UNIT I: BACTERIAL CULTIVATION AND MAINTENANCES
INTRODUCTION:
* The way to study the microorganisms, satisfactorily is by growing them under more or less well-defined conditions as cultures in the laboratory on artificial media. To achieve a good growth, the exact physical and nutritional requirements must be known.
* Any nutrient preparation employed to grow microorganisms in laboratory is termed as Culture Medium.
* In preparing a culture medium for any microorganisms, the primary goal is to provide a balanced mixture of the required nutrients, at a concentrations that will permit good growth.
* Additionally the cultivation of microorganisms requires careful control of various environmental factors which normally are maintained within narrow limits (pH and Temp.).
* Microbiological culture media are made up of various nutrient substances supporting the growth of particular type of microorganism based on its nutritional category.
* Some media contain solutions of inorganic salts and may be supplemented with one or more organic compounds where as other media are prepared from complex ingredients such as extracts or digests of plant and animal tissues.
* Different microorganisms require different nutrient material for its growth, thus culture media vary in form and composition depending on the species to be cultivated.
PROPERTIES OF GOOD CULTURE MEDIA:
A good culture media should possess the following characteristics:
1. Must give a satisfactory growth from a small inoculums and ideally from a single cell.
2. Should give a rapid growth.
3. Should be easy to prepare.
4. should be reasonably cheap.
5. Should be easily reproducible.
6. Should make it possible to study all the characteristics of the microorganism in which we are interested or to be demonstrated.
Types of Media :
Cultivation of bacteria means the process of growing them on a particular media. The media contains all the necessary nutritional requirements for growth and are termed as culture media. Several media are available which are classified on the basis of .
1. Nature of ingredients and
2. Application and functions.
Types of media on the basis of nature of ingredients :
On the basis of nature of ingredients there are two main types of media :
a) Living Media and
b) Non-living media or bacteriological media.
a) Living Media :
* These are the media which contains living cells. Living media are used for cultivation of viruses, Rickettsia & Chlamydiae.
* These organisms are obligate intracellular parasites and require living cells to grow.
* Living media are of three types :
i) Embryonated Chicken eggs (e.g. Fertile chicken eggs)
ii) Tissue culture (e.g. Cell culture, Monkey kidney cells, Hela cells)
iii) Animals (e.g. Mice, guinea pig, rabbit, hamster, etc).
b) Non-living media or bacteriological media :
b) Non-living media or bacteriological media are the media which contains nonliving materials like pure chemicals or natural ingredients. These media are of various types :
i) Synthetic or chemically defined media
ii) Non-synthetic media or crude media or complex media
iii) Natural
iv) Selective
v) Differential
vi) Enriched
vii) Enrichment
viii) Assay
ix) Minimal
x) Maintenance and
xi) Transport media.
Synthetic Media (chemically defined media)
* These media are also called as chemically defined media because exact chemical composition of these media is known. They are made up of the compound of known chemical composition.
* As the exact chemical nature of all the ingredients is known, it is possible to duplicate the same medium to a high degree of accuracy.
* They have special uses in research and industry.
* These are empolyed where it is desired to know the effect of certain compounds on an organisms.
* These are artificial media of exactly known, reproducible composition and hence called synthetic or chemically defined media.
* For example, a defined medium used for Thiobacillus thiooxidans is as follows :
CaCl2 0.25g
KH2PO4 2g
(NH4)2SO4 3.0g
MgSO4 0.5g
Sulfur powder 10.0g
Distilled water 1000ml
This provides all the needed elements and a source of energy. KH2PO4 serves as a buffer for maintaining the suitable pH. It also serves as source of phosphorous and potassium. The powdered sulfur is a source of energy. It is oxidised to H2SO4. Carbon is obtained as CO2 from atmosphere. Nitrogen is provided by ammonium salt from this simple solution the organisms is able to synthesize proteins, carbohydrates, DNA, RNA, etc.
Application :
1. To know the effect of certain compound on an organisms.
2. To determine nutritional requirements of bacteria.
3. To study metabolism of certain compounds in an organism.
Non-Synthetic media or complex media:
* Non synthetic medias are those whose exact chemical composition is not known.
* This is the usual culture media used by the bacteriologists for cultivation of microorganisms in the laboratory.
* It is practically impossible to prepare two identical sets of the same medium from differect batches of the same ingredients.
* Some of the ingredients used in these media are beef extract, Yeast extract, Peptones, blood, serum, casein hydrolysate etc.
