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  • 1. PHYSIOLOGY OF BACTERIA DR.BHARTI BAIS PROFESSOR
  • 2. INTRODUCTION  Bacteria because of their rapid- growth, reproduction and mutation rates as well as their ability to exist under adverse conditions are of immense importance.  Physiology includes chemical composition, physico-chemical properties, metabolism, respiration, reproduction, and growth of microorganisms.
  • 3. Bacterial growth Definition- An orderly increase in all chemical constituents by which there is replication of all cellular components, organelles and protoplasmic components from nutrients. Growth requires 3 complex processes Metabolism Regulation Cell division
  • 4. Metabolism • Definitions – Metabolism: The processes of catabolism and anabolism – Catabolism: The processes by which a living organism obtains its energy and raw materials from nutrients – Anabolism: The processes by which energy and raw materials are used to build macromolecules and cellular structures (biosynthesis)
  • 5. Microbial metabolism For surviving bacteria must have an efficient system for generating energy The common energy form =>ATP(Adenosine Triphosphate) Catabolism (Dissimilation) Pathways that breakdown organic substrates (carbohydrates, lipids, & proteins) to yield metabolic energy for growth and maintenance.
  • 6. Breakdown Proteins to Amino Acids, Starch to Glucose Synthesis Amino Acids to Proteins, Glucose to Starch
  • 7. Microbial metabolism Anabolism (Assimilation) Assimilatory pathways for the formation of key intermediates and then to end products (cellular components). It is highly complex. The bacterial cell synthesizes itself and generate energy for many activities as many as 2000 chemical reactions
  • 8. Microbial metabolism Fueling Biosynthesis Polymerisation and assembly Fueling reactions Provide cell with energy and precursor metabolites used in biosynthetic reactions First step is the capture nutrients from environment is by Passive diffusion  Facilitated diffusion Active transport Group translocation
  • 9. Microbial metabolism  Fueling reactions (contd)  Passive diffusion Passive diffusion is the process in which molecules move from a region of higher concentration to one of lower concentration  The rate of passive diffusion is dependent on the size of the concentration gradient between a cell’s exterior and its interior e.g. glycerol
  • 10. Microbial metabolism Fueling reactions I. Energy-independent mechanisms: A. Simple/Passive diffusion- is an energy independent process, No membrane protein is involved;  Movement of a solute is from a region of higher concentration to a lower concentration; can never have a higher concentration of solute inside cell than outside. Few molecules enter by this process (H2O, O2, CO2, NH3, weak acids and bases, and certain hydrophobic molecules).
  • 11. Microbial metabolism  B. Facilitated Diffusion- is an energy independent process, dependent on carrier proteins. called permeases, which are embedded in the plasma membrane  Rate of solute permeability increases with the concentration gradient more rapidly, and at a lower concentration of the solute molecule, than in passive diffusion.  Because a carrier aids the diffusion process it is facilitated diffusion  A major permease in bacteria is an aquaporin that helps to move water in and out of the cell
  • 12. Group of proteins- They facilitate transport by changining their shape or conformation when they pick up the molecule When they move the molecule to the opposite side of the membrane they again change their shape upon release of the molecule they have transported Facilitated Diffusion
  • 13. Membrane
  • 14. • II. Energy-dependent Mechanisms A. Active Transport- a process that can move a solute from a low concentration environment to a high concentration environment (i.e., against its concentration gradient). To accomplish this requires an expenditure of energy and specific membrane proteins. There are two broad categories of active transport systems found in Bacteria I. Ion driven transport systems - The movement of molecules across the membrane at the expense of a previously established ion gradient (H+ , K+ or Na+ gradient). The three transport strategies
  • 15.  II. ATP Binding dependent systems- depends on the energy from ATP hydrolysis to mediate the movement of solutes across the membrane  High affinity of solute transport comes from the affinity of the binding protein for the specific substrate.  Binding protein-dependent transport systems belong to a large group of transporters known as the ABC (ATP-binding cassette) superfamily of transport proteins.  ABC transports found in prokaryotes( most common in aerobic organisms )and eukaryotes are, related to each other in sequence and structural organization
  • 16. Microbial metabolism  Fueling reactions (contd)  Active transport  It employ special substrate binding proteins, which are located in the periplasmic space of gram-negative bacteria  Or are attached to membrane lipids on the external face of the gram positive plasma membrane.  E. coli transports a variety of sugars (arabinose, maltose, galactose, ribose) and amino acids (glutamate, histidine, leucine) by this mechanism
  • 17. Microbial metabolism B. Group Translocation- a process that can move solutes from a low to a high concentration environment  Requires a number of proteins (cytoplamic and membrane) in which a molecule is transported into the cell while being chemically altered The best-known group translocation system is the phosphoenol pyruvate: sugar phosphor transferase system (PTS) by which certain sugars are transported into cells.  Chemical energy in the form of phosphorenolpyruvate (PEP) is reqired, and molecule is modified (phosphorylated by PEP) as it moves across membrane
  • 18. Microbial metabolism Biosynthesis is the creation of order much energy is required for biosynthesis. Although most ATP dedicated to biosynthesis is employed in protein synthesis.  ATP is also used to make other cell constituents.
