Methods of Obtaining Cultures of Microorganisms | Microbiology

The following points highlight the three main methods of obtaining cultures of microorganisms. The methods are:- 1. Culture Collections 2. Isolation 3. Genetic Alteration. Learn about:- 1. Methods of Obtaining Pure Culture 2. Pure Culture Techniques 3. Isolation of Pure Culture Techniques 4. Microbial Culture Techniques 5. Methods Used to Isolate a Pure Culture.

Method # 1. Culture Collections:

1. Cultures and Patents:

The simplest method of obtaining cultures of mi­croorganisms is to acquire subcultures from cultures previously isolated. Throughout the world there exist numerous culture collections ranging from very small private collections to large public collections which may maintain several thousand strains of microorganisms.

Private collections are usually associated with specialized research groups in industries, uni­versities, or institutes and are maintained solely for the use of these groups. Cultures in this type of collection are not usually distributed generally; however, specific requests from interested scientists are often honored.

The need to preserve and maintain microorganisms for industry, gov­ernment, teaching, etc., has led to the organization of several large public culture collections. Well known collections of this type include the Com­monwealth Mycological Institute (CMI), Agricultural Research Service Culture Collections (NRRL), American Type Culture Collection (ATCC), and the Centralbureau voor Schimmelculturen (CBC).

From an industrial point of view, the NRRL and ATCC deserve special consideration. These two organizations have been recognized by the United States Patent Office as official depositories of cultures, both domestic and foreign, which are an essential part of a patented process.

The American Type Culture Collection is a non-profit organization which preserves and distributes cultures of microorganisms and animal cells to all areas of scientific investigation and instruction. In addition to culture preservation, research is carried out in the areas of comparative microbiol­ogy, microbial systematics, and improved methods of characterization and preservation of cultures.

This organization is supported by fees for cultures and services and by grants and contracts. ATCC has available two cata­logues. One lists the approximately 18,000 strains of bacteria, bacterio­phages, fungi, protozoa, and algae. The second lists animal cell lines, ani­mal viruses, chlamydiae, and rickettsiae. These catalogues may be ob­tained for a fee from the American Type Culture Collection.

Development of the Agricultural Research Service Culture Collection began in the early 1900s and started primarily as a combination of a collection begun by Charles Thom and the microbial collection of the former USDA Bureau of Chemistry and Soils. The ARS collection has grown from its early existence by acquiring special collections, patent culture deposits, and other donations both solicited and unsolicited.

Presently, the culture collection maintains over 17,000 strains of molds, bacteria, yeasts, and actinomycetes. The ARS does not publish a catalogue of organisms held in its collection, but may distribute free of charge cultures to scientists in industry and education both within and outside of the United States at the discretion of the curator of the collection.

Policy regarding the deposit and distribution of cultures from the ARS culture collection is detailed in “Cul­ture collections of microorganisms”, and “Sources and management of microorganisms for the development of a fermentation industry”.

2. Noted World Collections:

Throughout the world there are many culture collections, and an extensive list has been compiled by Martin and Skerman (1972) No attempt will be made here to list all of the industrially important culture collections. However, it does seem proper to list a few collections where one may obtain cultures for industrial research.

3. Shipping:

Each year thousands of cultures of microorganisms are shipped from country to country and within countries. Many laws and regulations exist governing the shipment of microorganisms; and it is important to be aware of these regulations. For the majority of microorgan­isms for the fermentation industry, little hazard to public health or agricul­ture is involved during shipment. However, shipment should conform to regulations by both the country of origin and the country of destination.

It is necessary to determine the pathogenicity of an organism in order to know which regulations affect its shipment. The standards used by the United States Government for evaluating the pathogenicity of microorgan­isms and for classifying etiological agents on the basis of hazard have been published.

The following general criteria are used for this classification:

Class 1:

Agents of none or minimal hazard under ordinary conditions of handling.

Class 2:

Agents of ordinary potential hazard. This class includes agents which may produce disease of varying degrees of severity from accidental inoculation or other means of cutaneous penetration, but which can be con­tained by ordinary laboratory methods.

Class 3:

Agents involving special hazard or agents obtained from outside of the United States which require a federal permit for importation unless they are specified for a higher classification. This class includes pathogens which require special conditions for containment.

Class 4:

Agents that require the most stringent conditions for their con­tainment because they are extremely hazardous to laboratory personnel or may cause serious epidemic disease. This class includes Class 3 agents from outside the United States when they are employed in entomological experi­ments or when other entomological experiments are conducted in the same laboratory area.

