MAINTENANCE-PRESERVATION OF PURE CULTURES
Associated with the study of pure cultures is the problem of maintaining them for various periods in a viable condition. This is necessary so as to eliminate genetic instability, protect against contaminants and retain its characters. Most bacteriological laboratories usually maintain a large collection of pure cultures, frequently refered to as the stock culture collection.
Microbial species isolated and characterised by microbiologists are deposited in these centres. The stock cultures generally retain all the characteristics, Stock culture collection centres have been established throughout the world to help microbiologists in obtaining pure cultures for various studies. Some of the centres also help in classifying a newly isolated organisms.
1. Maintenance methods for stock cultures :-
Once a pure culture is obtained it must be kept pure. Oneof the most frequent ways in which erroneous results and conclusions are obtained in microbiology is by the use of faulty or contaminated cultures. Cultures of organisms of interest that are maintained in the laboratory for study and reference are called stock cultures. The stock cultures must be maintained so that it is free from contamination, retains viability, and remains true to type. The most common way of maintaining stock cultures is either on agar slants or in agar stabs.
(A) Agar slants:
The slants are inoculated and incubated until reasonable growth is obtained, and then they are usually placed in a refrigerator, care being taken that the culture tubes are well sealed (with melted wax) so that they will not dry out. With hardy organisms such as spore formers, Gram-positive cocci, yeasts, and fungi, agar slant cultures maintained in this way may retain viability for weeks or months. It is the usual pre to transfer cultures to fresh slants at regular intervals (Sub-culturing).
(B) Agar stabs :-
For some organisms agar stabs are better for preserving viability tha agar slants. The tube for an agar stab, called an agar deep, is allowed to stand vertically while the agar is solidifying rather than at an angle as for slants. Inoculation is done with a straight needle, after being touched to the culture, is stabbed into the agar all the way to the bottom. During incubation, the culture
grows in a thin zone along the line of the stab, The advantage of agar stab procedure is that waste products such as acids that are produced by the organisms spread out into the agar from the zone of growth and are diluted. Accumulation of waste products is one of the main reasons for the dying off of mary stock cultures. Although less total growth is obtained in a stab than on slant, ‘enough growth occurs so that new cultures can be initiated when needed. It is usually inadvisable to maintain stock cultures on agar slants; they become contaminated relatively easily, since so much surface is exposed to the ai
2. METHODS OF PRESERVATION
A considerable amount of research has been performed to develop improved
conditions for the preservation of bacteria. Not all species respond in a similar . manner to a specific condition or process. A method useful for one species may not be applicable to another. It is also essential that the method of preservation and maintenance conserve all the characteristics of the species as they were at the time of preservation.
There are basically two major methods for preservation and maintenance of cultures –
(a) Methods in which organisms are in a state of continuous metabolism and,
(b) methods in which organisms are in a state of suspended metabolism,
1. ORGANISMS IN CONTINEOUS METABOLISM STATE:
It depends on repeated subculturing of organisms onto fresh nutrient medium when the formerly used medium gets dried or is used up. This includes preservation of organisms by following methods:
(A) PERIODIC TRANSFER TO FRESH MEDIA :-
All microbiology laboratories preserve microorganism on agar slants. The slants are incubated for 24 hours or more, and then stored in a refrigerator. These cultures are periodically transferred to fresh media. The time interval at which transfers are made varies with the organism and the conditions of growth.
(B) OVERLAYING CULTURES WITH MINERAL OIL-
The agar slants are inoculated and incubated until good growth appears. They are then covered with sterile mineral oil to a depth of 1 cm. above the tip of the slanted surce. This method has the unique advantage that one ca remove some of the growth under the oil with a transfer needle, inoculate a fresh medium and still preserve, the initial culture. The simplicity of this method makes it extremely attractive. Some species have been preserved satisfactorily for 15-20 years by this methou.