* It can support the growth of wide variety of bacteria and other organisms.
* There-fore, it is employed as a routine culture media for growth of heterotrophs having none or little growth requirements.
* For examples , the complex medium used to grow either Escherichia coli or Leuconostoc mesenteroides is a follows:
Glucose 15g
Yeast extract 5g
Peptone 5g
KH2PO4 2g
Distilled 1,000ml
pH 7.0
* Non- synthetic media often employ digests of casein (milk protein), soybeans, beef, yeast cells or any of a number of highly nutritions but chemically underfined substances.
* Such digets are available commercially in powdered form and can be weighed out rapidly and dissolved in distilled water to prepare a medium.
AGAR-AGAR :
Agar agar (commonly called as agar) is used as the most effective solidifying agent.
Properties of Agar are:
* Derived from certain seaweeds.
*Melts at 95°C and solidifies at 42°C.
* Remains firm at temperatures of incubation.
* Heat labile material can be added to it even at temperature of 45°C thus retaining their potency.
* Bacteriologically inert.
* Resistant to action of all medically important bacteria.
* Easily available.
* Economical.
Natural media:
Natural media are those which are used on the basis of experience and not on the basis of exact composition. Such media are made from natural materials like milk, urine blood, fruit or vegetable juices etc. They are contain soluble organic and inorganic compounds that satisfy nutritional requirements of many microorganisms. These are convenient and inexpensive. Their exact composition is not known.
Types of media based on application and functions:
Microbiologists use basis types of media for cultivation of bacteria, but some specific organisms require specific nutrients e.g. vitamins and other growth promoting substances. Such organisms are known as fastidious heterotrophs. The specific media are useful in identifications, enumeration and isolation of certain types of heterotrophs. On the basis of application and function, the media are classified as:
Selective media :
* Selective media are the media which contain selective agents that permit growth of desired organism and inhibit growth of others.
* Examples of common selective media are azide blood agar, MacConkeys agar, deoxycholate citrate agar.
* For examples, addition of sodium azide at specific concentration into media inhibits gram negative bacteria and selectively allows growth of stapylococcus and streptococcus species.
* Mac Conkeys agar contains selective substance, the bile salt (sodium taurocholate), this substance inhibits growth of non intestinal organisms and allows growth of intestinal organisms.
Differential media :
* Media containing reagents or chemical which differentiate growth of one kind of organism for other kinds are known as differential media.
* The common example of differential media are Mac-Conkey's agar, Eosin methylene blue agar and blood agar.
* The contain dyes, reagents or chemicals which allow the observe to distinguish between types of bacterial colonies developed after incubation.
* For examples, if raw sewage is streaked or spread on eosinmethylene blue (EMB) agar some bacteria produce brilliant green colonies, others gummy, pink colonies with dark centres.
* Blood agar medium is a good differential medium.
* If a mixture of bacteria is inoculated on a blood agar medium, some of the bacteria may hemolyse (destroy) the red blood cells while others do not show hemolytic reactions.
* The observer may see a clear zone of hemolyzed red blood cells around certain colonies colonies of bacteria while the non-hemolyzed bacterial colonies do not develop such a clear zone around them.
Enrichment media :
* Enrichment media are usually liquid media the contain a specific nutrient that will support growth of a desired organisms or inhibitory substances that will suppress the growth of undesired organisms and thus create a favorable environment for desired organisms.
* Enrichment media is that in which nutritional environment is adjusted in such a manner that it enhances selectively the growth of a certain bacterial types within a given mixed population.
* This approach constitutes a powerful tool for the bacteriologist in the isolation and identification of pure cultures from an initially mixed population of bacteria.
* This method is used for those microbes which are in small numbers in the sample and whose growth is slow than other species present.
* The principle of enrichment culture is to control the nutrients and culture conditions (temperature, air supply, light, pH, etc) in such a way that is suits only to the given species.
* If a medium containing salt solution cellulose as sole carbon source is incubated with a sample of garden soil and incubated at 25-30°C, will enrich only cellulose producing organism.
* Cultures of other soil bacteria may be selectively enriched by varying the composition of the medium and the conditions of incubation.
* e.g. Nitrogen Free Mannitol Broth [NFMB].
Enriched media :
* Addition of nutritionally rich ingredient like blood, serum or extract of plants or animal tissues, vitamins to the basic medium like nutrients agar, result in media called enriched media.
* The provide additional nutrients to the fastidious heterotrophs.
* Examples of enriched media are blood agar, egg ,media.
