technique used for pure culture isolation of bacteria.
The streak plate, spread plate and pour plate techniques can be made more effective for isolating specific kinds of bacteria using selective or differential culture media. It is also possible to treat the specimen before plating to eliminate unwanted bacteria. For example, to isolate spore forming bacteria, the sample can be first exposed to 85° C temperature for 5 mins. and then plated.
Since sporeformers are heat resistant, only nonsporelarmers will be destroyed, and any colony that develop is likely to be sporeforming bacterial species.
4th Method of isolating pure culture
4. SERIAL DILUTION PURE CULTURE TECHNIQUE:-
An organism that predominates in a mixed culture can be obtained as a pure culture by serial dilution. A mixed culture is serially diluted in the test tubes. of sterile medium to the point of extinction in the number of cells so that the last tube contains only a single organism. A single cell is sufficient to initiate growth in an appropriate medium. The purity of the culture isolated in this fashion is confirmed by a plating procedure. This technique was originally described by Lister. The major drawback of this technique is that one can never be sure that the last dilution tube will always contain a pure culture.
Figure.4 Serial Dilution Method
5th Method of isolating pure culture
5. ENRICHMENT PURE CULTURE TECHNIQUE:-
This is one of the special methods used for the isolation of a specific type of microorganism from a mixed population. This approach to the isolation of microorganisms from soil was introduced by Beijerinck and Winogradsky between 1890 and 1900. The method is often used if the microorganism to be isolated is present in relatively small numbers, or its growth is slow with respect to other species present in the mixed sample.
The enrichment culture provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the conditions of incubation. This will favour the growth of the particular type of microorganism, but will be unsuitable for the growth of other types. The procedure increases the proportion of the desired microorganism by application of the law of natural selection.
Let us assume that we want to isolate bacteria capable of utilizing cellulose, a complex polysaccharide. If we inoculate nutrient agar directly with the sample, our chances of finding cellulose-degrading bacteria will be limited. The rapidly growing common bacteria, present in large number in the specimen, will overgrow the desired type. Thus we employ the enrichment culture technique and prepare liquid medium consisting of inorganic salts, an inorganic nitrogen source and cellulose as the only source of carbon.
Bacteria unable to utilize cellulose will not grow. Allow this medium to incubate for few days with sample and then transfer a small amount from it into another flask of fresh medium of the same composition. Since cellulose is the sole carbon source available, in the successive transfers only organisms capable of utilizing cellulose grow. A sample from the medium can then be streaked on a medium of the same composition solidified with agar. The cellulose degrading bacterium will develop into colonies. The enrichment culture technique is shown in fig.5.
Thus this technique can help in the isolation of practically any desired organism from the natural environment. This technique is particularly very useful in isolating organisms which can degrade a variety of chemical pollutants that acculumate in the environment especially in soil and water.Some cultural conditions employed for enrichment cultures of various typical organisms are mentioned in Table.Some cultural conditions employed for enrichment cultures.
Figure 5.ENRICHMENT PURE CULTURE TECHNIQUE.
SPECIAL ISOLATION METHODS:-
The methods discussed are the most commonly used methods for majority of microbes. There are also some special methods which are used only for restricted type of microbes. These methods include:
6th Method of isolating pure culture
6. USE OF SELECTIVE MEDIA/ PURE CULTURE MEDIA
Selective medium contains specific chemicals which suppress or kills unwanted types of microorganisms, while allows the desired type to multiply For example, crystal violel or brilliant green inhibit gram positive bacteria and thus help in the isolation of gram negative bacteria. Similarly, sodium azide at specific concentrartion into media selectively isolates latic acid bacteria. The choice of selective agents and their concentrations varies with the kind of organisms to be isolated and the kinds or organisms to be suppressed. Thus, certain dyes are sued for brucellas, cetrimide for Pseudomonas aeruginosa tetrazolium for halophytic bacteria, bile salts for intestinal organisms etc.
7th Method of isolating pure culture
7.USE OF DIFFERENTIAL MEDIA:
These media contain dyes, reagents or chemicals which allow the observer to distinguish between types of bacterial colonies, after incubation, on the basis of colony charactiristics. For example, on a differential medium such as Eosin- Methylene Blue Agar, Escherichia coli and related species produce colonies with a brilliant green metallic sheen. However, on the same medium
Enterobacter aerogenes and related species produce pink colonies with dark centres.
8th Method of isolating pure culture
8. SINGLE CELL ISOLATION :-
The ideal way to obtain a pure culture from a mixture of microorganisms would be to pick up individual cells of the desired type. Individual cells of algat and protozoa, pieces of algal and fungal filaments or spores of fungi, cas be removed from natural materials, such as water rotting vegetation and soil suspensions. The material is placed in a watch glass or a petridish and observed through the microscope. Filaments can be removed from the material with forceps, and single cells or spores with capillary pipettes & transferred to the medium for growth.
The MICROMANIPULATOR is a special equipment which can be used in conjunction with a microscoope to pick a single bacterial cell from a hanging drop preparation. The micromanipulator has micrometer adjustments by means of which micropipettes or other instruments can be moved in any directions. A series of hanging drop preparations of diluted culture are placed on a special sterile cover slip by a micropipette which has a bent tip.
A second micropipette is inserted from below into one of the hanging drops which has been observed to contain only a single bacterium. The cell can be drawn into the pepelle by gentle suction and then transferred to a tube of broth; to get pure culture. As the equipment is expensive, this method is not generally used for isolation. Further, its manipulation is very tedious and requires a skilled operator. This technique is reserved for use in highly specialised studies.
9th Method of isolating pure culture
9.USE OF LABORATORY ANIMALS :-
Some animals are known to be extremely susceptible to specific organisms. For example, the mouse can be injected with a mixture of many organisms, but if a virulent Precumococcus is present in it, it is quite possible for the natural destructive forces of the mouse to dispose of all of the injected bacteria except the Pneumococcus.
This organism and its progeny may eventually destroy the mouse. An examination of the peritoneal cavity of the mouse immediately after death will frequently reveal a pure culture of the Pneumococcus. The guinea pig serves as a similar filter for separating the Mycobacterium tuberculosis bacterium from sputum of the suspected patient Thus, a susceptible animal can be used to obtain pure culture of some pathogens
10th Method of isolating pure culture
10. ISOLATION OF ANAEROBES :-
Anaerobic bacteria can be isolated either by pour-plate technique using a medium containing a reducing agent such as thioglycolate and incubating under anaerobic conditions or using the roll tube technique. In the roll tube technique an appropriate dilution of the inoculum is added to the molten, reduced agar medium and the tube is rolled in such a way that a thin layer of medium is formed on the inner wall of the tube.
The tubes are sealed with butyl stoppers and gassed with oxygen-free CO2 or Nitrogen. The advantage of this method is that complete anaerobic conditions are maintained with less risk of contamination and drying during the prolonged incubation periods which is necessary because of the slow growth of obligate anaerobes. So this are the methods of isolating pure culture in microbiology.