(C) STORAGE IN STERILE SOIL:-
This method is widely used for preserving spore forming bacteria and fungi Spore suspensions are added to sterile soil (sterilized for 2-3 hours at intervals of 1-2 days) and the mixture is dried at room temperature and stored in refrigerator Bacterial cultures maintained by this procedure have been found viable for 70-80 years.
(D) SALINE SUSPENSION-
Sodium chloride in high concentrations is frequently used as an inhibitor of bacterial growth. Bacteria are suspended in one per cent salt solution (Sublethal concentration) in screw cap tubes to prevent evaporation, The tubes are stored at room temperature. Whenever needed, the transfers are made on agar slants and incubated.
2.ORGANISMS IN SUSPENDED METABOLISM STATE:-
This depends on some sort of drying or preservation at low temperature to a level at which the metabolism is slowed down or stopped, but revival of culture is possible. It includes
(A) DRYING IN VACUUM
The organisms are dried over calcium chloride in a vacuum and are then stored in the refrigerator. The organisms survive longer than when air dried.
(B) LYOPHILIZATION (Freeze – Drying)
Lyophilization is the most effective procedure for the preservation of cultures.. Many species of bacteria preserved by this technique have remained viable and unchanged for more than 20 years. In this process the cells are dried rapidly, while they are frozen. The bacterial suspension is placed in small vials, which are then immersed in a mixture of dry ice and alcohol at a temperature of -78° C. The vials are immediately connected to a high-vaccum line. This dries the organisms while still frozen.
Finally the vials are sealed off in a vacuum with a small flame. These cultures can then be stored for years at 4°C. This method is also employed for the preservation of sera, toxins, enzymes and other biological materials To revive microbial cultures, it is merely necessary to break open the vial aseptically, add a suitable sterile medium, and after incubation make further transfers. This method greatly reduces the danger of contamination. An additional advantage is the requirement of a small storage area.
Figure.Lyophilization process of preservation of microorganisms.
[A] Small cotton-plugged vials containing frozen suspensions of microorganisms are attached to a condenser and a high-vaccum pump. This system dehydrates the specimen while it is in the frozen state.
[B] After the specimen is dehydrated, the tubes containing the vials are sealed while still under vaccum. Details of an individual lyophilized specimen are shown enlarged. This!! lyophilized culture of microorganisms will remain viable for years.
(C) USE OF LIQUID NITROGEN-
The ready availability of liquid nitrogen (temperature of -196 C) has provided the microbiologists with another means Tor maintaining the cultures. In this procedure the cultures are frozen with a protective agent (glycerol or dimethyl sulfoxide) in scaled vials. The frozen cultures are then kept in liquid nitrogen refrigerator. This process has been successful with many specimens which cannot be maintained by lyophilizaiton
(D) STORAGE IN SILICA GEL :-
Both bacteria and yeast can be stored in silica gel powder at low temperature for a period of 1-2 years. In this method, finely powdered, heat sterilized and cooled silica powder is mixed with a thick suspension (Paste) of cells, mixed and stored at low temperature.. The basic principle in this technique is quick dessiction at low temperature which allows the cells to remain viable for a longer period.
3. Role Culture Collection centres :-
A culture collection either can be a specialized collection of a restricted group of organisms or it can be a generalized collection that attempts to maintain a wide range of organisms of interest The purpose of a culture collection is to maintain cultures in a viable contaminant free state and to have them available for immediate shipment at nominal cost to research workers, teachers students, or industry.The individual worker thus need not maintain cultures that he uses rarely.
The maintenance of a large and diverse culture collection is not simple, since different organisms must be stored in different ways. A high degree of skill and knowledge is necessary. The price charged for a stock culture may often seem high, but it should be noted that the purchaser is paying for the immediate availability of a contaminant- free culture. It must be emphasized, however, that the investigator should not accept blindly the reliability and purity of a stock culture received from a culture collection centre. Immediately upon receipt, the culture should be carefully checked, and if it does not meet specifications, it should be returned.
