Chapter 3 bio 300 obe

1. 1 Chapter 3 : Techniques in microbiology

2. BIO 300 BIOLOGICAL TECHNIQUES AND SKILLS SARINI BINTI AHMAD WAKID FACULTY OF APPLIED SCIENCE CHAPTER 3 TECHNIQUES IN MICROBIOLOGY Chapter 3 : Techniques in microbiology 2

3. What is Microbiology? The science and study of microorganims What is Microorganisms? Microorganisms are minute living things that individually are usually too small to be seen with unaided eye. There are four major kinds of microbes: bacteria, fungi, protists and viruses. Chapter 3 : Techniques in microbiology 3

4. History and scope of microbiology The Importance of Microorganisms      medical and most populous group of organisms and are found everywhere on the planet play a major role in recycling essential elements source of nutrients and some carry out photosynthesis benefit society by their production of food, beverages, antibiotics and vitamins causative agents of some important diseases Chapter 3 : Techniques in microbiology 4

5. Microorganisms: - Microorganisms are everywhere; almost every natural surface is colonized by microbes, from body to ocean. Some microorganisms can live hot springs, and others in frozen sea ice. - Most microorganisms are harmless to humans; You swallow millions of microbes every day with no ill effects. In fact, we are dependent on microbes to help us digest our food. - Microbes also keep the biosphere running by carrying out essential functions such as decomposition of dead animals and plants. They make possible the cycles of carbon, oxygen, nitrogen and sulfur that take place in terrestrial and aquatic systems. - Microorganisms have also harmed humans and disrupted society over the millennia. -They sometimes cause diseases in man, animals and plants. -They are involved in food spoilage. Chapter 3 : Techniques in microbiology 5

6. - Organisms divided into 5 Kingdoms: • Monera – all procaryotes • Protista – unicellular or colonial eucaryotic cells lacking true tissues; includes algae, protozoa & simpler fungi • Fungi – eucaryoutic; includes molds, yeasts and mushrooms • Plantae – multicellular •Animalia - multicellular • Scope of Microbiology: - Microbiology has an impact on medicine, agriculture, food science, ecology, genetics, biochemistry, immunology, and many other fields. - Many microbiologists are primarily interested in the biology of microorganisms, while others focus on specific groups; - Virologists - viruses - Bacteriologists - bacteria - Phycologists – algae - Mycologist -fungi Chapter 3 : Techniques in microbiology 6

7. - Medical Microbiology: deals with diseases of humans and animals; identify and plan measures to eliminate agents causing infectious diseases. - Immunology: study of the immune system that protects the body from pathogens. - Agricultural Microbiology: impact of microorganisms on agriculture; combat plant diseases that attack important food crops. - Food and Dairy Microbiology: prevent microbial spoilage of food & transmission of food-borne diseases (e.g. salmonellosis); use microorganisms to make food such as cheeses, yogurts, pickles, beer, etc. - Industrial Microbiology: using microorganisms to make products such as antibiotics, vaccines, steroids, alcohols & other solvents, vitamins, amino acids, enzymes, etc. - Genetic Engineering: Engineered microorganisms used to make hormones, antibiotics, vaccines and other products. - Since viruses are acellular and possess both living and nonliving characteristics, they are considered neither prokaryotic nor eukaryotic. They will be discussed in separate section of the course. Chapter 3 : Techniques in microbiology 7

8. Classification of microbes Bacteria Chapter 3 : Techniques in microbiology 8

9. BACTERIA  ARCHAEBACTERIA   Lack peptidogycan in cell walls Live in extreme environments  EUBACTERIA     Includes most bacteria Most have one of three shapes May be divided into up to 12 phyla Classification is controversial Chapter 3 : Techniques in microbiology 9

10. TYPES OF ARCHAEBACTERIA Methanogens living in sewage Thermoacidophilies Living in hot springs Extreme halophile living in the Great Salt Lake Chapter 3 : Techniques in microbiology 10

11. Archaebacteria  Live in extreme locations: Oxygen-free environments  Concentrated salt-water  Hot, acidic water  Chapter 3 : Techniques in microbiology 11

12. Eubacteria - Heterotrophs  Found everywhere  Parasites: live off of other organisms  Saprobes: live off of dead organisms or waste (recyclers) Chapter 3 : Techniques in microbiology 12