Class 5:

Foreign animal pathogens that are excluded from the United States by law or whose entry is restricted by USDA administrative policy.

With the exception of industries involved in producing antisera, vaccines, pharmaceuticals, etc., the majority of organisms used by industry would fall into Class 1. Regulations regarding this class are limited primarily to the need to protect the United States mail from damage which could arise from broken shipping containers. Organisms that belong to Classes 2-5 require special permits and packaging procedures to ensure against the hazards which would result from improper handling of these more dangerous micro­organisms.

The shipment of microorganisms which can be considered as plant patho­gens is governed by two federal statutes, the Plant Quarantine Act of 1912 and the Federal Plant Pest Act of 1957. These acts prohibit the importation and movement of plant pests, pathogens, vectors, and articles that might harbor these organisms unless authorized by the U.S. Department of Agri­culture.

Authorization comes in the form of a permit, and the following guidelines are used in determining permit requirements:

Types of Organisms Requiring Permits:

(1) Foreign plant pests known to be injurious to crops grown in the United States.

(2) Domestic plant pests regulated by federal and state quarantines.

(3) Non-regulated domestic plant pests if shipment is into an area of the United States where the pests do not occur.

(4) Pests of noxious plants.

Types of Organisms Not Requiring Permits:

(1) Pure colonies of plant pest predators and parasites; pure cultures of plant pest pathogens.

(2) Non-pest organisms.

U.S. Government departments involved with regulating the shipment of microorganisms are- Agriculture; Commerce; Health, Education and Wel­fare (now Education, and Health and Human Services); Transportation; Treasury; and the U.S. Postal Service.

Presently, rules and regulations are undergoing change and clarification, and the following should be contacted concerning present regulations and permits:

(1) For importation or interstate transport of agents which are animal pathogens:

(a) Chief Staff Veterinarian

(b) Organisms and Vectors

(c) Veterinary Services, APHIS, USDA

(d) Federal Building

(e) Hyattsville, MD 20782

(2) For importation and interstate movement of agents which are human pathogens:

(a) Center for Disease Control

(b) Attn. Office of Biosafety

(c) Atlanta, GA 30333

(3) For importation or interstate transportation of agents which are plant pests, pathogens, or vectors:

(a) U.S. Dept. of Agriculture

(b) Animal and Plant Health Inspection Service

(c) Plant Protection and Quarantine

(d) Federal Building

(e) Hyattsville, MD 20782

Federal requirements for the packaging of both pathogenic and non­pathogenic materials may be found in the code of Federal Regulations 39CFR21.3 and 42CFR72.25. Information concerning the postal regula­tions of other countries can be found in Postal Service Publication 42.

Method # 2. Isolation:

The ultimate sources of microorganisms for use in industrial processes are found in nature. Soil, living or decaying plant and animal matter, sewage sludge, etc., provide a wide spectrum of organisms suited to many purposes. Pure cultures of microorganisms can be selected from such sources by the use of several methods.

1. Plating Methods:

The use of media solidified with agar or some other suitable jelling agent has been successful for many years in obtaining cultures of many microorganisms. The process is very useful for yeasts, bacteria, fungi, and other microorganisms which produce a distinct colony on solidified media, and pure cultures can usually be obtained by using one of the following variations.

i. Streak Plate:

A streak plate is prepared by dipping a sterile glass rod, loop, or bent needle into a medium containing a suspension of cells of the desired organism. The rod or wire is then used to make a series of parallel non-overlapping streaks across the surface of the medium. As successive streaks are made the number of cells is diluted to such a point that the final streaks will usually yield separate and distinct colonies.

ii. Pour Plate:

A second and very useful method is the pour plate. The process consists of adding a portion of the medium containing the desired cells into an agar medium which is still liquid but cooled to about 45°C. If the cell concentration is not known, a portion of the liquid agar may be serially diluted with additional agar medium and finally poured into sterile petri dishes.

This results in the formation of a solidified medium with colonies scattered throughout the medium and not just on the surface. This allows for fairly easy separation. A second series of isolations should be made from colonies of the streak or pour plate. This ensures that a pure culture is prepared.

iii. Dilution:

This technique is widely used for obtaining pure cultures of many types of organisms. The process is carried out by evenly dispersing a sample of material in sterile water by use of a blender, homogenizer, or by vigorous shaking. The resulting suspension is then diluted to such a point that plating out a small portion of it will yield a number of colonies on each plate which can easily be separated and removed to fresh media. Variations of this process exist but lead to the same result.

iv. Antibiotics:

A very useful addition to the plating methods is the incor­poration of substances which are inhibitory to certain groups of organisms. Some antibiotics, such as penicillin, are toxic for most prokaryotic organ­isms but not for eukaryotic organisms. Therefore, the use of penicillin is very helpful to reduce or eliminate the number of bacterial contaminants when yeasts or molds are isolated. Conversely, such compounds as nystatin can be used to inhibit eukaryotic organisms when prokaryotic organisms are being isolated.