  • 19. Bacterial chemical components • Water: free water and compound water. • Inorganic salt: phosphus, potassium magnesium, calcium, nitrium,etc. • Protein: 50%-80% of dry weight according bacterial kinds and age. • Sugar: mainly distributing in cell wall and capsule. • Lipids: composed of lipid, fatty acid, wax, etc. • Nucleic acid: RNA and DNA.
  • 20. FACTORS AFFECTING GROWTH OF BACTERIA 1. Nutrients 2. Temperature 3. Hydrogen ion concentration ( pH ) 4. Oxygen/Carbon dioxide Requirements 5. Osmotic pressure 6 Other factors
  • 21. BACTERIAL NUTRITION  Every organism must find in its environment all of the substances required for energy generation and cellular biosynthesis.  The chemicals and elements of this environment that are utilized for bacterial growth are referred to as nutrients or nutritional requirements  Water is the principal constituent of bacterial cells, 80% of the total weight
  • 22. NUTRIENT REQUIREMENTS OF BACTERIAL GROWTH 1. Water 2. Carbon source 3. Nitrogen source 4.Minerals 5.Growth factors : that a cell must have for growth but cannot synthesize itself. Such as amino acids, purines, pyrimidines and vitamins
  • 23. Carbon source Carbon is needed for energy Classification based on their source of carbon Autotrophs Autotrophs are obtaining energy from sunlight or chemosynthetic breaking down inorganic substances for energy  Autotrophs (lithotrophs): use CO2 as the C source  Photosynthetic autotrophs: use light energy  Chemolithotrophs: use inorganic
  • 24. Carbon source Heterotrophs (organotrophs): Unable to synthesize own metabolites Depend on preformed organic compounds and derive energy from breaking down complex organic compounds (eg. glucose) for growth. Nutritional needs are variable- monosaccharides - glucose, galactose, fructose, ribose, etc. disaccharides - sucrose , lactose organic acids - succinate, lactate, acetate amino acids - glutamate, arginine alcohols - glycerol, ribitol fatty acids
  • 25. Carbon source  Saprophyte - bacteria that feed on decaying material and organic wastes  Parasite-- absorbing nutrients from living organisms  Clinical Labs classify bacteria by the carbon sources (eg. Lactose) & the end products (eg. Ethanol,…).
  • 26. Nitrogen  Nitrogen atoms of amino acids purines, pyrimidines etc come from ammonium ion NH4.in amino acids, proteins  Most bacteria decompose proteins  Some bacteria use NH4 + or NO3 −  A few bacteria use N2 in nitrogen fixation  This can be done either by fixing atmospheric nitrogen e.g. Rhizobium sp. Azobacter.
