Purification & preservation

Microorganisms in nature occur only as mixed cultures. A culture that is grown from a single cell is called as pure culture. The pure cultures are necessary to study the colony characteristics, morphology, biochemical characteristics and immunological reactions of particular bacteria, fungi or Actinobacteria.

Three methods viz. (i) Streak plate method, (ii) Pour plate method and (iii) Spread plate method are commonly employed for obtaining pure cultures of bacteria. The general principle involved in this technique is to dilute or divide the mixed population so that the single cells are separated and develop in to single colonies. The individual colonies are picked up and grown as pure culture i.e. growth derived from a single cell.

Streak plate method

Streaking is rapid and ideally a simple process of isolation dilution. The technique is done by diluting a comparatively large concentration of bacteria to a smaller concentration. The decrease of bacteria should show that colonies are sufficiently spread apart to effect the separation of the different types of microbes. Streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. Aseptic techniques are used to maintain microbiological cultures and to prevent contamination of the growth medium. There are many different types of methods used to streak a plate. Picking a technique is a matter of individual preference and can also depend on how large the number of microbes the sample contains. The three-phase streaking pattern, known as the T-Streak, is recommended for beginners. The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. The inoculation loop is first sterilized by passing it through a flame. When the loop is cool, it is dipped into an inoculum such as a broth or patient specimen containing many species of bacteria. The inoculation loop is then dragged across the surface of the agar back and forth in a zigzag motion until approximately 30% of the plate has been covered. The loop then is re-sterilized and the plate is turned 90 degrees. Starting in the previously streaked section, the loop is dragged through it two to three times continuing the zigzag pattern. The procedure is then repeated once more being cautious to not touch the previously streaked sectors. Each time the loop gathers fewer and fewer bacteria until it gathers just single bacterial cells that can grow into a colony. The plate should show the heaviest growth in the first section. The second section will have less growth and a few isolated colonies, while the final section will have the least amount of growth and many isolated colonies.

Spread plate and pore plate techniques have already described in Isolation chapter.

Preservation

Once a microorganism is isolated in a pure form, it is sub cultured on plates or agar slants at regular intervals to maintain viability. The interval between sub culturing which varies between every four weeks or months depends on the storage conditions and on the growth rate of organisms. The organism can be sub cultured on maintenance media especially designed to allow low growth rates and extend the culture's life. Storing cultures in a refrigerator, at a temperature of 4°C, which slows growth, protects from damage due to evaporation of medium, and preserves the culture. To preserve micro-organisms, it is necessary to reduce their metabolism to a minimum. As a result, the processes that lead to ageing and death are slowed down and the microorganism can be maintained in its inactive state for several years. Lyophilization (freeze-drying) or freezing in liquid nitrogen are used for preserving the microorganisms where they can retain viability for several years.

There are several methods available for maintenance of pure cultures, the choice of the method to be used depending upon the purpose, size of collection and the laboratory. Some of the commonly used methods are:

A. Use of refrigerator or cold room storage

Live cultures on a culture medium can be successfully stored in refrigerators or cold rooms maintained at 4°C. The bacterial and fungal cultures in their appropriate media as slant cultures or stab cultures will be stored in the refrigerators for 1 to 3 months. Generally the metabolic activities of the microorganisms will be greatly slowed down at this temperature, but it is not low enough to stop metabolism completely. Thus growth will occur slowly, nutrients will be utilized and waste products produced, which will eventually kill the microorganism. So regular sub culturing is necessary, for bacteria it ranges from 2-3 weeks and for fungi from 3-4 months.

B. Overlaying cultures in the mineral oil

This is a simple and most economical method of preserving bacteria and fungi where they remain viable for several years at room temperature. In this method, sterile liquid paraffin is poured over a slope culture of the microorganism and stored upright at room temperature. The layer of paraffin prevents dehydration of the medium and by ensuring anaerobic conditions, the microorganisms remain in a dormant state.

This method is very simple, economical and proved to be very successful for storing cultures of bacteria and fungi. The microbes remain viable for several years at room temperature. During revival or transfer from oiled cultures, a loopful of the culture is removed, inoculated on an agar slant (in case of fungi) or a broth tube (in case of bacteria).

C. Preservation at –40°C in glycerol (Glycerol stocks)

Cultures can be preserved for several years in glycerol at –40°C in a deep freeze. In this method approximately 2 ml of the glycerol solution is added on to the agar slope culture, emulsify the culture by shaking. The culture as liquid broth can also be stored in this method. Approximately 1 ml of actively growing culture, mixed with 0.5 ml of sterile glycerol (autoclaved) is transferred into an ampoule which are placed in a mixture of industrial methylated spirit and carbon dioxide and are freezed rapidly to –70°C. Ampoules are removed from the mixture and placed directly into a deep freeze at –40°C. During transfer from these stock cultures, tubes are placed in water bath at 45°C for few seconds or until the suspensions melt and are aseptically streaked onto agar plates.

D. Liquid nitrogen method (storage at low temperature)

Freezing in liquid nitrogen at temperatures of –196°C also suspends metabolism of cells and these survive unchanged for long periods. In this method cell suspension in the presence of a stabilizing agent, such as glycerol, or dimethyl sulfoxide (DMSO), that prevents the formation of ice crystals which may kill frozen cells, is sealed into small ampoules and stored in liquid nitrogen refrigerator (-196°C). Most species of bacteria can remain viable for 10 to 30 years or even more without undergoing change in their characteristics.

The easiest, most effective and efficient way to maintain mold isolates indefinitely is on sterile distilled water.

In culture collection centers, the cultures of micro-organisms are usually preserved as freeze-dried (also called lyophilized) cultures and supplied in ampoules.

E. Freeze-drying method

Freeze-drying (lyophilization) is the rapid dehydration of organisms while they are in a frozen state. Most of the microbes are protected from the damage caused due to water loss by this method. Since metabolism requires water, the organisms are in a dormant state and can retain viability for over 30 years unchanged in their characteristics. In this technique, the culture is rapidly frozen at –70°C and then dehydrated by vacuum and the tubes containing freeze-dried cultures are sealed and stored in the dark at 4°C in refrigerators. Freeze-drying method is used for storing of cultures in the National/International Culture Collection Centres of the world as dehydrated (lyophilized) cultures retain their viability for several years.

F. Maintenance of mold cultures on distilled water

Mold cultures are usually maintained at 4°C in refrigerators in teaching and research laboratories. These cultures are regularly sub-cultured to fresh potato dextrose agar slants, every two weeks to six months, depending upon the type of the mold. Successive sub-culturing of molds often leads to the production of sterile mutant strains or death of the organisms. The easiest, most effective and efficient way to maintain mold cultures indefinitely, without change in isolate characteristics, is on sterile distilled water. Although the isolates on distilled water remain viable for several years, ideally cultures are revived every two years and a fresh distilled water suspension is made from a typical sporulating colony.