Since culture collections often maintain thousands of different cultures, it is only natural that errors may occur, leading to the distribution of faulty cultures. (Culture collection centres also render the services of identification and confirmation of any new isolate obtained by researches.)
Organisms are listed in culture collections by species name, but they are also often given numerical designations. Not all pure cultures of the same species are identical in all ways. Each isolate of the same species is usually designated as a strain, and one strain is distinguished from another by use of arbitrary numbers or letters. The culture number used by the culture collection is unique number referring to a single strain of the species. Since strains of the same species may differ in important ways, it is necessary to specify the exact strain number when referring to work carried out with a specific stock culture.
A number of countries have good culture collection centres which are listed in Mid-appendix Catalogues are available from the culture collection centres.
WORLD WISE STATUS OF CULTURE COLLECTION CENTRES
The increasing use of microorganisms for various purposes has made it obligatory that the culture of great value should be maintained to provide viable and stable cultures. Hence all microbiologists need to preserve cultures for a variety of reasons. The role of maintenance of cultures is performed by the culture collections. The basic functions of culture collections, therefore, are collection, maintenance, and supply of reliable, authentic cultures. In addition to the above basic activities, culture collections provide a number of other services also.
There are 566 culture collections listed in their directory by the World Data Centre on Microorganisms (WDCM) and in India there are more than 50 culture collections and National Collection of Industrial Microorganisms (NCIM), Pune is the largest amongst these collections and American Type Culture Collection (ATCC), USA is the largest in the world.
The diversity of culture collections and wide interest of their users has led to need to coordinate activities and World Federation for Culture Collections (WFCC) provides this international coordination.
The collection, maintenance,characterization and distribution of cultures generates a great deal of information that must be effectively documented. For this purpose Microbial Strain Data Network (MSDN) has established link through computers between the culture collections all over the world and specific information and retrieval of data can be obtained. Characterization, identification, preservation and patent services make an important contribution to the development of biotechnology and the biotechnological importance of culture collections can be exploited only if information on the properties of holdings is known, properly documented and easily accessible.
LIST of some Important Culture collection centres.
(A) For all types of microorganisms (Algae, Moulds, Yeast, Bacteria, Actionomycetes, Protozoa, Animal Viruses, Bacteriophages, Cell lines):-
(1) American Type Culture Collection [ATCC] 12301 Parklawn Drive,
Rockville, Maryland, 20852, USA
(2) Carolina biological Supply Company [Commercial Service] 2700 York Road. Burlington, North Carolina 27215, USA
(B) For Protozoa and Algae :-
(3) Culture Collection of Protozoa and Algae. 36 storey’s way, Cambridge University, Cambridge CB3 ODT England
(4) General Biological Supply House (Commercial service ] 8200 S. Hoyne
Avenue, Chicago, U.S.A.
(C) For Algae :
(5) The Culture collection of Algae and Microorganisms. Institute Applied Microbiology (IAM), Uni. of Tokyo, 1- Chome, Yoyoi,Bunkyo- Ku, Tokyo, Japan.
(6) University of Texas culture collection of Algae, Uni. of Texas, AustiTexas, U.S.A.
(D)For Fungi :-
(7) International Mycological Institute (Formerly, CMI) Bakeham Lase, Egham, Surry TW 209 TY, U.K. (8) Centralbureau voor Schimmelcultures (CBS) Osterstaat 1, Baam,
(9) IHEM Culture collection Place Croiok due sud 3-B-1348, Louvain-la-Neuve-Belgium.
(E) For Yeast :-
(10) National collection of Yeast cultures. Brewing Industry Research Foundation, Lyttel Hall, Nutfiel, Near Redhill, Surrey, U.K.
(F) For Actinomyceters :.
(11) Institute for Research of New Antibiotics, USSR Academy of Medical
Sciences, Bolshaia Pirogovskaia 11, Moscow, Russia. (12) Botanical Department, Uni. of North Carolina, Coker Hall, Chapel Hill, North Carolim 27514, U.S.A.