13. Eubacteria: Photosynthetic Autotrophs    Photosynthetic: make their own food from light Cyanobacteria: blue-green, yellow, or red ponds, streams, moist areas Eubacteria: Chemosynthetic Autotrophs   Get energy by breaking down inorganic substances like sulfur and nitrogen Make nitrogen in the air usable for plants Chapter 3 : Techniques in microbiology 13

14. Structure of Bacteria  Two parts to Bacteria Structure: Arrangement  Shape  Chapter 3 : Techniques in microbiology 14

15. Arrangement  Paired: diplo  Grape-like clusters: staphylo  Chains: strepto Chapter 3 : Techniques in microbiology 15

16. Shape  Rod: bacillus  Spheres: coccus  Spirals: spirillum Chapter 3 : Techniques in microbiology 16

17. Examples Streptococcus: chains of spheres  Staphylospirillum: Grapelike clusters of spirals  Streptobacillus: Chains of rods  Chapter 3 : Techniques in microbiology 17

18. BASIC SHAPES OF EUBACTERIA ROD-SHAPED SPHERICAL SPIRILLA Chapter 3 : Techniques in microbiology 18

19. Most Species of Eubacteria may be Grouped Based on Staining  Gram-Negative     Lack thicker layer of peptidoglycan Stain pink Endotoxins Gram-Positive    Thicker layer of peptidogycan Stain purple Exotoxins (released when bacteria die) Gram-positive Gram- negative Chapter 3 : Techniques in microbiology 19

20. Classification of microbes FUNGI Chapter 3 : Techniques in microbiology 20

21. WHAT ARE FUNGI?  Fungi are not classed as animals or plants, they have a Kingdom of their own to which they belong.  They range from being just a single cell, like the yeasts, to others that cover hundreds of acres of land.  Most fungi are said to be filamentous. This is because the main body of the fungus is made up of thin, thread-like filaments that are called hyphae, which form the mycelium. Chapter 3 : Techniques in microbiology 21

22. KINGDOM FUNGI To date, 100,000 species of fungi have been discovered.  People that study fungi are called Mycologists. It is thought that there are over one million species still to be found.  Fungi are not able to produce their own food as plants do.  Fungi are said to be SAPROTROPHS, because they live on dead organic matter such as leaves and wood.  To obtain nutrients fungi secrete special digestive enzymes which degrade organic material outside the mycelium. The degraded compounds can then be ingested. The fungi that most people are familiar with are those that form fruit bodies or mushrooms. Fungi can live in many habitats including the arctic, tropical rainforest, fresh and salt water. However, most fungi live in soil. Chapter 3 : Techniques in microbiology 22

23. Traits of Fungi   They are either:  Saprobes – feed on material from previously living things (shoes, dead trees, dead animals etc.) or  Parasites – which eat or derive there energy from living things. To reproduce, they  send out spores instead of seeds.  Carry pieces of broken hyphae to new places  Form Buds in which a small part of the parent grows into a new organism. . Chapter 3 : Techniques in microbiology 23

24. Traits of Fungi  Most are multicellular  Some like yeasts are unicellular Chapter 3 : Techniques in microbiology 24

25. Examples of Fungi  Bread Mold Chapter 3 : Techniques in microbiology 25

26. What are True FUNGI?         Eukaryotic organisms Heterotrophic, lacking chlorophyll Obtain nutrients via enzyme secretion and absorption of resulting byproducts Cells walls containing chitin and beta glucans Glycogen as primary food storage Can reproduce both sexually and asexually Heterotrophic – as such can consume almost any carbonaceous substrate including jet fuel and wall paint Biggest role is in the recycling of dead plant material Chapter 3 : Techniques in microbiology 26

27. From The Fungi Name Trail by Liz Holden & Kath Hamper Chapter 3 : Techniques in microbiology 27

28. Chapter 3 : Techniques in microbiology 28

30. Fungal Ecology Saprobe decomposer of all terrestrial organic matter (and some aquatic matter) Pathogen purveyor of plant and animal disease Mycorrhizae symbiosis of plant and fungus (fungi) Chapter 3 : Techniques in microbiology 30

31. Mycorrhizae      The term mycorrhiza, which literally means fungus-root first applied to fungus-tree associations described in 1885 95% of all plant species Symbiotic associations that form between the roots of most plant species and fungi characterized by bi-directional movement of nutrients where carbon flows to the fungus and inorganic nutrients move to the plant Chapter 3 : Techniques in microbiology 31

32. Helpful Fungi        Food – mushrooms Used to make cheese – Blue Cheese Used to make wine, beer, and whiskey (Yeast) Used to make bread rise Used to make soy sauce from soy beans Used to break down materials and recycle wastes and dead organisms Used to make certain drugs (ex. Penicillin) Chapter 3 : Techniques in microbiology 32

