MICROBIAL CULTIVATION

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MICROBIAL CULTIVATION AND ISOLATION TECHNIQUES

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By: arvane (21 month(s) ago)

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MAEER’S, MAHARASHTRA INSTITUTE OF PHARMACY,PUNE-38. MICROBIAL CULTIVATION Prepared By Prof. S. A. Polshettiwar M.Pharm, D.I.T.(C-DAC), PhD.,F.I.E.A

Cultivation: 

Cultivation The process of growing microorganisms in culture by taking bacteria from the infection site (in vivo or environment) and grow them in artificial environment in the laboratory (in vitro).

MICROBIAL CULTIVATION : 

MICROBIAL CULTIVATION Bacteria may require adequate nutrition , optimum pH , temperature and oxygen for growth and multiplication. Suitable artificial media containing sources of carbon, nitrogen, hydrogen, oxygen, phosphorous and other elements such as sodium, potassium, magnesium, iron and growth factor (Vitamins) in very small amounts have been used for cultivation of microorganism.

MICROBIAL CULTIVATION : 

MICROBIAL CULTIVATION When microorganisms are cultivated in the laboratory, a growth environment called a medium is used. The medium may be purely chemical (a chemically defined medium), or it may contain organic materials, or it may consist of living organisms such as fertilized eggs. Microorganisms growing in or on such a medium form a culture. A culture is considered a pure culture if only one type of organism is present and a mixed culture if populations of different organisms are present. When first used, the culture medium should be sterile, meaning that no form of life is present before inoculation with the microorganism.

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Definition: it is artificially prepared media which contains all essential nutrient to provide for growth and multification of particular microorganism. There are two different kinds of media, defined media which contain peptone , and undefined composition. The majority of the commonly used culture media are commercially available as dehydrated products. Which are reconstituted by the addition of distilled water and then sterilised in the Culture media

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Nutrient media This is an undefined medium because the amino acid source contains a variety of compounds with the exact composition being unknown. Nutrient media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections. Physcomitrella patens plants growing axenically on agar plates ( Petri dish , 9 cm diameter). An undefined medium (also known as a basal or complex medium) is a medium that contains: Defined media (also known as chemically defined media or synthetic media) Differential medium

General Characteristics of culture Media : 

General Characteristics of culture Media Must give satisfactory and rapid growth from single culture Should maintain pH during storage Reasonably cheap and easily available Maintain sterility throught expt.

Media for Purpose: 

Media for Purpose 1-Media for isolation 2-Selective or inhibitory media 3-Enrichment media 4-Media for maintenance 5-Media for determining nutritional requirements or ability to use a substrate 6-Media for characterization 7-Media for screening 8-Media for microbiological assay of vitamins and amino acids 9-Non-nutrient basal media

Uses of Media: 

Uses of Media Cultivation and isolation Identification Sterility test Preservative efficacy test Evaluation of disinfectant To check the antimicrobial susceptabilty

Classification of M. O. on the basis of nutritional requirement: 

Classification of M. O. on the basis of nutritional requirement 1.Source of energy: derive their energy from sunlight are called Phototrophs Obtained energy from chemical reaction called as chemotrophs ( E.coli) 2. Source of electron: bacteria reduced inorganic compounds as a electron donars are called as lithotrophs .( e.g- Pseudomonas), organic compound called as organotrophs . 3.source of corbon: autotrophs 4. source of Nitrogen Source of sulphur, Phosphorus, mineral salt and growth factor.

COMMON INGREDIENTS OF MEDIA : 

COMMON INGREDIENTS OF MEDIA Agar Peptone Yeast extract Meat Extract Water NaCl

COMMON INGREDIENTS OF MEDIA : 

COMMON INGREDIENTS OF MEDIA Agar: Solidifying agent Peptone: Source of Nitrogen Yeast extract: as a source of vitamins. Meat Extract: Vitamins, corbohydrates. Water: uptake of nutrients NaCl: To maintain isotonicity

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Microbial Growth and Culture Media Solid Media : Nutrient material that contains a solidifying agent (plates, slants, deeps). The most common solidifier is agar, first used by Robert Koch. Unique Properties of Agar: Melts above 95 o C. Once melted, does not solidify until it reaches 40 o C. Cannot be degraded by most bacteria. Polysaccharide made by red algae. Originally used as food thickener (Angelina Hesse ).

