Culture Media, Isolation,presesrvation

REVIEWER

Types of Bacteriological Culture Medium Types of Bacteriological Culture Medium Culture media are solutions containing all of the nutrients and necessary physical growth parameters necessary for microbial growth. All microorganisms cannot grow in a single culture medium and, in fact, many can't grow in any known culture medium. Organisms that cannot grow in artificial culture medium are known as obligate parasites. Mycobacterium leprae, rickettsias, Chlamydias, and Treponema pallidum are obligate parasites. Bacterial culture media can be distinguished on the basis of composition, consistency and purpose. Classification based on consistency 1.

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Solid medium solid medium is media containing agar (at a concentration of 1.5-2.0%) or some other, mostly inert solidifying agent. Solid medium has physical structure and this allows bacteria to grow in physically informative or useful ways (e.g. as colonies or in streaks).solid medium is useful for isolating bacteria or for determining the characteristics of colonies. Semisolid media They are prepared with agar at concentrations of 0.5% or less. They have soft custard like consistency and are useful for the cultivation of microaerophilic bacteria or for determination of bacterial motility. Liquid (Broth) medium These media contains specific amounts of nutrients but don’t have trace of gelling agents such as gelatin or agar. Broth medium serves various purposes such as propagation of large number of organisms, fermentation studies, and various other tests. eg. Sugar fermentation tests, MR-VR broth.

Classification based on the basis of composition 1.

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Synthetic or chemically defined medium A chemically defined medium is one prepared from purified ingredients and therefore whose exact composition is known. Non synthetic or chemically undefined medium

Non-synthetic medium contains at least one component that is neither purified nor completely characterized nor even completely consistent from batch to batch. Often these are partially digested proteins from various organism sources. Nutrient broth, for example, is derived from cultures of yeasts. Synthetic medium may be simple or complex depending up on the supplement incorporated in it. A simple non-synthetic medium is capable of meeting the nutrient requirements of organisms requiring relatively few growth factors where as complex non-synthetic medium support the growth of more fastidious microorganisms. Classification based on the basis of purpose/ functional use/ application many special purpose media are needed to facilitate recognition, enumeration, and isolation of certain types of bacteria. To meet these needs, numerous media are available. On the basis of their application or function, they are classified as follows, 1. General purpose media/ Basic media Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar considered basal medium. These media are generally used for the primary isolation of microorganisms.

2. Enriched medium (Added growth factors):

Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched media. Blood agar is preparing by adding 5-10% (by volume) to a basal medium such as nutrient agar or other blood agar bases. Chocolate agar is also known as heated blood agar or lysed blood agar. 3. Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the pathogen. Various approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these. a. Selective medium Principle: Differential growth suppression Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of others (i.e., they select for certain microbes).Solid medium is employed with selective medium so that individual colonies may be isolated.

Examples of selective media include: Thayer Martin Agar used to recover N.gonorrhoeae contains Vancomycin, Colistin and Nystatin. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contain 10% NaCl. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% Potassium tellurite.

McConkey’s Agar used for Enterobacteriaceae members contains Bile salt that inhibits most gram positive bacteria. Pseudosel Agar (Cetrimide Agar) used to recover P.aeruginosa contains cetrimide (antiseptic agent). Crystal Violet Blood Agar used to recover S.pyogenes contains 0.0002% crystal violet. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating Malachite green. Wilson and Blair’s Agar for recovering S.typhi is rendered selective by the addition of dye Brilliant green. Selective media such as TCBS Agar used for isolating V. cholerae from fecal specimens have elevated pH (8.5-5.6), which inhibits most other bacteria. b. Enrichment culture/ Medium Enrichment medium is used to increase the relative concentration of certain microorganisms in the culture prior to plating on solid selective medium. Unlike selective media, enrichment culture is typically used as broth medium. Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone water are used to recover pathogens from fecal specimens. 4. Differential/ indicator medium: Differential appearance:

Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media are called differential media or indicator media Differential media allow the growth of more than one microorganism of interest but with morphologically distinguishable colonies. Examples of differential media include:

Mannitol salts agar (mannitol fermentation = yellow) Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis) Mac Conkey agar (lactose fermenters, Pink colonies whereas non- lactose fermenter produces pale or colorless colonies. TCBS (Vibrio cholera produces yellow colonies due to fermentation of sucrose) 5. Transport media: Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory factors. Cary Blair medium and Venkatraman Ramakrishnan (VR) medium are used to transport feces from suspected cholera patients. Sach’s buffered glycerol saline is used to transport

feces from patients suspected to be suffering from bacillary dysentery. Pike’s medium is used to transport streptococci from throat specimens. 6. Anaerobic media: Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation –reduction potential and extra nutrients.

Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced. Before use the medium must be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin. Robertson cooked meat that is commonly used to grow Clostridium spps medium contain a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate. Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the medium. Under reduced condition, methylene blue is colorless. 7. Assay media These media are used for the assay of vitamins, amino acids and antibiotics. E.g. antibiotic assay media are used for determining antibiotic potency by the microbiological assay technique.

Other types of medium includes Media for Enumeration of Bacteria, Media for characterization of Bacteria, Maintenance media etc.

Techniques of isolation and Enumeration of Bacteria Enumeration of microorganisms is especially important in dairy microbiology, food microbiology, and water microbiology.

Direct Microscopic count/ Total cell count direct microscopic counts are possible using special slides known as counting chambers, consisting of a ruled slide and a cover slip. It is constructed in such a manner that the cover slip, slide, and ruled lines delimit a known volume. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted.

Bacteria can be counted easily and accurately with the petroff-Hausser counting chamber. This is a special slide accurately ruled into squares that are 1/400 mm2 in area; a glass cover slip rests 1/50 mm above the slide, so that the volume over a square is 1/20,000 mm3i.e.

1/20, 000, 000 cm3. If for example, an average of five bacteria is present in each ruled square, there is 5 x 20,000,000 or 108, bacteria per milliliter. A suspension of unstained bacteria can be counted in the chamber, using a phase-contrast microscope.

The formula used for the direct microscopic count is: The number of bacteria per cc = The average numbers of bacteria per large doublelined square X The dilution factors of the large square (1,250,000) X The dilution factor of any dilutions made prior to placing the sample in the counting chamber, e.g., mixing the bacteria with dye Advantage of Direct Microscopic count 1. 2. 3.

Rapid, Simple and easy method requiring minimum equipment. Morphology of the bacteria can be observed as they counted. Very dense suspensions can be counted if they are diluted appropriately.

Limitations of Direct Microscopic count 1. 2.

Dead cells are not distinguished from living cells. Small cells are difficult to see under the microscope, and some cells are probably missed.

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Precision is difficult to achieve A phase contrast microscope is required when the sample is not stained. The method is not usually suitable for cell suspensions of low density i.e. < 107 Cells per ml, but samples can be concentrated by centrifugation or filtration to increase sensitivity. Viable count/ plate count technique

Dilution:

