Preservation and Maintenance of Microorganisms-I�

Dr.Jitender Kumar

Dept. Biotechnology

HMV, Jalandhar

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Source: A Text Book OF Microbiology By -

Dubey and Maheshwari (S.Chand Publisher)

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Introduction

• Industrially important microorganisms are often mutants, and the condition of low metabolism in which they are kept, limits their tendency to revert to their low- yielding ancestors.
• In some circumstances organisms are maintained for comparatively short periods of days in an active state in which they are immediately ready for use in fermentations; such organisms are called working stock.
• In many breweries, for example, the producing yeasts are reused sometimes for up to eight runs or more before being discarded.
• In the interval between inoculations such yeasts are regarded by some workers as working stocks.
• It must be borne in mind that working stocks stand the chance of contamination and/or mutation, two serious problems inherent in industrial fermentations.

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Strain Preservation

• Strain preservation involves methods like cryopreservation, lyophilization (freeze-drying), and short-term refrigeration or subculturing to maintain the viability and genetic characteristics of microbial or cellular strains for future use. 
• The goal is to slow metabolic activity, prevent contamination, and avoid mutations to preserve the strain's original traits and product-producing capabilities. 
• Popular techniques include storage in liquid nitrogen with cryoprotectants like glycerol, and freeze-drying cultures in ampoules for long-term stability. 

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Introduction�

• Industrial microbiological establishments usually keep a collection of the microorganisms which possess the gene pools for producing the goods manufactured by the establishment.
• This stock of organisms is known as a culture collection and ensures a regular supply of organisms to be used in the manufacturing process.
• Organisms in a culture collection are maintained in a low metabolic state in which replication of the cells is kept to a minimum or even entirely restricted.

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CULTURE COLLECTIONS CENTRES�

• Culture collections maintained by industrial establishments are usually specialized and store mainly those used in that particular organization.
• Some national culture collections handle a wide variety of org., of whatever kind.
• The best known in this category is the American Type Culture Collection (ATCC).
• Other collections are specialized and may handle:
• only pathogenic microorganisms, such as:
• the National Collection of Type Cultures (NCTC) in Colindale, London, UK
• industrial microorganisms, such as:
• National Collection of Industrial Bacteria (NCIB) in Aberdeen, Scotland.

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CULTURE COLLECTIONS CENTERES

• Still others almost exclusively handle one type of organism such as:
• Center vor Braunsveitzer (CBS) in Holland, which handles fungi exclusively.
• Many universities all over the world have culture collections which reflect their range of microbiological interests.
• Culture Collections around the world are linked by the World Federation of Culture Collections (WFCC).
• The WFCC is an affiliate of the International Union of Microbiological Societies (IUMS) the organization which links national microbiological societies world wide.

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NAME OF CULTURE COLLECTIONS CENTRES�

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• Culture Collections are organized on regional and international basis for the exchange of cultures and ideas and include the:
• National Agriculturally Important Microbial Culture Collection (NAIMCC, India),
• BCCCM (Belgium Co-ordinated Collections of Microorganisms),
• ECCO (European Culture Collection Organization),
• JFCC (Japanese Federation of Culture Collections),
• MICRO-NET (Microbial Information Network of China),
• MSDN (Microbial Strain Data Network, UK),
• UKNCC (United Kingdom National Culture Collection),
• USFCC (United States Federation of Culture Collections, USA)

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TYPES OF CULTURE COLLECTIONS�

• Culture collections may be specialized and in-house such as those in industrial establishments.
• Others are public and have the function of acquiring, identifying, preserving and distributing microorganisms and for a fee will supply cultures for in teaching, research or to industry.
• Such culture collections receive cultures from all over the world and thus serve the overall purpose of maintaining worldwide microbiological biodiversity.
• In addition to making available organisms for industrial use, the major culture collections serve the important function of acting as depositories for microorganisms mentioned in the patenting of microbiological processes.

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MAINTENANCE OF CULTURE COLLECTIONS�

• An industrial process may be initiated with organisms obtained through the Patent Office in connection with a patent.
• Often only one vial of such an organism is usually available.
• Once growth has been obtained from that vial the organism should be multiplied and stored in one or more of the several manners described later for the preservation of primary stock organisms in a Culture Collection.

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HANDLING CULTURE COLLECTIONS

• Several replicates are stored immediately for fear of contamination while tests are carried out to ascertain its potential for fulfilling the expected activity.
• If the tests show that the expected antibiotic or other desired metabolite is being produced in the expected quantity then stored organisms are retained.
• The stocks of those organisms which proved negative at first sampling should not be discarded in a hurry because further examination may show that poor productivity was due to factors extrinsic to the organism such as an inadequate medium.
• Date of transfer, the medium and the temperature of growth, etc., must all be carefully recorded to afford a means of assessing the effect of the preservation method.