  • 27. Nitrogen Human pathogens e.g. Kleb.pneumoniae and Clostridium sp. can also fix atmospheric nitrogen Or by Nitrate reduction which can be assimilatory—nitrate--nitrite-- hydroxylamine ammonia -- NH3  Dissimilatory When nitrate serve as an alternative electron acceptor to O2(anaerobic) and NO2 and N2 produced
  • 28. Nitrogen Nitrogen - ammonium ion (NH 4 + ) is end product of all nitrogen assimilation pathways. Pathway depends on organism. 10% dry weight is N (proteins, nucleic acids) Inorganic source a.ammonia: NH 3 (actually NH 4 + ) glutamate and glutamine biochemical pathways b. nitrogen (N 2 ) fixation - Azotobacter, Klebsiella, Rhizobium Nitrogenase N 2 NH 3 (NH 4 + ) glutamate and glutamine c. nitrate (NO 3 - ) or nitritre (NO 2 - ) nitrate reductase nitrite reductase (i) nitrate reduction: NO 3 NO 2 NH 3 (NH 4 + ) (ii) denitrification: NO 3 - N 2 (under anaerobic conditions; use NO 3 - as electron acceptor, give off N 2 )
  • 29. Sulfur source A component of several coenzymes thiamine, biotin and amino acids. Most microorganisms can use sulfate (SO4 2- ) as the S source.  Phosphorus source  A component of ATP, nucleic acids, coenzymes, phospholipids, teichoic acid, capsular polysaccharides; also is required for signal transduction.  Phosphate (PO4 3- ) is usually used as the P
  • 30. Mineral source Required for enzyme function. For most microorganisms, it is necessary to provide sources of K+ , Mg2+ , Ca2+ , Fe2+ , Na+ and Cl- .  Many other minerals (eg., Mn2+ , Mo2+ , Co2+ , Cu2+ and Zn2+ ) can be provided in tap water or as contaminants of other medium ingredients. Uptake of Fe is facilitated by production of siderophores (Iron-chelating compound, eg. Enterobactin).
  • 31. Growth factors Or bacterial Vitamins organic compounds (e.g., amino acids, sugars, nucleotides) a cell must contain in order to grow but which it is unable to synthesize May be essential when growth does not occur in their absence Accessory when they enhance growth without being absolutely necessary Identical with the vitamins necessary for mammalian nutrition, particularly belonging to group B – thiamine, riboflavine, nicotinic acid, pyridoxine, folic acid & vitamin B12
  • 32. Oxygen requirement
  • 33.  Obligate aerobes  They grow in presence of air. Oxidative phosphorylation takes place and uses oxygen as hydrogen acceptor. Can not carry out fermentation e.g. Cholera vibrio, Pseudomonas and Myco.tuberculosis 2O2 - + 2H+ SOD H2O2+O2 2H2O2 catalase 2H2O +O2 H2O2+A H2 peroxidase 2H2O+A peroxidase destroy H2O2 to H2O using NAD to NADH with simultaneous oxidation of Oxygen Requirements
  • 34. Facultative anaerobes Can grow in the presence and absence of oxygen Have SOD and catalase e.g. Bacillus anthracis and Enterbacteriaceae Microaerophilic Grow well in low concentrations of oxygen. Killed by higher concentrations of oxygen have superoxide dismutase enzyme e.g.Campylobacter, Helicobacter Aertolerant can survive in presence of air but can not grow have enzyme superoxide dismutase e.g. Cl.tertium,Cl.histolyticum  Use fermentation in presence and absence of oxygen
  • 35.  ANAEROBIC BACTERIA  The hydrogen receptors in respiration are nitrates and sulphates  They die on exposure to oxygen  This is due to the lack of the enzyme cytochrome and cytochrome oxidase catalase and superoxide dismutase which prevents them from destroying the free radicals formed on exposure to oxygen. e.g C.tetani.,C.novy,C.botulinum,Bacteroides
  • 36. Carbon dioxide  All bacteria require small amounts of carbon dioxide for growth for  Structural organic molecules, energy source  Chemoheterotrophs use organic carbon sources  Autotrophs use CO2  Some bacteria like Brucella,gonococcus,meningococcus require much higher levels of carbon dioxide (5 – 10%)  They are called as capnophilic
  • 37. TEMPERATURE  Optimum Temperature : The temperature at which maximum bacterial growth occurs above and below which growth is blocked.  Range of temperature a range in that organism can grow  Psychrophilic: < 20 degrees (even at -7 o C)  Mesophilic: 25-40 o C . include all human pathogens and opportunists.  Thermophiles: 55-80 o C
  • 38. Temperature and Growth Physical requirementsPhysical requirements TemperatureTemperature
  • 39. Temperature ranges for microbial growth
  • 40. Thermophiles-These are Archaebacteria from hot springs and other high temperature environment Some can grow above boiling temperature of water. They are anaerobes ,performing anaerobic respiration. Thermophiles contain genes for heat stable enzymes of great value in industry and medicine. An example is Taq polymerase, the gene isolated from Thermus aquaticus from Yellow stone park hot springs. Taq polymerase is used to make large number of copies of DNA sequences in a DNA sample used in medicine ,biotechnology and biological research. Annual sale of Taq polymerase is roughly about half billion dollars.