MEDIA CONSTITUENTS: 

MEDIA CONSTITUENTS Agar Peptone Yeast extract Blood Plasma Serum Bile Bile salts Gelatine Carbohydrates Defined media

Composition of culture media: : 

Composition of culture media: Bacteria infecting humans (commensals or pathogens) are chemoorganoheterotrophs. When culturing bacteria, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat. Hence, an artificial culture medium must provide all the nutritional components that a bacterium gets in its natural habitat. Most often, a culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors. Besides these, the pH of the medium must be set accordingly. Some of the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract and malt extract. Classification: Bacterial culture media can be classified in at least three ways; Based on consistency, based on nutritional component and based on its functional use. 1) Classification based on consistency: Culture media are liquid, semi-solid or solid and biphasic. A) Liquid media: These are available for use in test-tubes, bottles or flasks. Liquid media are sometimes referred as “broths” (e.g nutrient broth). In liquid medium, bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on the surface of undisturbed broth. Bacillus anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen © Sridhar Rao P.N (www.microrao.com)in penumococci. Long chains of Streptococci when grown in liquid media tend to entangle and settle to the bottom forming granular deposits. Liquid media tend to be used when a large number of bacteria have to be grown. These are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can be used to obtain viable count (dilution methods). Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria can not be detected.

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B) Solid media: Any liquid medium can be rendered by the addition of certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent. It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium. Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95oC (sol) and solidifies at 42oC (gel), doesn’t contribute any nutritive property, it is not hydrolyzed by most bacteria and is usually free from growth promoting or growth retarding substances. However, it may be a source of calcium & organic ions. Most commonly, it is used at concentration of 1-3% to make a solid agar medium. New Zealand agar has more gelling capacity than the Japanese agar. Agar is available as fibres (shreds) or as powders. C) Semi-solid agar: Reducing the amount of agar to 0.2-0.5% renders a medium semi-solid. Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains (U-tube and Cragie’s tube). Certain transport media such as Stuart’s and Amies media are semi-solid in consistency. Hugh & Leifson’s oxidation fermentation test medium as well as mannitol motility medium are also semi-solid. D) Biphasic media: Sometimes, a culture system comprises of both liquid and solid medium in the same bottle. This is known as biphasic medium (Castaneda system for blood culture). The inoculum is added to the liquid medium and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid medium. This obviates the need for frequent opening of the culture bottle to subculture. Besides agar, egg yolk and serum too can be used to solidify culture media. While serum and egg yolk are normally liquid, they can be rendered solid by coagulation using heat. Serum containing medium such as Loeffler’s serum slope and egg containing media such as Lowenstein Jensen medium and Dorset egg medium are solidified as well as disinfected by a process of inspissation.

TYPES OF CULTURE MEDIA: 

TYPES OF CULTURE MEDIA Based on the physical state Liquid medium : ( Absence of Agar) Without agar. for the proliferation of bacteria. e.g. Fluid Thioglycolate medium Solid medium: 1.5 to 2.5% agar. for the isolation and identification of bacteria e.g., slant, Petri dishes/plates. e.g. Nutrient agar Semisolid medium: 0.3-0.5% agar. for the observation of bacterial motility and preservation of bacteria. Eg . Nutrient Broth

Bacterial growth patterns : 

18 Bacterial growth patterns In liquid medium : Superficial growth; Turbidity/diffuse; Precipitate growing; (sediment) In solid medium : Confluent growth / Smear; Colony : a cluster of microorganisms growing on a solid medium. It is directly visible and arises from a single cell.

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19

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20 In semi-solid medium: Only grow along the line of inoculation Grow diffusely

Streak-plate technique : 

21 Streak-plate technique four-area streak plate technique IV II I 1/5 I 1/10 Rotate plate 90  Flame loop Rotate 90  Rotate 90  III 1/4 Flame loop

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22 Slant inoculation

Liquid medium inoculation: 

23 Liquid medium inoculation

Colony- (clone): 

Colony- (clone) Colony - A bacterial population derived from one bacterial cell. The cells within the colony have identical, genus, species, genetic and phenotypic characteristics. Pure bacteria - derived from a single colony. Selection of a pure colony -most important for bacterial identification

TYPES OF CULTURE MEDIA: 