(G) For Bacteria :-
(13) The Wellcome Bacterial Foundation Wellcome Foundation Ltd, Longley court,
Beckenham, Kent, UK. (14) National Collection of Industrial Bacteria
Torry Research Station, P.O. Box 31, 135 Abhey Road,Aberdeen AB 9 8 DG. Scotland.
(15) National collection of plant Pathogenic Bacteria Plant Pathology Laboratory
Milton Road, Harpenden, Hert fordshire, Scotland.
(16) National collection of Dairy organisms
National Institute for Research in Dairying
Shinfield, Reading Berkshire, U.K.
(17) The Upjohn Stock Culture Collection The Upjohn Company, 301 Henrietta street, Kalamazoo, Michigan 49001, U.S.A.
(18) LMG collection, Laboratorium voor Microbiologic Rijks Universiteit Gent
K.L. ledeganck straat 35, B-900 Gent, Belgium.
(19) Culture collection Laboratory of Microbiology Technical University, Julianalaan 67 A, Delft, Netherlands.
(20) Institute Pasteur, 25 Rue du Docteur Roux, 75724 PARIS Cedex 15, France.
(21) Centre de Collections de Types Microbeins, 19, Rue Cesar-Roux, Lausanne, Switzerland.
(22) Microbiological Type Culture collection, 4- 54,Juso-Nishinocho, Higashiyodogawa-ku, Osaka, Japan.
(23) Culture collection Unit, Northen Utilization Research Branch,
U.S. Dept of Agriculture, Peoria 5, Illinois, U.S.A.
(24) National collection of Type- cultures, Central Public Health Laboratory, Colindale Avenue, London, UK.
Adress your inquiry to “The curator of above culture collection centres centres for taking services from them.
(II) In India
(A) For all types of microorganisms :-
(1) National collection of Industrial Microorganisms [NCIM] C.S.I.R. National Chemical Laboratory, Poona – 411008 Helping Person :- Dr.LI. Sutar
(2) Microbial Type Culture Collection and Gene Bank [MTCC] Institute of Microbial Technology [IMTECH] Post Box No. 1304, sector 39-A, Chandigarh- 160 014 Telephone -45108, 450004 Fax: 40985, 28032 Email: tapan (a) imtech. ernet in Telex: 395 7395 IMT-IN Helping Person Dr. Swarnjit Sing Cameotra, Dy. Director.
(B) For Fungal Cultures :-
(3) Herbarium Cryptogame Indie Orientalis Division of Mycology and Plant Pathology,
L.A.R.I., New Delhi 110 012
Helping person – Dr. S.P. Lal.
(C) For Yeast :-
(4) Fermentation Technology Laboratory, Indian Institute of Science, CV. Raman Road, Bangalore 560 012
(D) For Actinomycetes: (helping only in identification services)
(5) Department of Microbiology, Bose Institute, 93/1, Acharya Prafulla Chandra Road, Calcutta – 700 009
(E) For Cyanobacteria :-
(6) National Facility for Marine Cyanobacteria,Bharathidasan University, Tiruchirapall; – 620 024
Helping Person Dr. G. Subramanian, Director.
The use of pathogenic microorganism in testing programs for potential laboratory technicians has posed a dilemma since the practice was fint instituted. Proponents suggest that real situations must be duplicated if the laboratory technician is to be judged competent to make accurate conclusions under routine conditions. Opponents counter that handling pathogens creates an unnecessarily dangerous situation for the inexperienced technician.
The problem was brought to the fore in 1979 when the centere for Disease control reported a total of 19 cases of typhoid fever from 14 different states where typhoid bacilli were used in proficiency examinations. Seven cases were students and twelve were laboratory teachers (1) In all instances, the laboratory cultures were detrermined to be the source of bacilli. The CDC called for renewed caution in laboratory practices and the use of less- virulent organisms as substitutes. It also suggested that more attention be paid to the sign that says:
“No Eating, Chewing, smoking or Drinking in the Lab.”
Would you like to follow above instruction ? Yes, I hope positively from all of you.