  • 41. H- ION CONCENTRATION  Optimum pH :The pH at which a bacterium grows best  Range of pH  The majority of pathogenic bacteria grow best at pH 7.2-7.6
  • 42. pH  Neutrophiles ( 5 to 8 ) Many bacteria grow best at neutral pH.  (pH 7.2-7.6)  Acidophiles ( below 5 ) Bacteria can be acidophilic grow at low pH eg. Helicobacter, T.B. pH 6.5-6.8  Alkaliphiles ( above 8.5 ) grow in alkaline conditions V. cholerae pH 8.4-9.2
  • 43. Halophiles These are salt-loving bacteria usually Archaebacteria growing in salt lakes but some Eubacteria also are halophilic as some Vibrios.They are aerobes perform aerobic respiration. Extreme halophiles can live in extremely salty environment, most are photo synthetic autotrophs using a pigment bacteriorhodopsin for photosynthesis that uses all light except for purple light making the cell appear purple.
  • 44. Osmotic Effect: Bacteria are more tolerant to osmotic variation can grow in varying concentration of salt and sugars than most other cells due to the mechanical strength of their cell walls and certain oligosaccharides. Osmotic strength provided by Na2HPo4 and K2HPo4 in medium. If transferred from one lower to higher need adaptation and it influences the growth.
  • 45. Osmotic pressure High osmotic pressure (hypertonic) removes water causing plasmolysis – inhibits growth i.e. salt as preservative Low osmotic pressures (hypotonic) cause water to enter and can cause lysis NaCl 0.85% NaCl 10% H2O Plasma membranePlasma membrane Cell wall
  • 46. (a) At beginning of osmotic pressure experiment (b) At equilibrium (c) Isotonic solution — no net movement of water (d) Hypotonic solution — water moves into the cell and may cause the cell to burst if the wall is weak or damaged (osmotic lysis) (e) Hypertonic solution — water moves out of the cell, causing its cytoplasm to shrink (plasmolysis) Glass tube Rubber stopper Rubber band Sucrose molecule Water molecule Cellophane sack Cytoplasm Solute Plasma membrane Cell wall Water
  • 47. OTHER FACTORS  Light: Cultures die if exposed to light  Moisture and Drying: Water is an essential component of protoplasm and hence drying is lethal to cells.  Osmotic Effect: Bacteria are more tolerant to osmotic variation than most other cells due to the mechanical strength of their cell walls.
  • 48. Kinetics of growth • Bacteria divide by binary fission - log function during the period of maximum rate of growth - exponential phase - continuing growth in optimal condition • Generation time in vitro- – is 20 minutes in Vibrio cholerae (from 1 cell in 2 days give cell mass 4000 times that of earth) – 14 hours in Mycobacterium tuberculosis – mammalian cell 8 hours • in vivo generation time of bacteria is longer - forces of host defense and nutritional limitations
  • 49. BINARY FISSION Most bacterial cells reproduce asexually by binary fision, a process in which a cell divides to produce two nearly equal-sized progeny cells. Binary fision involves three processes: Increase in cell size (cell elongation), DNA replication Cell division
  • 50. Generation time  It is the time interval between the two cell division or time required for a population of bacteria to double in number under optimum conditions  minimum in log phase depends on species  many common bacteria : 20~60 min  most common pathogens in the body : 5-10 hours  Pseudomonas 8-9 mts  Vibrio and C.perfringenes 8-10 mts  Staphylococcus 23mts, Streptococci 30 mts  Myco.tuberculosis 18 hrs,Myco.leprae 20 days
  • 51. BACTERIAL GROWTH CURVE  Lag: Increase in cell size  Log: Increase in cell number  Stationary: Viable cells = dead cells  Decline: Viable cells < dead cells
  • 52. LAG PHASE  Time required for adaptation to the new environment  Increase in the size of the cells  Duration varies with the size of inoculum, nature of culture medium & environmental factors such as temperature
  • 53. LOG PHASE OR EXPONENTIAL PHASE • The cells start dividing and their number increases exponentially. • Contant generation time high metabolic activity • Most susceptible to antimicrobial agents
  • 54. STATIONARY PHASE  Depletion of nutrients and the accumulation of toxic products.  Increased resistance to UV light etc.  If spore bearing spore forms.  The viable count remains stationary as an equilibrium exists between the dying cells and newly formed cells.