TYPES OF CULTURE MEDIA B. Depending on oxygen requirement i. Aerobic media e.g. MacConkey’s Broth i. Anaerobic media e.g. Roberson’s cooked medium C. Depending on chemical composition i. Simple or Basal media ii. Synthetic or defined media iii. Nonsynthetic or complex media

TYPES OF CULTURE MEDIA: 

TYPES OF CULTURE MEDIA D. Depending on Functional type: i. Enriched media ii. Enrichment media iii. Selective media iv. Indicator media v. Differential media vi. Assay media vii. Transport media viii. Storage media

SPECIAL MEDIA: 

SPECIAL MEDIA ENRICHED MEDIA: (Liquid form) Prepared for fastidious micoorganism by addition of substances such as blood, serum and egg to basal medium. e.g. Blood Agar ( Streptococcus ), Chocolate Agar ( Neisseria, haemophilus)

Blood agar plate (BA): 

Blood agar plate (BA) Nutrient agar with 5% sheep blood Cultivation of fastidious and non fastidious bacteria. Differential – Identify hemolysis - Some bacteria secrete enzymes that lyse red blood cells (hemolysins) such that a clearing around the colony appears. b hemolysis- complete clearing (white hemolysis) a hemolysis – incomplete clearing (green hemolysis) g hemolysis- no hemolysis

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II. ENRICHEMENT MEDIA: When a Specific substances is added in a liquid medium which medium inhibits the growth of unwanted bacteria and favours the growth of wanted bacteria. e.g. Tetrathionate broth (inhibits the growth of E.Coli), allow Salmonella

MacConkey Agar (MAC) : 

MacConkey Agar (MAC) Selective and differential medium. Selective - Gram positive bacteria are inhibited by the presence of bile salts and crystal violet inhibitors in the medium Most of gram negative bacteria will grow. Differentiate - Between Gram negative bacteria by their ability to ferment lactose . Pink colonies- Bacteria that ferment lactose (precipitation of some salts in media by acid production). Pale colonies- Non fermenters

Eosine Methylene blue (EMB), : 

Eosine Methylene blue (EMB), Differentiatial between lactose fermenting and non fermenting enteric bacteria

Tellurite Glycine Agar (TGA): 

Tellurite Glycine Agar (TGA) Selective- Tellurite glycine and lithium inhibit most bacteria Preferential growth of Staphyloccoci coagulase positive (Staphyloccocus aureus)

Blood agar plates are often used to diagnose infection. On the right is a positive Streptococcus culture; on the left a positive Staphylococcus: 

Blood agar plates are often used to diagnose infection. On the right is a positive Streptococcus culture; on the left a positive Staphylococcus

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III. SELECTIVE MEDIA: ( Solid form) favor the growth of particular microorganism and inhibit the growth of others.. e.g. MacConkeys agar ( E.Coli), Jensen Agar medium ( Mycobacterium Tuberculosis)

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IV. INDICATOR MEDIA :. e.g. Wilson and Blair S. Typhi Reduces sulphite to sulphide in the presence of glucose and the colonies have black metallic shine.

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DIFFERENTIAL MEDIA : Used to distinguish between different types of bacteria based on some observable characteristics. e.g. Blood agar medium( Hemolytic) MacConkeys medium ( Lactose fermenter & non-lactose fermentor ) These media provide environments in which different bacteria can be distinguished from one another. For instance, violet red bile agar is used to distinguish coliform bacteria such as Escherichia coli from noncoliform organisms. The coliform bacteria appear as bright pink colonies in this media, while noncoliforms appear a light pink or clear.

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Certain media are both selective and differential. For instance, MacConkey agar differentiates lactose-fermenting bacteria from nonlactose-fermenting bacteria while inhibiting the growth of Gram-positive bacteria. Since lactose-fermenting bacteria are often involved in water pollution, they can be distinguished by adding samples of water to MacConkey agar and waiting for growth to appear.

INDICATORS: 

INDICATORS pH indicators are incorporated in some culture media and give visual evidence of pH changes occurring during the growth of bacteria. Indicators for this purpose must be non-toxic in the concentration used.