33. Harmful Fungus     Cause food spoilage Cause plant disease such as rusts, Dutch Elm Disease, and mildew Cause Human diseases such as Ring Worm, Athlete’s Foot, Thrush, lung Infections, and Yeast Infections Destroy leather, fabrics, plastics, etc. Chapter 3 : Techniques in microbiology 33

34. Food Spoilage Chapter 3 : Techniques in microbiology 34

35. Ringworm Chapter 3 : Techniques in microbiology 35

36. Athlete’s Foot – Tinea pedis Chapter 3 : Techniques in microbiology 36

37. Thrush Chapter 3 : Techniques in microbiology 37

38. Fungal Lung Infection Chapter 3 : Techniques in microbiology 38

39. Yeast Infections Chapter 3 : Techniques in microbiology 39

40. Fungus Destroying Leather Chapter 3 : Techniques in microbiology 40

41. Classification of microbes ALGAE Chapter 3 : Techniques in microbiology 41

42. Characteristics      Range in size from microscopic to single celled organisms to large seaweed Autotrophic Form the reproductive structures – gametangia or gamete chambers Aquatic and have flagella at some point in life Often contain pyrenoids, organelles that synthesis and store starch Chapter 3 : Techniques in microbiology 42

43. IDENTIFY THE TYPE OF ALGAE Chapter 3 : Techniques in microbiology 43

44. ALGAE MICROALGAE MACROALGAE Unicellular -body is only comprised of one cell Multicellular -differentiated structures within cells to perform photosynthesis, flotation, anchorage and others. Chapter 3 : Techniques in microbiology 44

45. Classification of algae Algae are classified into seven major groups:        Chrysophyta (golden brown algae) Cyanobacteria (blue green algae) Pyrrophyta (dinoflagellates) Euglenophyta (Euglenoid) Rhodophyta (red algae) Chlorophyta (green algae) Phaeophyta (brown algae) Chapter 3 : Techniques in microbiology 45

46. BACILLARIOPHYCEAE Examples: Chaetoceros sp., Coscinodiscus sp., Asterionella sp., Cymbella sp., Frustulia sp. Chapter 3 : Techniques in microbiology 46

47. CHRYSOPHYCEAE Examples: Dinobryon sp., Synura sp., Chapter 3 : Techniques in microbiology 47

48. CYANOBACTERIA Examples: Anabaena sp., Oscillatoria sp., Nostoc sp., Chapter 3 : Techniques in microbiology 48

49. PYRROPHYTA Examples:Peridinium sp., Ceratium sp. Chapter 3 : Techniques in microbiology 49

50. Phylum Euglenophyta    1000 species of Euglenoids Have both plantlike and animal-like characteristics Fresh water Chapter 3 : Techniques in microbiology 50

51. EUGLENOPYHTA Examples: Euglena sp., Phacus sp., Lepocinclis sp., Strombomonas sp. Chapter 3 : Techniques in microbiology 51

52. RHODOPHYTA     Some are single-celled, others are macroscopic and multicellular. Mostly marine algae The larger species typically grow attached to a hard substrate or occur as epiphytes on other algae. Contain chlorophyll a and d, but appear red due to accessory pigments,phycocyanin and phycoerythrin. Chapter 3 : Techniques in microbiology 52

53. RHODOPYHTA Examples: Porphyra sp., Batrachospermum sp Chapter 3 : Techniques in microbiology 53

54. CHLOROPHYTA Examples: Cosmarium sp., Closterium sp., Spirogyra sp., Caulerpa racemosa. Chapter 3 : Techniques in microbiology 54

55. “Kelp Forests” Chapter 3 : Techniques in microbiology 55

56. USES O ALGAE           Agar Alginates Energy source - Biofuel Fertilizer Nutrition – Health food Pollution control – treatment sewage Pigments – chemical dyes, coloring agents. Stabilizing subtances Cosmetics Animal Feed Chapter 3 : Techniques in microbiology 56

57. Classification of microbes VIRUS Chapter 3 : Techniques in microbiology 57

58. INTRODUCTION TO VIRUSES  Virus means "veleno". Viruses are basically a way a form of genetic information insures its continued survival. They are entities which reproduces their DNA/RNA within living cells utilizing mechanisms of cells for this. Chapter 3 : Techniques in microbiology 58