  • 55. DECLINE PHASE  The population decreases due to cell death because of nutritional exhaustion, toxic accumulation and in the case of autolytic bacteria, due to lytic enzymes.  Pneumococcus 2-3 days Esch coli month
  • 56. Log phase Lag phase growth curve 0 5 15 20 25 3010 5.5 6.0 8.5 8.0 7.5 7.0 6.5 9.0 DeclineViable No Tim e Log No Of cells Stationary phase Total No
  • 57. MEASUREMENT OF GROWTH  Determination of bacterial cell mass  Dry weight. Separate bacteria by filtration or centrifugation and then weigh: weighing after drying the bacteria is a more accurate assessment than wet weight. .  Opacity (turbidity) of bacterial suspensions measured spectrophotometrically  Nitrogen mass -Measurement of cell constituents, eg. Protein colorimetric  Measurement of cell constituents, eg. (proportional to protein), ATP, muramic acid.
  • 58. MEASUREMENT OF GROWTH (Cont) Total counts, including both living and dead bacteria, 1 Direct counts by microscopy using: - Petroff’s- Hausser counting chambers -Coulter counter or electron counter -a known volume of culture drawn through probe No counted size can also be known - a known volume of bacterial suspension filtered onto the surface of a membrane filter, stained with acridine orange and viewed and counted using epifluorescence microscopy - a known volume spread on a slide stained and counted
  • 59. Direct counts by microscopy
  • 60. Direct counts by microscopy
  • 61. MEASUREMENT OF GROWTH (Cont)  .Viable counts in which cells that are able to grow in the conditions provided are counted  Spread plates Dilutions of bacteria may be spread onto the surface of agar  Pour plate incorporated into molten, cooled agar medium which is then poured into Petri dishes In both cases colonies are counted after incubation (counts are expressed as colony forming units, CFU's, per ml of bacterial suspension). The medium used will depend on species and strain of organisms .
  • 62. Pour plate
  • 63. Measuring Growth • Direct Counts – Petroff-Hauser Chamber • Serial Dilution – 10-fold serial dilutions • MPN (Most Probable Number) – Put 10, 1, and 0.1 ml into 10-mls broth • Repeat 5 times per volume – Statistical accurate sampling – Public Health Standards are written for MPN
  • 64. Serial Dilution 10-fold serial dilutions
  • 65. Serial dilution and colony counting Also know as “viable cell counts” Concentrated samples are diluted by serial dilution The diluted samples can be either plated by spread plating or by pour plating Diluted samples are spread onto media in petri dishes and incubated Colonies are counted. The concentration of bacteria in the original sample is calculated (from plates with 25 – 250 colonies, from the FDA Bacteriological Analytical Manual). A simple calculation, with a single plate falling into the statistically valid range, is given below: ml)inplated,lumefactor)(vo(dilution countedcolonies# sampleoriginalin ml CFU = If there is more than one plate in the statistically valid range of 25 – 250 colonies, the viable cell count is determined by the following formula: V**...])*1.0()*1[( C ml CFU dnn 121 ++ = ∑ Where: C = Sum of all colonies on all plates between 25 - 250 n1= number of plates counted at dilution 1 (least diluted plate counted) n2= number of plates counted at dilution 2 (dilution 2 = 0.1 of dilution 1) d1= dilution factor of dilution 1 V= Volume plated per plate
  • 66. MPN (Most Probable Number)
  • 67. Measuring Growth- cont’d • Turbidity – Spectrophotometer – Scale • %Transmittance • Optical Density or Absorbance • Filtration – 0.45 - 0.2 um sizes – Grid Pattern on Filter – Standards for Public Health • 0 E.coli / 100 ml of water – Also used for sterilization
  • 68. ●Membrane filtration – Used for samples with low microbial concentration – A measured volume (usually 1 to 100 ml) of sample is filtered through a membrane filter (typically with a 0.45 μm pore size) – The filter is placed on a nutrient agar medium and incubated – Colonies grow on the filter and can be counted
  • 69. Turbidity Spectrophotometer
  • 70. Turbidity Spectrophotometer
  • 71. Spectrophotometer
  • 72.  Plating -approx. dilution of clinical material is doneand placed on a solid media with help of calibrated loop and number of colonies counted  Colony number × dilution factor .Done for urine and water  A known volume of bacterial suspension filtered onto the surface of a membrane filter and transferred to a suitable solid media incubated and colonies are counted  Release of radiometric CO2 by labeled substrate  Detection of labeled precursor in DNA ,RNA or protein  Essay of specific enzyme MEASUREMENT OF GROWTH (Cont)