Principles of Isolation: 

Principles of Isolation 1. Isolation methods a- Clinical history. b- Nature of specimen. c- Suspected pathogen. d- Direct smear (microscopy). e- Cultural methods 2. Microscopy 3. Cultural methods

Pure Culture of Bacteria : 

Pure Culture of Bacteria Definition: culture started from a single cell Purpose: to isolate and maintain a single type of bacteria Methods: Sterilization -- removing all contaminants Aseptic technique --handling cultures and materials Growth medium -- providing for cell nutrition Techniques: Streak plate (twice) -- most common Spread plate (primarily used for counting) Goal: to dilute bacteria

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In order to work with microorganisms in the laboratory, it is desirable to obtain them in pure cultures. Pure cultures of bacteria can be obtained by spreading bacteria out and permitting the individual cells to form masses of growth called colonies. One can then pick a sample from the colony and be assured that it contains only one kind of bacteria. Cultivating these bacteria on a separate medium will yield a pure culture.

Culturing and Isolation Techniques: 

Culturing and Isolation Techniques Bacteria require a constant nutrient supply to survive and grow. Acquire nutrients from their surroundings (free-living) or from a host (parasites). Artificial media is used to grow bacteria in a lab (in vitro). Agar is extracted from marine algae. A carbohydrate that cross-links to form a semi-solid mesh. Melts at 100 ° C, solidifies at 42 ° C, but will remain a liquid at 60 ° C. Most pathogenic bacteria have an optimum growth temperature of 37 ° C (human body temp.) Organisms grown in broth cultures are apparent through the turbidity that the large numbers of cells produce in the broth. On agar, a solid medium, the bacterial cells form masses called colonies after about 18 – 24 hours of growth. Colonies represent one viable cell or Colony Forming Unit (CFU) that came to rest on the agar surface. This cell or CFU divides many times to form visible colonies on the agar. Isolated colonies, meaning those not touching other colonies, represent clones of the original cell or CFU since all the cells in the colony were derived from one cell or CFU and are genetically identical. Isolated colonies are considered to be pure cultures of a particular bacterial species and strain.

PURE CULTURE TECHNIQUES: 

PURE CULTURE TECHNIQUES Streak plate method Spread plate techniques Pour plate techniques i. tube dilution ii. Serial dilution Micromanipulator Roll tube technique

Advantages of pure culture techniques: 

Advantages of pure culture techniques To isolate microorganism from heterogeneous or mixed population To study morphology of bacteria To study role played by specific microorganisms. For identification of species Cultivation and Multiplication

Streak plate method: 

Streak plate method Principle:  By spreading a large amount of bacteria over the large surface area of a plate, the amount of bacteria is diluted until individual cells are spread on the surface of the plate.  From these individual cells a single colony arises.  All of the cells in this colony are genetically identical In order to identify bacteria, it is necessary to obtain a pure culture. This is one by using the streak-plate method. Bacterial cells are spread over the surface of an agar plate in a continuous dilution, so that the cells will be separated from each other. When the plate is incubated, those individual cells will grow into colonies that originated from a single cell.

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Plating out techniques

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Contamination of a streak plate resulting from leaving the plate open too long

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Contamination of a streak plate resulting from leaving the plate open too long

Streak Plates: 

Streak Plates Allow for the growth of isolated colonies on the surface of the agar. Used to isolate clones of a particular bacterial species/strain. An isolated colony, one that is not touching any other colonies, is assumed to be a pure culture. May observe colony morphology that can be used to help identify the bacterial species. Colonies of the same organism may grow differently on different media, e.g. the shape, color, growth pattern of the colony may differ on other types of media. Colony Morphology Characteristics Colony color Type of hemolysis (if grown on Sheep Blood Agar) Form Elevation Margin

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Streak Isolation on Nutrient Agar - Trypticase Soy Agar (TSA)

Pure culture isolates (colonies): 

Pure culture isolates (colonies)

Related answers: Why is streak plate technique important? so that we can dilute the consentrated bacteria (or whatever) into forming a single colony that can be idntifiied with the naked eye What is streak dilution plate technique? The streak plate technique is a method of diluting bacteria down suficiently so that the will grow as single colonies. The technique varies from individual to individual so much so that you can... Disadvantage of the streak plate technique? A disadvantage of the streak plate technique could be colony isolation problems. If the streaking technique is not done properly or if there is too much of an organism present on the inoculating loop... How could your streak plate technique improved? Try holding the loop at a different angle or try getting a smaller amount (a small amount of bacteria will go along way). What is the purpose of using streak plate technique? isolate a single cell by dilution : 