59. VIRUS COMPOSITION  Viruses are unique from all other life forms in that they can contain ONLY ONE FORM OF NUCLEIC ACID. Some viruses use RNA as their genetic material and other use DNA, but NEVER do they contain both. Further, this nucleic acid polymer may either exist as DOUBLE STRANDED (DS) DNA or RNA or as SINGLE STRANDED (SS) DNA or RNA. Each of these characteristics is a constant for a particular virus and is part of it description. The nucleic acid polymer may contain as few as 4 to 7 genes for very small viruses to 150 to 200 genes for very large viruses. In some viruses the nucleic acid exists in more that one molecule. Some viruses contain a few enzymes and some contain none, but no viruses contain the large numbers of enzymes found even in the smallest bacteria. Chapter 3 : Techniques in microbiology 59

60.  All virus are covered with a PROTEIN COAT. This protein coat is mainly composed of a FEW TYPES of proteins of which there are many copies per virus; something like the individual threads in a shirt. These identical protein subunits are called CAPSOMERES and they are made so that they spontaneously come together (ASSEMBLE) in a PREDETERMINED way to produce the virus coat which is called the CAPSID.  If a virus has ONLY a protein capsid covering it, it is termed a NAKED CAPSID VIRUS or a NAKED VIRUS. However, some viruses pick up a lipid membrane from the host cell when it is released, that surrounds the capsid. The lipid membrane is called an ENVELOPE and such viruses are termed ENVELOPED VIRUSES. Chapter 3 : Techniques in microbiology 60

61. Virus Structure  Size   Basic shape    Rod-like “Spherical” Protective Shell - Capsid      17 nm – 3000 nm diameter Made of many identical protein subunits Symmetrically organized 50% of weight Enveloped or non-enveloped Genomic material   DNA or RNA Single- or double-stranded Chapter 3 : Techniques in microbiology 61

62. Virus Structure  Virus capsids function in:   Packaging and protecting nucleic acid Host cell recognition   Protein on coat or envelope “feels” or “recognizes” host cell receptors Genomic material delivery   Enveloped: cell fusion event Non-enveloped: more complex strategies & specialized structures Chapter 3 : Techniques in microbiology 62

64. Culture Media Culture is the term given to microorganisms that are cultivated in the lab for the purpose of studying them. Medium is the term given to the combination of ingredients that will support the growth and cultivation of microorganisms by providing all the essential nutrients required for the growth (that is, multiplication) in order to cultivate these microorganisms in large numbers to study them. Chapter 3 : Techniques in microbiology 64

65. Laboratory culture: pure culture - Contaminants = other microorganisms present in the sample - history of the pure culture: - Koch employed gelatin as solidifying agent - Walter Hesse adopted agar - Petri (1887) invented Petri-dish - culture medium: - rich/selective Confluent mixture - growth inhibitors 1 Isolated colony - liquid/solid - temperature -Nutrients: - carbon, nitrogen, elements ... -Aseptic technique: - sterilization of medium and equipment 4 - proper handling Chapter 3 : Techniques in microbiology 2 3 65

66. Among the different kinds of microorganisms the two groups that can be grown in cultures are bacteria and fungi. Algae and protozoa require many different nutrients in minute quantities that are difficult to anticipate and prepare in the lab. These organisms have different nutritional requirements and thus various kinds of culture media have been developed. Primary ingredients required by all living organisms include: a carbon source, water, minerals, and a nitrogen source. Chapter 3 : Techniques in microbiology 66

67. Living cells need nutrients required for their structure (biosynthesis) as well as nutrients to provide them with energy to perform all of their various life processes. Nutrients are acquired from the environment in which they live in their natural habitat. Most of these nutrients are then processed within the cell through a variety of metabolic pathways to be incorporated in different ways. The process of building complex structures from simple building blocks is called anabolism. The process of breaking up complex materials to harvest the energy in them is called catabolism. The ability to use particular compounds is dependent upon the genetic makeup (DNA) that the cells have. Chapter 3 : Techniques in microbiology 67

68. Since there are different kinds of organisms that can be grown in culture media with varying needs, culture media have also been formulated with different ingredients. Culture media may be found in one of three states: liquid (called broth) semi-solid solid. Media are solidified by the addition of solidifying agents such as agar (inert compound). Varying the concentration of agar will yield varying degrees of solidification. Chapter 3 : Techniques in microbiology 68

