Isolation and Cultivation of Microorganisms

In this article we will discuss about the isolation and culture of microorganisms.

Isolation of Microorganisms:

Microorganisms occur in natural environment like soil. They are mixed with several other forms of life. Many microbes are pathogenic. They cause a number of diseases with a variety of symptoms, depending on how they interact with the patient. The isolation and growth of suspected microbe in pure culture is essential for the identification and control the infectious agent.

The primary culture from natural source will normally be a mixed culture containing microbes of different kinds. But in laboratory, the various species may be isolated from one another. A culture which contains just one species of microorganism is called a pure culture. The process of obtaining a pure culture by separating one species of microbe from a mixture of other species, is known as isolation of the organisms.

Methods of Isolation:

There are special techniques employed to obtain pure cultures of microorganisms. In few cases it is possible to secure pure culture by direct isolation or direct transfer. This can be done only in those situations in which pure culture occurs naturally. Kinds of specimens taken for culturing will depend on the nature and habitat of microbes.

Different pathogens can be isolated from body tissues and fluids such as blood, urine, sputum, pus, faces, spinal fluid, bile, pleural fluids, stomach fluids etc. In the blood stream of a patient suffering with typhoid fever, the bacteria Salmonella typhosa may be present.

A pure culture of this bacterium may be obtained by drawing blood sample using a sterilized hypodermic syringe and treating the blood with anticoagulant such as heparin and potassium oxalate. The presence of the anticoagulant prevents the pathogenic microbe from entrapping in fibrin clot. The sample of the blood may be inoculated into a suitable medium.

Following isolation methods are employed to isolate microbes from mixed cultures:

1. Streaking

2. Plating

3. Dilution

4. Enriched procedure, and

5. Single cell technique.

1. Streaking:

This is most widely used method of isolation. The technique consists of pouring a suitable sterile medium into sterile petriplate and allowing the medium to solidify. By means of a sterile loope or straight needle or a sterile bent glass-rod a small amount of growth preferably from a broth culture or bacterial suspension is streaked back and forth across the surface of agar until about one third of the diameter of the plate has been covered.

The needle is then flamed and streaking in done at right angles to and across the first streak. This serves to drag bacteria out in a long line from the initial streak. When this streaking is completed the needle is again flamed and streaking is done at right angles to the second streak and parallel to the first.

2. Plating:

It includes diluting of a mixture of microorganisms until only a few hundred bacteria are left in each millilitre of the suspension. A very small amount of the dilution is then placed in a sterile petriplateby means of a sterile loop or pipette. The melted agar medium is cooled to about 45°C and is poured into plate. The microorganism and agar are well mixed. When the agar is solidified the individual bacterium will be held in place and will grow to a visible colony.

3. Dilution:

This method is used for the microorganisms which cannot be easily isolated by streaking or plating method. Sometimes when several organisms are present in a mixture, with one organism predominating, the predominating form may be isolated by this method. For example, when raw milk is allowed to sour at room temperature it will, at the time of curding, have a mixture of microorganisms with high percentage of Streptococcus lactis.

If 1 ml of the sour milk is taken into a tube containing 9 ml. of sterile milk (in which no organisms are present) then 1 ml. of this mixture is transferred with a sterile pipette into a second tube of sterile milk and the procedure is repeated i.e. from second to third tube, third to fourth tube until a series of about 10 tubes are inoculated. By this serial dilution, the chances are that a pure culture of S. lactis will be obtained.

4. Enrichment Procedure:

This procedure involves the use of media and conditions of cultivation which favour the growth of the desired species. For example, when a man suffers with typhoid, the intestinal discharge posses small number of Salmonella typhosa when compared with E. coli and other forms.

It is almost impossible to isolate the typhoid organisms because they represent only a fraction of a per cent of the total microorganisms present. The media are therefore derived, which allow the rapid growth of the desired organisms, at the same time inhibiting the growth of other microorganisms.

5. Single Technique:

This is one of the most ideal and difficult method of securing pure culture. In this method a suspension of the pure culture is placed on the under-side of a sterile cover-glass mounted over a moist chamber on the stage of the micros­cope.

While looking through the microscope, a single cell is removed with the help of sterile micropipette and transferred to a small drop of sterile medium on a sterile cover-glass and is mounted on a sterile hanging drop slide, which is then incubated at suitable temperature. If the single cell germinates in this drop, few cells are transferred into a tube containing sterile culture medium which is placed in the incubator to obtain pure culture originated from single cell.

Other methods:

The isolation of anaerobic microorganisms is very difficult. There are certain special techniques by which these organisms are isolated.

Cultivation of Microorganisms:

For cultivating microbes in laboratory, we require culture media. The various mixtures of nutritive substances used for the laboratory cultivation of microorganisms are collectively known as culture media. The culture media serve as soil in which bacteria are planted for the purpose of study.

Culture Media:

Culture media must contain all the essential nutrients required by the organism for its growth and reproduction. A suitable source of energy, building materials and growth factors must be supplied in adequate amounts.

