Microbial cultivation- Pharmaceutical Microbiology
- 2. 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). 2
- 3. 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. 3
- 4. 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. 4
- 5. NUTRITIONAL REQUIREMENTS • Water • Carbon • Nitrogen and sulphur • Oxygen • Organic growth factors: purines, pyrimidines, vitamins 5
- 6. 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. • An undefined medium (also known as a basal or complex medium) • Defined media (also known as chemically defined media or synthetic media) 6
- 7. • 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 autoclave. • Nutrient media • This is an undefined medium because the amino acid source contains 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. 7
- 8. 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. 8
- 9. 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
- 10. USES OF MEDIA Cultivation and isolation Identification Sterility test Preservative efficacy test Evaluation of disinfectant To check the antimicrobial susceptabilty 10
- 11. COMMON INGREDIENTS OF MEDIA • Agar • Peptone • Yeast extract • Meat Extract • Water • NaCl 11
- 12. 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(Micronutrients and Macronutrients) • NaCl: To maintain isotonicity 12
- 13. 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 95oC. Once melted, does not solidify until it reaches 40oC. Cannot be degraded by most bacteria. Polysaccharide made by red algae. Originally used as food thickener (Angelina Hesse). 13
- 14. 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 14
- 15. 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. 15
- 16. TYPES OF 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. 16
- 17. • 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 thin film called ‘surface pellicle’ on the surface of undisturbed broth. • . Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen 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 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 Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria can not be detected. 17
- 18. • 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 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 nutritive property, it is not hydrolyzed by most bacteria and is 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. 18
- 19. • 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 fermentationm 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 is added to the liquid medium and when subcultures are to be 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 19
- 20. BACTERIAL GROWTH PATTERNS • In liquid medium: Superficial growth; Turbidity/diffuse; Precipitate growing; (sediment) 20 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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- 22. In semi-solid medium: • Only grow along the line of inoculation • Grow diffusely 22
- 23. 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 23
- 26. 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 26
- 27. TYPES OF CULTURE MEDIA B. Depending on oxygen requirement i. Aerobic media e.g. Mac Conkey’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 27
- 28. 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 28
- 29. SPECIAL MEDIA I. ENRICHED MEDIA: (Liquid form) Media is enriched by addition of substances such as blood, serum and egg to basal medium to facilitate growth of particular microorganism. e.g. Blood Agar ( Streptococcus), ChocolateAgar (Neisseria,haemophilus) 29
- 30. BLOOD AGAR PLATE (BA) • Nutrient agar with 5% sheep blood • 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 30
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- 32. 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 32
- 33. 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 33
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- 35. EOSINE METHYLENE BLUE (EMB), • Differentiatial between lactose fermenting and non fermenting enteric bacteria 35
- 36. TELLURITE GLYCINE AGAR (TGA) • Selective- Tellurite glycine and lithium inhibit most bacteria • Preferential growth of Staphyloccoci coagulase positive (Staphyloccocus aureus) 36
- 37. BLOOD AGAR PLATES ARE OFTEN USED TO DIAGNOSE INFECTION. ON THE RIGHT IS A POSITIVE STREPTOCOCCUS CULTURE; ON THE LEFT A POSITIVE STAPHYLOCOCCUS 37
- 38. 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) 38
- 39. 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. 39
- 40. V. 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. 40
- 41. • 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. 41
- 42. 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 42
- 43. • 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. 43
- 44. PURE CULTURE TECHNIQUES • Streak plate method • Spread plate techniques • Pour plate techniques i. tube dilution ii. Serial dilution • Micromanipulator • Roll tube technique 44
- 45. 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 45
- 46. 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. 46
- 48. Contamination of a streak plate resulting from leaving the plate open too long 48
- 49. Contamination of a streak plate resulting from leaving the plate open too long 49
- 50. 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 50
- 51. Streak Isolation on Nutrient Agar - Trypticase Soy Agar (TSA) 51
- 52. 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 45oC • Transfer into Petri plate • Allow to solidify • And incubate 52
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- 55. ADVANTAGES • Surface and subsurface colonies are developed • Counting of colonies is possible 55
- 56. DISADVANTAGES • Tedious, time consuming and requires skill • MO subjected to hot shock b’cos liquid medium is maintained at at temp 45oC • Unsuitable for isolating psychrophile bacteria( damaged by the melted agar) 56
- 57. Two processes for isolating bacteria from a mixed culture. (a) The streak plate technique. (b) The pour plate technique. 57
- 58. 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 58
- 59. 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. 59
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- 62. 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. 62
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- 65. • 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 65
- 66. 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. 66
- 67. 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. 67
- 68. ADVANTAGES • Method makes one reasonably sure of the pure culture coming from single cell. • However device has to be used with skill and precision. 68
- 69. 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 CO2 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. 69
- 70. Fungi growing in axenic culture (ascomycetes) 70
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- 72. THANKYOU 72