69. Culture media may be classified as: Synthetic media (Defined) Complex (Non-synthetic) media Synthetic media contain only ingredients for which a complete chemical formula is known. Complex media contain at least one ingredient for which a chemical formula is not known (such as milk, egg, malt, animal tissues) Culture media can also be classified based on the function they perform in determining various characteristics of organism that are able to grow on/in them e.g. Differential, Selective media. Chapter 3 : Techniques in microbiology 69

70. Microbial growth media - chemically defined: highly purified inorganic and organic compounds in dest. H2O - complex (undefined): digests of casein, beef, soybeans, yeast, ... Chapter 3 : Techniques in microbiology 70

71. Microbial growth media Media *Complex Purpose Grow most heterotrophic organisms *Defined Grow specific heterotrophs and are often mandatory for chemoautotrophs, photoautotrophs and for microbiological assays *Selective Suppress unwanted microbes, or encourage desired microbes *Differential Distinguish colonies of specific microbes from others *Enrichment Similar to selective media but designed to increase the numbers of desired microorganisms to a detectable level without stimulating stimulating the rest of the bacterial population *Reducing Growth of obligate anaerobes Chapter 3 : Techniques in microbiology 71

72. Microbial nutrition Nutrients = chemical „tools“ a cell needs to grow/replicate Macronutrients = chemicals needed in large amounts Micronutrients = chemicals needed in small/trace amounts Autotrophy = CO2 can be sole C-source % of dry weight 50% 12% (sometimes non-essential) (sometimes non-essential) Chapter 3 : Techniques in microbiology 72

73. Media Provides Nutrients for Bacteria  Nutrient broth: liquid media (trypticase soy broth, TSB)  Nutrient agar: solid media (trypticase soy agar, TSA)    Agar slants (tubes) Agar plates Agar: polysaccharide isolated from red algae    Solid at room temp (25oC) Liquid at 100oC Provides framework to hold moisture & nutrients Chapter 3 : Techniques in microbiology 73

74. Microbial nutrition: Growth factors - organic compounds required by some bacteria - vitamins, amino acids, purines, pyrimidines - Streptoccus, Lactobacillus, Leuconostoc (lactic acid bacterium): complex vitamin requirements Chapter 3 : Techniques in microbiology 74

76. The ingredients in a medium will affect the chemical nature of the medium. This is important because organisms vary in their requirement for different environments. One such property is: pH (which is a measure of the amount of hydrogen ions in a particular medium). This has to be monitored during the preparation of media since this will influence the kind of organisms that are able to grow in the medium. The pH of the medium will thus determine which organisms are able to grow on the medium. For example, fungi prefer acidic media for their growth while bacteria grow on neutral pH media. Chapter 3 : Techniques in microbiology 76

77. The primary function of culture media is to be able to grow particular organisms on/in them. It is important that these media are devoid of any other living organisms. This is possible through the process of sterilization (a process by which all living organisms and their spore forms are killed and the medium is made sterile) Culture media are most commonly sterilized through the process of autoclaving (using high temperatures that will kill all living organisms under increased pressure for specified periods of time – in an appliance called the autoclave) Chapter 3 : Techniques in microbiology 77

79. Aseptic Techniques …protective clothing …hand washing …bench cleaning …loop flaming …pipettors Chapter 3 : Techniques in microbiology 79

80. The 5 I’s of Culturing Microbes 1. 2. 3. 4. 5. Inoculation: introduction of sample into a container of media Incubation: under conditions that allow growth Isolation: separating 1 species from another Inspection Identification Chapter 3 : Techniques in microbiology 80

81. Inoculation Chapter 3 : Techniques in microbiology 81

82. Incubation & Isolation Chapter 3 : Techniques in microbiology 82

83. Pure vs Mixed Cultures    Eschericia coli (white) Micrococcus luteus (yellow) Serratia marcescens (red) Chapter 3 : Techniques in microbiology 83

84. Isolation Technique Chapter 3 : Techniques in microbiology 84

86. Inspection & Identification Chapter 3 : Techniques in microbiology 86

87. Bacteria 1) Bacillus = Rod shaped. (pl. bacilli) (diplobacilli, streptobacilli) 2) Coccus = Round shaped. (pl. cocci) (diplococci, streptococci, staphylococci) 3)Spiral = Spiral shaped (spirilla, vibrio, spirochete) Chapter 3 : Techniques in microbiology 87

88. Bacteria may appear as single cells or in groups:     These terms describe typical bacteria groupings: 1) diplo = paired cells 2) strepto = long chains 3) staphylo = grape-like clusters Chapter 3 : Techniques in microbiology 88