Related answers: Why is streak plate technique important? so that we can dilute the consentrated bacteria (or whatever) into forming a single colony that can be idntifiied with the naked eye What is streak dilution plate technique? The streak plate technique is a method of diluting bacteria down suficiently so that the will grow as single colonies. The technique varies from individual to individual so much so that you can... Disadvantage of the streak plate technique? A disadvantage of the streak plate technique could be colony isolation problems. If the streaking technique is not done properly or if there is too much of an organism present on the inoculating loop... How could your streak plate technique improved? Try holding the loop at a different angle or try getting a smaller amount (a small amount of bacteria will go along way). What is the purpose of using streak plate technique? isolate a single cell by dilution

Pour Plate Method: 

Pour Plate Method Another method of separating bacteria is the pour plate method. With the pour plate method, the bacteria are mixed with melted agar until evenly distributed and separated throughout the liquid. The melted agar is then poured into an empty plate and allowed to solidify. After incubation, discrete bacterial colonies can then be found growing both on the agar and in the agar. In this method, the mixed culture is diluted directly in tubes of liquid (cooled ) agar medium. Inoculum is maintained in liquid state at temp 45 o C Transfer into Petri plate Allow to solidify And incubate

Advantages: 

Advantages Surface and subsurface colonies are developed so isolation of microaierophiliic bacteria is possible Counting of colonies is possible

Disadvantages: 

Disadvantages Surface and subsurface colonies are developed Tedious, time consuming and requires skill MO subjected to hot shock b’cos liquid medium is maintained at at temp 45 o C Unsuitable for isolatating psychrophile bacteria( damaged by the melted agar)

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Two processes for isolating bacteria from a mixed culture. (a) The streak plate technique. (b) The pour plate technique .

Serial dilution method: 

Serial dilution method As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions

Spread plate techniques: 

Spread plate techniques Used for isolation of diluted mix population of microorganism so that individual colonies can be isolated. In this techniques microorganism are spread over the solidified agar with a sterile glass rod or spreader. The theory behind this techniques is that as that as the petridish is rotated at some stage single cell will be deposited from a clump of cell. Some of these cells will be separated from each other by a distance sufficient to allow the colonies to develop on agar medium.

Spread plate techniques: 

Spread plate techniques

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The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution. The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. For convenience, suppose we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms i.e., 100 microorganisms/ml of the liquid medium

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If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10 microorganisms/ ml. If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism. If this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism.

Advantages: 

Advantages Simple method Used to isolate the microorganism from any sample like: Milk,food,soil etc. Embedded colonies are developed hence microbial count is possible. Microorganism are not exposed to higher temperature. Disadvantages: Chances of contamination If the sample is not spread properly or evenly then isolation is possible.

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MICROMANUPULATOR Device that can pick up single cell from a colony of mixed culture. These are used in conjunction with microscope to pick up a single bacterial cell from hanging drop techniques. The single microbial cell is gently sucked into the micropipette and transferred on to a large drop of sterile medium on another cover slip.

Advantages: 

Advantages Method makes one reasonably sure of the pure culture coming from single cell. However device has to be used with skill and precision.

5. Roll tube techniques: 

5. Roll tube techniques Used for isolation of stringent anaerobes In this method stoppered anaerobic culture tube is used for isolation which has been coated with a prereduced agar medium containing oxygen free nitrogen. When stopper is removed the tube is kept anaerobic by continuously flushing oxygen free CO 2 from a gas canula. Inoculation is done with a transfer loop held against the agar surface as the tube is being rotated by a motor. Inoculation starts from the bottom and draws the loop gradually upward. After inoculation the tube is restoppered and and incubated anaerobically to get well isolated colonies.

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Fungi growing in axenic culture ( ascomycetes )

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Results + = Positive (Black coloration more than halfway down the agar within 72 hours) - = Negative (Less than half the agar turns black) V = Variable

Expected Question?????: 

Expected Question????? 1.Explain the different methods used for isolation of cultures. 2.Explain- Mac Coonkey’s broth is only selective and not differential media.(2004) 3.Which method would you prefer for isolation of Thermophiles and why? 4.Write advantages and disadvantages of pour plate technique. 5.Write advantages and disadvantages Streak plate method

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Thank you