* Blood agar is used for cultivation of streptococcus pyogens, chocolate agar is suitable for Haemophilus influenza. Serum medium is suitable for coryenbacterium diphtheriae.
ASSAY MEDIA:
* Certain media of prescribed composition have profound influence on the bacterial cells with respect to formation of enzymes, toxins, antibiotics and other products.
* Such media are called assay media or media for special purposes.
* An "assay" represents analysis to determine the presence, absence, or quantity of one or more components.
* Example: if assay medium of folic acid is to be prepared, the medium should supply all the requirements of a given species except the vitamin folic acid.
* The given species will not grow on this medium in absence of folic acid.
MAINTENANCE MEDIA:
* These media are different from growth media and are required to maintain the viability and physiological characteristics of bacteria.
* Maintenance media is a specialized media used to maintain the growth and development of bacteria.
* Nutrients supplemented in the maintenance media are sufficient enough to sustain the viability of the organism and not the growth because growth is followed by death.
* Medium in which bacteria are stored as "stock culture" for longer periods to provide a source of viable cultures are called maintenance medium.
* Example: Yeast extract mannitol agar medium.
TRANSPORT MEDIA:
These are special media devised for transporting the specimens containing delicate organisms, which may not survive the time taken for transporting the specimen to the laboratory. or may be overgrown by the non pathogens. E.g. Peptone water (pH 9.8) for Vibrio and Buffered glycerol saline for Enteric bacteria.
MINIMAL MEDIA:
A culture medium for microorganisms that contains the minimum nutrient required for growth of the wild type strain or It is a medium containing only inorganic salt, carbon source and water is called as minimal medium. These medium normally contains minimum nutrients possible for growth, generally without growth factors.
BUFFER IN CULTURE MEDIA:
* An buffer is a solution containing either a weak acid and a conjugate base or weak base and a conjugate acid . or Buffer is a solution which resists changes in pH when acid or alkali is added to it.
* Many bacteria produce metabolic acid that lower the pH and inhibit their growth. To prevent this, buffer that produce a pH equilibrium are added to culture media to neutralize these acids.
* Biological buffers are typically added to culture media to maintain the pH of a culture and to avoid undesirable fluctuations of pH due to cell metabolism that may have negative effect on cell parameters.
* e.g. Acetate buffer [Acetic acid and Sodium acetate]
* Phosphate buffer [K2HPO4 and KH2PO4].
ANAEROBIC CULTIVATION: Media & Methods of Cultivation
* Obligate anaerobes are the microorganisms that do not require oxygen for growth.
* In fact, oxygen kills or inhibits their growth.
* In absence of oxygen they grow by fermentation, anaerobic respiration, bacterial photosynthesis or methanogenesis.
* Examples include Clostridium haemolyticum, Clostridium botulinum, Clostridium tetani etc.
*Cultivation of anaerobes requires removal of oxygen from environment.
* The oxygen from culture environment is removed either by combining it with reducing agents or by mechanical exclusion.
* Many of the anaerobic procedures used, employ combination of both,
*Anaerobic conditions can be accomplished by using: (a)Anaerobic media (RCMM) (b)Candle jar, (c)Anaerobic jar (d) Roll tube (e) Anaerobic glow box.
ANAEROBIC MEDIA:
* Anaerobic media such as Thioglycolate agar/broth, Robertson’s Cooked meat media [RCMM] are commonly used for the cultivation of anaerobes.
* These media contains reducing agents like sodium thioglycolate and cysteine.
* Cooked meat broth is an excellent medium because it contains many reducing agents as well as nutrients. Reducing agents react with oxygen and reduce it to water and make the medium anaerobic.
* To ensure complete anaerobic condition, cultures are incubated in anaerobic jar.
CANDLE JAR:
* The candle jar is a large screw-capped airtight glass container.
* The culture plates and candle is placed inside the jar. * The candle is lit and the jar is sealed.
* The candle will burn and reduce the oxygen concentration.
* The jar atmosphere has less than 10% oxygen and about 3% carbon dioxide.
* The candle jar is commonly used to create microaerophilic conditions.
ANANROBIC JARS:
* All anaerobic jars have same basic principle. The oxygen present in the jar is removed by reaction with hydrogen added in presence of catalyst.The oxygen combines with hydrogen to from water.
* There are two types to anaerobic jars: (a) Brewer’s Jar (b)Gaspak anaerobic jar.
THE BREWER’S JAR :
* The Brewers jar is convenient for small scale growth studies.