89. Bacterial growth Growth rate = ∆cell number/time or ∆cell mass/time Chapter 3 : Techniques in microbiology 1 generation Growth = increase in # of cells (by binary fission) generation time: 10 min - days 89

90. Bacterial growth: exponential growth Generation time = 30 min Chapter 3 : Techniques in microbiology 90

91. Bacterial growth: exponential growth Semilogarythmic plot Straight line indicates logarithmic growth Chapter 3 : Techniques in microbiology 91

92. Bacterial growth: calculate the generation time t g= n t = time of exponential growth (in min, h) g = generation time (in min, h) n = number of generations The generation time is the time needs the culture population to double Chapter 3 : Techniques in microbiology 92

93. Bacterial growth: calculate the generation time t g= n t = time of exponential growth (in min, h) g = generation time (in min, h) n = number of generations Chapter 3 : Techniques in microbiology 93

94. Bacterial growth: calculate the generation time t = time of exponential growth (in min, h) g = generation time (in min, h) n = number of generations t g= n Nt = N0 x 2 n Nt = number of cells at a certain time point N0 = initial number of cells n = number of generations Chapter 3 : Techniques in microbiology 94

95. Bacterial growth: calculate the generation time t = time of exponential growth (in min, h) g = generation time (in min, h) n = number of generations t g= n Nt = N0 x 2 Nt = number of cells at a certain time point N0 = initial number of cells n = number of generations n logNt = logN0 + n x log2 logNt - logN0= n x log2 n= logNt - logN0 log2 n = 3.3 x (logNt - logN0) Chapter 3 : Techniques in microbiology 95

96. Bacterial growth: batch culture Chapter 3 : Techniques in microbiology 96

97. Batch culture: Lag phase no Lag phase: Inocculum from exponential phase grown in the same media Lag phase: Inocculum from stationary culture (depletion of essential constituents) After transfer into poorer culture media (enzymes for biosynthesis) Cells of inocculum damaged (time for repair) Chapter 3 : Techniques in microbiology 97

98. Batch culture: exponential phase Exponential phase = log-phase Maximum growth rates „midexponential“: bacteria often used for functional studies Chapter 3 : Techniques in microbiology 98

99. Batch culture: stationary phase Bacterial growth is limited: - essential nutrient used up - build up of toxic metabolic products in media Stationary phase: - no net increase in cell number - „cryptic growth“ - energy metabolism, some biosynthesis continues - specific expression of „survival“ genes Chapter 3 : Techniques in microbiology 99

100. Batch culture: death phase Bacterial cell death: - sometimes associated with cell lysis - 2 Theories: - „programmed“: induction of viable but non-culturable - gradual deterioration: - oxidative stress: oxidation of essential molecules - accumulation of damage - finaly less cells viable Chapter 3 : Techniques in microbiology 100

101. Measurement of microbial growth A. Weight of cell mass B. number of cells: - Total cell count - Viable count - Dilutions - turbidimetric Chapter 3 : Techniques in microbiology 101

102. total cell count A. Sample dried on slide B. Counting chamber: Limitations: - dead/live not distinguished - small cells difficult to see - precision low - phase contrast microscope - not useful for < 106/ml Chapter 3 : Techniques in microbiology 102

103. viable cell count synonymous: plate count, colony count 1 viable cell  1 colony cfu = colony forming unit Advantage: high sensitivity; selective media Optimal: 30 – 300 colonies per plate ( plate appropriate dilutions) spread plate method: pour plate method: Bacteria must withstand 45°C briefly Chapter 3 : Techniques in microbiology 103

104. dilutions Example: 3 h culture of E. coli in L-broth How do I determine the actual number? Chapter 3 : Techniques in microbiology 104

105. Turbidimetric measurements Relationship between OD and cfu/ml must be established experimentally Exponential culture of E. coli in L-broth: 1 OD = ca. 2-3 x 109 cfu/ml Chapter 3 : Techniques in microbiology 105

106. Continuous culture: the chemostat steady state = cell number, nutrient status remain constant Control: 1. Concentration of a limiting nutrient 2. Dilution rate 3. Temperature  Independent control of: - Cell density - Growth rate Chapter 3 : Techniques in microbiology 106

107. Factors affecting microbial growth • • • • • Nutrients Temperature pH Oxygen Water availability Chapter 3 : Techniques in microbiology 107

108. NEXT CLASS: Chapter 4 TECHNIQUES IN BIOCHEMICAL ANALYSIS THANK YOU Chapter 3 : Techniques in microbiology 108

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