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STORAGE OF CULTURE COLLECTIONS

• Culture collections may be specialized and in-house such as those in industrial establishments.
• Others are public and have the function of acquiring, identifying, preserving and distributing microorganisms and for a fee will supply cultures for in teaching, research or to industry.
• Such culture collections receive cultures from all over the world and thus serve the overall purpose of maintaining worldwide microbiological biodiversity.
• In addition to making available organisms for industrial use, the major culture collections serve the important function of acting as depositories for microorganisms mentioned in the patenting of microbiological processes.

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Microbial Preservation Methods Based on the temperature �

• Preservation on agar with ordinary refrigeration (4 – 10°C)
• Organisms growing on suitable agar at normal growth temperatures attain the stationary phase and begin to die because of the release of toxic materials and the exhaustion of the nutrients.
• Agar-grown organisms are therefore refrigerated as soon as adequate growth is attained as to preserve them.

Aerobic organisms

• Agar slants: Aerobic organisms may be grown on agar slants and refrigerated at 4 – 10°C as soon as they have shown growth.
• Petri dishes: Aerobic organisms may also be stored on Petri dishes.
• The plates may be sealed with electrical tapes to prevent the plates from drying out on account of evaporation.

Anaerobic organisms: Anaerobic organisms may be stored on agar stabs which are then sealed with sterile molten petroleum jelly

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PRESERVING MICROORGANISMS�

• Methods employed in the preservation of microorganisms all involve some limitation on the rate of metabolism of the organism.
• A low rate of spontaneous mutation exists during the growth of microorganisms, about once in every 10⁹ division.
• Lowering the metabolic rate of the organism will further reduce the chances of occurrence of mutations.
• The principles involved in preserving microorganisms are:
1. reduction in the temperature of growth of the organism;
2. dehydration or desiccation of the medium of growth;
3. limitation of nutrients available to the organism.
• All three principles lead to a reduction in the organisms metabolism.

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Microbial Preservation Methods at low temperature�

• Preservation on agar with ordinary refrigeration (4 – 10°C)
• Organisms growing on suitable agar at normal growth temperatures attain the stationary phase and begin to die because of the release of toxic materials and the exhaustion of the nutrients.
• Agar-grown organisms are therefore refrigerated as soon as adequate growth is attained as to preserve them.

Aerobic organisms

• Agar slants: Aerobic organisms may be grown on agar slants and refrigerated at 4 – 10°C as soon as they have shown growth.
• Petri dishes: Aerobic organisms may also be stored on Petri dishes.
• The plates may be sealed with electrical tapes to prevent the plates from drying out on account of evaporation.

Anaerobic organisms: Anaerobic organisms may be stored on agar stabs which are then sealed with sterile molten petroleum jelly

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Preservation on agar with ordinary refrigeration�

• The advantage is that agar storage methods are inexpensive because they do not require any specialized equipment.
• The disadvantages are:
• The organisms must be sub-cultured at intervals which have to be worked for each organism, medium used, laboratory practice, etc.
• This is because the temp. of refrigeration is not low enough to limit growth completely.
• Consequent on regular sub-culturing is the possibility that contaminations and or mutations may occur.
• The third disadvantage is that Petri dishes occupies a lot of space in the refrigerator when compared with agar slants.
• But even agar slants are too bulky in comparison with the small vials in which lyophilized (freeze-dried) cultures are stored.
• Since plates occupy a lot of space, test tubes are usually preferred for storage in refrigerators.
• Process of sub-culturing is tedious apart from possibility of contamination and mutation.
• When petroleum jelly is used as a seal, the arrangement can be messy.

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Microbial Preservation Methods�

• Oil overlay
• With the method of oil overlay whose function is to limit oxygen diffusion many bacteria, especially anaerobes and facultative, and fungi survive for up to three years, and most of them for at least one year.
• Medium for storing organisms on agar
• The nature of the medium on which organisms are stored is of importance.
• A medium prepared from natural components rather than a chemically defined material is preferable, since a defined medium may, because it lacks some components present in the natural components, select for organisms specifically capable of growing on it.
• A stock culture medium should also not be unduly rich in carbohydrates such as glucose which will lead to early production of acid and hence possible early microbial death.
• Where glucose is used, such as for lactic bacteria, the medium should be buffered with calcium carbonate.

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References

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A TEXT BOOK OF MICROBIOLOGY BY DUBEY AND MAHESHWARI GENERAL MICROBIOLOGY BY STANIER.

• EXPANDING HORIZON BIOTECHNOLOGY BY B.D.SINGH.
• GENERAL MICROBIOLOGY BY P.TAURO.
• MODERN FOOD MICROBIOLOGY BY JAMES JAY.

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• Thanks