with both the spread plate and pour plate methods, it is important that the number of colonies developing on the plates not be too large because on crowded plates some cells may not form colonies and some colonies may fuse, leading to erroneous measurements. It is also essential that the number of colonies not be too small, or the statistical significance of the calculated count will be low. The usual practice, which is the most valid statistically, is to count colonies only on plates that have between 30 and 300 colonies. The number of bacteria in a given sample may be usually too great to be counted directly. To obtain the appropriate colony number, the sample be counted must almost always diluted in such a manner that single isolated bacteria form visible isolated colonies , the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution. Several 10-fold dilutions of the sample are commonly used. In most cases, serial dilutions are employed to reach the final desired dilution. However, if the organism normally forms multiple cell arrangements, such as chains, the colony-forming unit may consist of a chain of bacteria rather than a single bacterium. In addition, some of the bacteria may be clumped together. The development of one colony can occur even when the cells are in aggregates. i.e. cocci in clusters (staphylococci), chains (streptococci), or pairs (diplococci), the resulting counts will be lower than the number of individual cells. Each colony that can be counted is called a colony forming unit (CFU). By extrapolation, this number can in turn be used to calculate the number of CFUs in the original sample rather than number of bacteria per milliliter. The assumption made in this type of counting procedure is that each viable cell can yield one colony. There are two ways of performing a plate count: the spread plate method and the pour plate method. Generally, one wants to determine the number of CFUs per milliliter (ml) of sample. To find this, the number of colonies (on a plate having 30-300 colonies) is multiplied by the number of times the original ml of bacteria was diluted (the dilution factor of the plate counted). For example, if a plate containing a 1/1,000,000 dilution of the original ml of sample shows 150 colonies, then the number of CFUs per ml in the original sample is found by multiplying 150 x 1,000,000 as shown in the formula below: The number of CFUs per ml of sample = The number of colonies (30-300 plate) X The dilution factor of the plate counted In the case of the example above 150 x 1,000,000 = 150,000,000 CFUs per ml This method is used to count only live (viable) cells. A viable cell is defined as one that is able to divide and form off springs, and the usual way to perform a viable count is to determine the number of cells in the sample capable of forming colonies on a suitable agar medium. For this reason, the viable count is often called the

plate count, or colony count. This method is used to enumerate bacteria in milk, water, foods, soils; cultures etc and the number of bacteria are expressed as colony-forming units (CFU) per ml. Advantage of plate count method this method is used routinely and with satisfactory results for the estimation of bacterial populations in milk, water, foods, and many other materials. 1.

Its sensitivity (theoretically, a single cell can be detected), and it allows for inspection and positive identification of the organism counted. 2. It is easy to perform and can be adapted to the measurement of populations of any magnitude. 3. It is sensitive method, since small numbers of organisms can be counted. Eg. If a specimen contains as few as one bacterium per ml, one colony should develop up on the plating of 1 ml Limitation of plate count technique (1) Only living cells develop colonies that are counted; (2) clumps or chains of cells develop into a single colony; (3) colonies develop only from those organisms for which the cultural conditions are suitable for growth.

Types of Techniques; Pour plate technique, Spread plate technique and Streak plate technique Pour plate Technique A pour plate is a method of melted agar inoculation followed by petri dish incubation. A known volume (usually 0.1-1.0 ml) of culture is pipette into a sterile petri plate; melted agar medium is then added and mixed well by gently swirling the plate on the table top. Because the sample is mixed with the molten agar medium, a larger volume can be used than with the spread plate. However, with the pour plate method the organism to be counted must be able to briefly withstand the temperature of melted agar, 45oC.

The cultures are inoculated into melted agar that has been cooled to 45oC. The liquid medium is well mixed then poured into a petri dish (or vice versa) Colonies form within the agar matrix rather than on top as they do when streaking a plate. Pour plates are useful for quantifying microorganisms that grow in solid medium. Because the "pour plate" embeds colonies in agar it can supply a sufficiently oxygen deficient environment that it can allow the growth and quantification of microaerophiles. Spread Plate Technique Spread plate technique is an additional method of quantifying microorganisms on solid medium. With the spread plate method, a volume of an appropriately diluted culture usually no greater than 0.1 ml is spread over the surface of an agar plate using a sterile glass spreader. The plate is than incubated until the colonies appear, and the number of colonies counted. Instead of embedding microorganisms into agar, as is done with the pour plate method, liquid cultures are spread on the agar surface.