So a culture medium must contain:

Since microorganisms show a considerable variation in their nutritional requirements, no single medium is suitable for growth of all of them.

The different types of culture media employed fall into three groups:

1. Defined or synthetic media:

These are the media prepared from chemical compounds. They are highly purified and specific, an investigator working in another laboratory can duplicate them.

2. Complex or non-synthetic media:

Media that are prepared from ingredients that have not been precisely defined. It contains hydrolysed proteins and vitamin extracts. This type of medium cannot be duplicated by another worker in another laboratory. Peptone is usually produced by boiling beef, by the hydrolysis of its protein. Casein peptone and milk peptone are also used in complex media as the source of amino acids and nitrogen.

All liquid media, whether complex or synthetic may be converted to solid media by adding either gelat in (a protein) or agar-agar, (a complex polysaccharide) extracted from red marine algae. The use of agar has an advantage. The most bacteria are unable to hydrolyze this molecule into more simple components. Since gelatin is a liquid at room temperature, the use agar allows the medium to remain in a solid form while microbes are growing on its surface.

3. Living cells:

These are used for the cultivation of viruses. For example, fertilized eggs incubated for 8 to 12 days are able to support the growth of many viruses.

In another classification culture media are grouped into following four types:

1. Natural media:

Includes substances occurring in nature, as 1) Milk 2) Eggs 3) Blood 4) Extract of plant and animal tissues.

2. Derived media:

Includes known substances but the chemical composition of each is unknown. Examples are 1. Nutrient broth (prepared by boiling beef to extract nutrients and adding an amino acid-nitrogen source.) 2. Nutrient agar 3. Nutrient gelatin.

3. Chemically defined media:

Exact chemical composition known.

4. Special media:

Include combinations of the other three types of media.

There are four categories of media used in laboratory:

They are:

1. Enrichment

2. Selective

3. Differential and

4. Propagation.

1. Enrichment media:

They are prepared with ingredients that will enhance the growth of certain microbes. Enrichment media encourage the growth of the suspected pathogen so that it will become the most pre-dominant type of microbe in the culture. Two types of enrichment media are blood agar and chocolate agar.

2. Selective media:

They are prepared with ingredients that inhibit the growth of unwanted microbes which might be in the specimen. The inhibitor may be an antibiotic, salt or other chemical. Mixed culture of microbes originally grown in enrichment media may be inoculated into selective media to isolate the desired microbe.

3. Differential media:

They are designed to differentiate among microbes. Different bacterial species may produce dissimilar colony colours when grown on differential agar. While in differential broth cultures, the media change colour. Differential media are used to confirm the identity of a microbe that has already been isolated by culturing in enrichment and selective media.

4. Propagation media:

They are used to propagate, or keep microbes growing for a long lime. Samples grown on these media may be taken for analysis. The most common propagation media are nutrient broth and agar.

Preparation of Media:

There are three main steps in the preparation of media:

(a) Preparation as solutions of chemicals and adjusting the pH.

(b) Dispensing the media, and

(c) Sterilization.

A broth is prepared by dissolving the appropriate amount of the components in distilled water and pH is adjusted by the addition of either dilute NaOH or Hcl. The broth is dispensed into clean rimless ‘Pyrex’ test tubes which are plugged with non-absorbant cotton wool plugs. The test tubes are placed in wire baskets which are covered with grease proof paper.

The media are sterilized by autoclaving at a temperature of 121 °C and a pressure of 151 b/in² for 15 minutes. But medium containing heat- sensitive substances are sterilized either by filtering the solution at room temperature, using bacteria-proof filter or by a process called Tyndallization.

In this method, the liquids are steamed for one hour a day on three consecutive days and the liquids are incubated at 25-30°C. During the first steaming, all the heat sensitive vegetative cells are killed, leaving only the spores. During the first incubation period, most of the spores germinate in to vegetative cells. These vegetative cells are killed by the second steam period.

In the second incubation period, the rest of the spores germinate into vegetative cells which are killed by the third steaming period. In this way, the liquids are sterilized without temperature rising above 100°C.

Maintenance of Pure Culture:

After obtaining the pure culture of a particular microbe, it may be grown and maintained as a pure culture in different ways:

1. The most common practice is to grow the culture on suitable medium until it reaches the stationary phase of growth, and then store in a refrigerator. If they are to be kept alive for a long period all culture must be transferred to a fresh sterile medium. Thus by successive transfer, a culture may be kept for an indefinite period.

2. A second method involves freezing of young culture and desiccating it under vaccum. The cells of a pure culture will remain viable for a long period of time if they are mixed with sterile blood serum, sterile skimmed milk, before freezing and drying. They dried cultures are kept in the sealed, evacuated tubes and are stored in cool places.

3. This method of maintaining pure culture is most suitable for spore forming species. The microorganisms are grown in pure culture in suitable media. A suspension of microorganisms is then transferred to cotton stoppered tubes of sterilized dry soil. The spores remain viable, though dormant, for long periods of time, in dry soil. The organism can be grown after a desired period, by transferring the soil into a suitable medium and incubating it under suitable temperature.