* The jar has a lid fitted with an outlet.
* Air is evacuated from the sealed jar containing the culture plates and is replaced with an oxygen free mixture of 85 % nitrogen, 10% hydrogen and 5 % carbon dioxide.
* A catalyst in the lid catalyzes the reduction of any traces of oxygen that may remain.
Gaspak Anaerobic jar:
* Gaspak anaerobic jar is a plastic or glass vessel fitted with a lid.
* The vessel contains and envelope that generates gas a pall adium catalyst holder attached to the underside of the lid of the jar and a pad soakedwith methylene blue (anaerobic indicator strip).
* Gaspak is most commonly used anaerobic system for cultivation of strict anaerobes.
* The plates and tubes to be incubated are placed in the jar and anaerobic condition is created by adding 10 ml water to a gas generator envelope that is placed in the jar just before the lid is clampled down tightly. The envelope contains sodium borohydride and sodium bicarbonate.
* Water reacts with these chemicals and produce hydrogen and carbon dioxide gases
* The hydrogen gas combines with free oxygen in the jar and produce water.
* Palladium coated alumina pellets attached to the underside of the lid catalyze the reaction.
* The concentration of carbondioxide in the jar is about 4-10%.
* Carbondioxide generated is required for growth by some anaerobes and stimulates the growth of others.
* A methylene blue indicator strip turns colorless when the oxygen has been removed.
ROLL TUBE METHOD:
* This method is used for isolation and cultivation of obligate anaerobes that cannot be recovered by using the anaerobic jar.
* The roll tube is essentially a stopered test tube containing oxygen free gas and a thin layer of pre –reduced agar medium on its inside surface.
* The medium in the tube is inoculated with a loop while the tube is rotated.
* The tube is opened during inoculation.
* The entry of oxygen into the tube is prevented by continuously flushing a stream of oxygen free gas (N2 of CO2) through it.
* Each tube represents a sophisticated anaerobic container.
Pure culture Techniques:
Culture: It is the term usually employed for deliberate growth of microorganism in laboratory.
Cultivation: it is the process of inducing microbes to grow. Pure Culture: “A culture containing only a single type (species) of microbes is called as pure culture.”
Colony: Growth of identical cells in mass is called as colony
“A culture containing a growth of a single kind of organism free from other organisms” is Called as Pure Culture.
Pure Culture Methods:
* To determine the characteristics of a particular selected organism, it is essential that the organism should be isolated and grown in the laboratory as a pure culture. Numbers of techniques have been employed for the isolation of microorganisms for the natural environment in pure culture.
* In order to obtain pure culture of any microorganism, method to be used must ensure the introduction of a single cell into a sterile growth medium in a suitable culture vessel. The small size of most microbes makes mechanical separation of single cells impossible.
* Though, recent development using micromanipulator has made this possible, but it is a difficult task and specialized instrument are needed. Consequently, other methods needs to be discovered which will effectively dilute a mixed sample so that single cells can be obtained that could be grown to get a pure culture.
* Earlier methods were based upon a dilution of the culture until an aliquot was likely to contain theoretically a single cell as judged from an initial count.
* However, such methods are tedious, unreliable and can only be used for the isolation of dominant organisms from the mixed culture. These methods are now rarely used.
* Microbiologists began to find the possibility of diluting mixed culture on a solid media.
* The sample is placed at one point on a sterile solid growth medium and then, using a sterile needle, the sample (or inoculum) is drawn several times over the surface (as in streak-plate method).
* Each streak represents a dilution process and eventually single cells are obtained along the streak.
*Each of these, on incubation, grows up into a separate colony, which can be used for pure culture. This, streak method was pioneered by Robert Koch.
* An alternative to streak method is pour plate method. Here the diluted sample is mixed with a previously melted agar growth medium at a temperature just above the solidifying point.
* The mixture is poured into a suitable vessel and is incubated. Each cell produces a colony within the agar.
* Following are the most commonly used methods of pure cultures in routine laboratory work:
(i) Streak plate method
(ii) Spread plate method
(iii) Pour plate method and
(iv) Single cell isolation
Streak Plate Method:
* Streak plates are prepared by streaking a loopfull of mixed culture over the surface of solid medium in a petridish with an transfer loop (nichrome wire loop) in such a way as to provide successive dilution. That is this procedure thins out the bacteria on the solid medium so that some of the bacteria are separated from each other and form isolated colonies on the surface of solid agar medium.
* After overnight incubation, colonies develop or originate from a single cell and thus creating pure culture.
* Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/ needle and restreaked onto fresh media to ensure purity. After overnight incubation, surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium.
Various methods of streaking:
a) Parallel non- overlapping streak method b)four quadrant streak method c)Back and forth streak method d) T- plate method.
i) Parallel non-overlapping streak plate method: In this method a sterile wire loop is dipped into a suitably diluted mixed culture suspension and then wire loop is used to make series of parallel, non- overlapping streaks on the surface of agar. The suspension is progressively diluted with each successive streak. Initial streaks may give confluent growth but latter streaks will give well isolated colonies.
ii) Four quadrant streak plate method: In this method, a loopful of mixed culture is placed on the surface of agar medium and streaked over a fourth of the agar plate surface. The wire loop is then flamed and another portion of the plate is streaket at right angles to original streaking. This will drug the bacteria from the streak, diluting the number present with a sterile loop. Similarly, a third streak is made in the same manner at right angles to the second streak, further diluting the bacteria. A fourth streak is made with a sterile wire loop at right angles to and cross the third streak. Initial quadrants give confluent growth but later quadrents gives well isolated colonies.
iii) Back & forth streak plate method: In this method, a loop ful suspension is streaked on the surface of the agar about half centimeter apart. At the terminal end, there will be isolation. Each isolated colony is the progeny of a single cell and hence a pure culture. This method has advantages that it is relatively simple, quick, takes little apparatus and works effectively in most instances. It has the advantages that one is not quite sure that each isolated colony came from a single cell.
iv) T-Plate method: In this method, agar plate is marked with three separate region as shown in figure as growth zone, sterile zone and streak zone. First a loop full of mixed suspension is taken aseptically and placed in the region marked as growth zone, then with the same loop, culture is streaked in the streaking zone without touching the intermediate sterile zone in the form of T alphabet. Now the loop is sterilized and streaking is done in a zig zak manner in the marked area to thin out the population to get well isolated colonies.
Advantages of Streak Plate Method:
* Relatively simple
* Require little material & apparatus
* Gives pure culture within a short time
* gives surface colonies.
Spread Plate Method:
* In this method the mixed culture of microorganisms is diluted in a series of tubes containing sterile liquid, usually, water or physiological saline (0.85% NaCl) so that few cells will be present per ml of sample. A drop of (0.01 ml) diluted liquid from each tube is placed on the surface of an sterile agar plate and spread evenly over the surface by means of a sterilized bent-glass
* rod (Spreader). The medium is now incubated for 24 hours.
* When the colonies develop on the agar plates, it is found that there are some plates in which well-isolated colonies are formed.
* This happens as a result of separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate.
* These well isolated colonies can be picked up for getting pure culture.
Pour Plate Method:
* This method involves plating of diluted samples mixed with melted agar medium.
* The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium (42-45°C) so as to permit a thorough distribution of bacterial cells within the medium.
* Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C).
* The bacteria and the melted medium are mixed well.
* The contents of each tube is poured into separate Petri plates, allowed to solidify, and then incubated for 24 hours.
* After incubation, bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies).
* These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity.
Pour plate method has certain disadvantages as follows:
* The picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and
* The microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms.
* However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture.
* The isolated colonies are picked up and transferred onto fresh medium to ensure purity.
Single Cell Isolation:
* The ideal way to obtain a pure culture from a mixed population of microorganisms would be to pick out individual cells of the kind wanted.
* Single cells can be isolated by a micromanipulator.
* The micromanipulator is a special equipment which can be used in conjugation with a microscope to pick a single bacterial cell from a hanging drop preparation and suspend that cell into sterile nutrient media so as to get pure culture.
* Droplets containing individual bacterial cells are deposited on a cover glass in a special chamber.
* The droplets are then transferred by sterile micropipettes to suitable nutrient media.
Significance of Pure Culture:
Following are some of the significant points of pure culture
1. With pure culture one can satisfactorily determine the characteristics of particular species of microorganisms.
2. pure culture makes easy the systematic study of the organism i.e. one can identify the organism up to species level only when it is in pure form.
3. Pure culture makes easy the study of cultural and biochemical characteristics of the organsism. i.e size, shape, elevation, surface, color etc.
4. Nutritional requirements of the organisms can be found out with ease and studied.
5. A pure culture is of great importance in the field of industrial microbiology for production of different important products like amino acids, vitamins, antibiotics etc.
6. Genetic characteristics of the organism can be studied only when the organism is in pure form ie pure culture.