Plating methods are extremely useful in the isolation of organisms from a natural habitat; however, it must be noted that microorganisms vary greatly in their nutritional, temperature, 0₂, osmotic, pH, and other growth requirements. Therefore, it is necessary to be careful in selecting a medium which will provide a suitable environment for the microorganism.

2. Single Cell:

Some regard a culture as a pure culture only if the isolation is visually from a single cell or spore. It is necessary to have a single cell isolated for some types of work such as genetic investigations. However, the need for single cell isolation in the maintenance of cultures for industrial use is rather limited. If there is a need for single cell isolates, this can be fulfilled by the use of a micro-manipulator. The instrument is very helpful for work requiring the preparation of many single cell isolates, but it is also very expensive.

Aside from the use of a micro-manipulator there are several other meth­ods available which require only equipment readily available in any labo­ratory. With fungi, for example, dilution plates can be prepared from spore suspensions and examinations made until germ tubes appear. A small portion of agar with one germ tube can then be removed to fresh medium.

3. Hypheal Tip:

Some fungi produce very few or no spores. For these organisms, isolates can be obtained by plating a single colony in the center of a petri dish. A tip from the radiating hyphae at the outer edge of a rapidly growing colony can then be cut and transferred to fresh medium.

4. Enrichment Culture:

This technique is a process by which a special environment is created which will allow for the selection of the desired organism, or group of organisms, from a mixture of many organisms. This may be accomplished by setting forth conditions which will allow the de­sired organism to outgrow the other organisms present or by inhibiting the growth of all organisms except those desired.

Enrichment techniques may be carried out in liquid culture or on agar plates. An advantage to plating methods is the separation of colonies which allows for easy final isolation. Generally, liquid cultures are used to prepare a sufficient population from which isolation may be completed on solid media.

Enrichment techniques may employ alteration in the energy source, nitrogen source, carbon source, trace elements, pH, temperature, osmotic pressure, oxygen tension, source of inoculum, or any other parameter to select for the desired organism. For example, to select for ability to degrade hydrocarbons, a medium is prepared which contains only hydrocarbons as the source of energy.

The medium can then be inoculated with soil, sewage sludge, etc., and incubated. After incubation, a portion of the medium is transferred to fresh hydrocarbon medium and allowed to incubate further. After several such transfers the majority of organisms remaining are capa­ble of degrading hydrocarbons. Pure cultures may then be isolate’ from the liquid culture.

Method # 3. Genetic Alteration:

Since the late 1930s the use of mutation as a method of strain improve­ment has been exploited in many industrial processes. Probably the most noted example of this type of work is the improvement in the production of penicillin. The basic procedure for producing mutants from microorganisms is relatively simple and involves exposing cells or spores to the action of a mutagen.

The exposure may take place on the surface of agar in a petri plate, in a liquid culture, or by any method which allows contact between the mutagen and the cell. The process usually kills 99% of the exposed cells. The remaining living cells can then be isolated, sub-cultured, and checked for the desired result. Several commonly used mutagens are available and fall into two large groups, chemical and radiation.

Chemical:

A wide variety of chemical compounds are capable of produc­ing mutations in microorganisms. The action of chemical mutagens may proceed in a number of ways. Some compounds cause the loss or addition of bases in DNA; some cause changes in the nucleic acids; while others are structurally closely related to nucleic acid bases and are incorporated into new strands of nucleic acid during replication.

Chemical mutagens do not act equally well on different organisms. The best combination of mutagen and method of treatment is usually arrived at from published literature plus trial and error. It should also be noted that many of the chemical mutagens are carcinogenic and should be used with great care.

Nitrous Acid:

This compound is one of the easiest and least harmful of the chemical mutagens. Treatment is usually effected by suspending the cells in an acidic buffer and adding sodium nitrite. Mutagenesis is easily controlled by varying the length of time the cells are exposed.

Ethyl Methane Sulphonate (EMS), Ethyl Ethane Sulphonate (EES), and Diethyl Sulphate (DES):

These compounds belong to a larger group known as alkylating agents and give relatively high yields of mutants at high survival rates. Cells of the organism to be treated are usually sus­pended in a neutral buffer, and the mutagen is added to the suspension. The mutagenic reactions may be stopped by using sodium thiosulfate or by dilution during plating.