An advantage of spreading a plate over the pour plate method is that cultures are never exposed to 45 oC (i.e. melted agar temperatures). Note: Surface of the plate must be dry, so that the liquid that is spread soaks in. volume greater than 0.1ml are rarely used because the excess liquid does not soak in and may cause the colonies to coalesce as they from, making them difficult to count. Streak Plate Technique For organisms that grow well on agar plate, streak plate is the method of choice for obtaining pure culture. The key principles of this method is that, by streaking, a dilution gradient (number of cells decrease as they move across the agar and away from the point of inoculation) is established across the face of the plate as bacterial cells are deposited on the agar surface. Because of this dilution gradient, confluent growth occurs on part of the plate where the bacterial cells are not sufficiently separated; in other regions of the plate where few bacteria are deposited separate macroscopic colonies develop that can easily be seen with naked eye. Each well isolated colony is assumed to arise from a single bacterium and therefore to represent a clone of a pure culture.

Purpose of Streak Plate Technique: The purpose of the streak plate is to obtain isolated colonies from an inoculum by creating areas of increasing dilution on a single plate. Isolated colonies represent a clone of cells, being derived from a single precursor cell.

Many different streaking patterns can be used to separate individual bacterial cells on the agar surface. Methods of making a streak plate to obtain pure cultures. 1. Loop is sterilized, and then a loopful of inoculums is removed from tube 2. A loopful of bacterial cells is streaked across the surface of a sterile solidified nutrient medium. 3. Following the initial streak, subsequent streaks are made at angles to it, the loop being sterilized between streaks. 4. The plates are than incubated under favorable conditions to permit growth of the bacteria. 5.

After incubation colonies appear along the points of the streak. It is from such well isolated colonies that pure cultures can usually be obtained.

Maintenance and Preservation of Pure Cultures of Bacteria Once a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure cultures free from contamination. Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium (sub culturing) to allow continuous growth and viability of microorganisms. The transfer is always subject to aseptic conditions to avoid contamination. Since repeated sub culturing is time consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time. In addition, there is a risk of genetic changes as well as contamination. Therefore, it is now being replaced by some modern methods that do not need frequent sub culturing. These methods include refrigeration, paraffin method, cryopreservation, and lyophilization (freeze drying). Periodic Transfer to Fresh Media Strains can be maintained by periodically preparing a fresh culture from the previous stock culture. The culture medium, the storage temperature, and the time interval at which the transfers are made vary with the species and must be ascertained beforehand. The temperature and the type of medium chosen should support a slow rather than a rapid rate of growth so that the time interval between transfers can be as long as possible. Many of the more common heterotrophs remain viable for several weeks or months on a medium like nutrient agar. The transfer method has the disadvantage of failing to prevent changes in the characteristics of a strain due to the development of variants and mutants. Refrigeration Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their

growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime. Paraffin Method/ preservation by overlaying cultures with mineral oil This is a simple and most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin in poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture can be preserved form months to years (varies with species). The advantage of this method is that we can remove some of the growth under the oil with a transfer needle, inoculate a fresh medium, and still preserve the original culture. The simplicity of the method makes it attractive, but changes in the characteristics of a strain can still occur. Cryopreservation Cryopreservation (i.e., freezing in liquid nitrogen at -196°C or in the gas phase above the liquid nitrogen at -150°C) helps survival of pure cultures for long storage times.

In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol or

Dimethyl Sulfoxide (DMSO) that prevent the cell damage due to formation of ice crystals and promote cell survival. This liquid nitrogen method has been successful with many species that cannot be preserved by lyophilization and most species can remain viable under these conditions for 10 to 30 years without undergoing change in their characteristics, however this method is expensive. Lyophilization (Freeze-Drying) In this method, the culture is rapidly frozen at a very low temperature (70°C) and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the microbes go into dormant state and retain viability for years. Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in refrigerators. Freeze-drying method is the most frequently used technique by culture collection centers. Many species of bacteria preserved by this method have remained viable and unchanged in their characteristics for more than 30 years.

Advantage of Lyophilization *Only minimal storage space is required; hundreds of lyophilized cultures can be stored in a small area

* small vials can be sent conveniently through the mail to other microbiology laboratories when packaged in a special sealed mailing containers * Lyophilized cultures can be revived by opening the vials, adding liquid medium, and transferring the rehydrated culture to a suitable growth medium.