N-Methyl-N’-Nitro-N-Nitrosoguanidine (NTG):

Of the chemical muta­gens, this compound is one of the most potent, and great care should be exercised during its use. NTG can produce a high yield of mutants coupled with a high survival rate under proper conditions. Large amounts of infor­mation on the conditions influencing the mutagenic and killing effects of NTG in E. coli and Streptomyces coelicolor have been reported by Adelberg et al. (1965) and Delic et al. (1970).

Base Analogues:

Compounds such as 5-bromouracil and 2-amino-purine have been used for mutagenesis. These compounds are often incor­porated into the DNA in place of thymine and adenine, respectively. The mutagenic effect appears to be the result of incorrect base pairing in cell generations sometime after the incorporation of the base analogue.

Acridine Compounds:

Compounds of this group have limited use on a routine basis. Acridines produce relatively few mutants in most systems but derivatives of acridines known as ICR (Institute of Cancer Research) compounds are known to produce a high yield of mutants in specific systems.

Radiations:

Several systems for mutagenesis have been developed using X-rays, y-rays, neutrons, ultraviolet light, etc. Radiation systems are relatively convenient. If handled properly they may be used to produce a high yield of mutants in some systems.

Ionizing Radiations:

Ionizing radiations such as X-rays, γ-rays, neu­trons, and other particles have been used successfully to produce mutations. This type of radiation is particularly useful whenever chemical agents or ultraviolet light is not effective. It should be noted that ionizing radiations often cause chromosomal breakage which may lead to undesirable struc­tural changes such as translocations and inversions.

Ultraviolet Light:

The use of light between the wavelengths of 200 and 300 nm is a particularly easy and safe way of producing mutagenesis in microorganisms. Low pressure mercury vapor lamps emit UV light at a wavelength which is very efficient in producing mutations. Users of UV light should be aware of the possibility of photo-reactivation upon exposure of treated cells to longer wavelengths of UV light and the visible spectrum. Of course, any user must also be aware of the damage which can be done to the unprotected eye.

Hybridization:

The use of genetic recombination in microorganisms is valuable for producing new strains of industrial importance. Genetic re­combination in fungi and yeast can be accomplished by obtaining haploid strains from ascospores, basidiospores, etc., and then mating to produce new strains. With some lower fungi, the parasexual cycle has been utilized for strain improvement, and bacteria have been hybridized by transduction, conjugation, and transformation.

The hybridization of yeasts and higher fungi requires 3 main steps sporulation, spore isolation, and hybridization. Sporulation is controlled by many factors such as media composition, age of the culture, temperature, etc. These factors have been reviewed by Fogel and Mortimer (1971).

Once sporulation has taken place, it is necessary to separate the asci from the non-sporulating cells. After the asci have been separated, the individual spores can then be obtained by one of several methods, such as ascus dissection. Hybrids can be prepared by mating spores, mating haploid cells, etc., to produce new strains.

Parasexuality was first described by Pontecorvo in 1952 and reviewed by him in 1956. Since that time this process has been found in many fungi, and utilized by some for the improvement of industrial strains. The parasexual cycle briefly consists of heterokaryotic mycelium in which fusion of nuclei takes place.

These nuclei may be like or unlike. After fusion the diploid nuclei multiply along with the haploid nuclei; and finally genetic sorting out takes place during the production of uninucleate conidia. Successful utilization of the parasexual cycle for increased production of penicillin has been accomplished. However, its use as a common methodology has never materialized.

Genetic recombination in bacteria differs from that of the sexual process of eukaryotes. In bacteria a portion of the genetic material of one cell (donor) is transferred to a second cell (recipient). The recipient cell, therefore, becomes diploid only in a section of its genetic complement. The process of recombination may proceed in 1 of 3 ways.

The first, transformation, is accomplished by the adsorption of a piece of double-stranded DNA (which had been released into the medium by a donor cell) onto the surface of a recipient cell. One strand of the DNA is degraded and the remaining strand is incorporated into the genetic material of the recipient cell.

The most recently discovered process for genetic recombination, trans­duction, requires a bacteriophage. In transduction a portion of double- stranded DNA from the donor cell is carried to the recipient cell by a bacteriophage. When infection by the bacteriophage takes place, the ge­netic material carried from the donor to the recipient is also incorporated to form a partial zygote.

In the third process, conjugation, the genetic material is transferred to the recipient by direct contact. A single strand of DNA is transmitted. This, portion of the genetic material may represent a major portion of the donor’s genome.