  • 73. Growth of bacterial culture Culture mediaCulture media Solution of nutrients thatSolution of nutrients that support the growth of bacteria is called culturesupport the growth of bacteria is called culture media.media.  Liquid or solidLiquid or solid Inoculation- introduction of live cells to aInoculation- introduction of live cells to a suitable sterile mediumsuitable sterile medium Population of bacterial cells referred as culturePopulation of bacterial cells referred as culture Pure culture –genetically homogenous orPure culture –genetically homogenous or axenic cultureaxenic culture Colony –viable mounds of bacterial massColony –viable mounds of bacterial mass single original cell divide and clone.single original cell divide and clone. If no 10If no 1066 cells/ml are visibly turbidcells/ml are visibly turbid
  • 74. Continuous cell culture system 1.Chemostat 2.Turbidostat Chemostat The fresh medium contains a limiting amount of an essential nutrient. Growth rate is determined by the rate of flow of medium through the culture vessel. To maintain bacteria in log phase GROWTH (Cont)
  • 75. Our Chemostat System
  • 76. Continuous cell culture system Turbidostat-- The turbidostat it has a photocell that measures the absorbance or turbidity of the culture in the growth vessel. Flow rate of media through the vessel is automatically regulated to maintain cell density. In this dilution rate varies and its culture media lacks limiting nutrient. It works at a high dilution rates.
  • 77. Synchronous culture Where all cells divide with in same time period -Manipulation of environment -Sorting out cells according to age and size - Filtration through stack filters -Filtration through membrane filters -Velocity sedimentation in density gradient like sucrose
  • 78. Cultivation methods Medium Basic media Rich media Enrichment media Selective media Differential media Agar: an acidic polysaccharide extracted from red algae For microbiologic examination Use as many different media and conditions of incubation as is practicable. Solid media are preferred; avoid crowding of colonies. For isolation of a particular organism Enrichment culture Differential medium Selective medium Isolation of microorganisms in pure culture Pour plate method Streak method For growing bacterial cells Provide nutrients and conditions reproducing the organism's natural environment.
  • 79. Biofilms Polysaccharide encased community of bacteria attached to a surface. Attachment of bacteria to a surface or to each other is mediated by glycocalyx. About 65% of human bacterial infection involve biofilms. Biofilms also causes problems in industry. Bioremediation is enhanced by biofilms. Bacterial growth in nature
  • 80. Biofilm: a community of microbes embedded in an organic polymeric matrix (glycocalyx, slime), adhering to an inert or living surface.
  • 81. Interaction of mixed communities A natural environment may be similar to a continuous culture. Bacteria grow in close association with other kinds of organisms. The conditions in bacterial close association are very difficult to reproduce in the laboratory. This is part of the reason why so few environmental bacteria have been isolated in pure culture
  • 82. Culture methods • Anaerobes differ in their sensitivity to oxygen and the culture methods employed reflect this - some are simple and suitable for less sensitive organisms, others more complex but necessary for fastidious anaerobes • Vessels filled to the top with culture medium can be used for organisms not too sensitive
  • 83. Culture methods • Most common adaptation of media is the addition of a reducing agent, e.g. thioglycollate, cysteine • Acts to reduce the oxygen to water, brings down the redox potential -300mV or less. • Can add a redox indicator such as rezazurin, pink in the presence of oyxgen - colourless in its absence
  • 84. Culture methods • Deep culture tubes can be used to test whether an unknown organism is anaerobic/facultative or aerobic • Thioglycollate added to culture medium, oxygen only found near top where it can diffuse from air -pattern of colony formation characteristic of organisms
  • 85. +500 mV - 300 mV Redox potential Culture methods
  • 86. Culture methods • Pyrogallic acid-sodium hydroxide method can be used, again relies on a chemical reaction to generate an anaerobic environment, but a catalyst rather than a reducing agent • Anaerobic jars (GasPak System) are sued to incubate plates in an anaerobic atmosphere, useful if brief exposure to oxygen is not lethal
  • 87. Culture methods
  • 88. P. aeruginosa Strict aerobe Enterococcus Facultative Grows aerobic or anaerobic. Bacteriodes fragilis
  • 89. Culture of strict anaerobes • For culture of strict anaerobes all traces of oxygen must be removed from medium and for many organisms sample must be kept entirely anaerobic during manipulations • Methanogenic archaea from rumen and sewage treatment plants killed by even a brief exposure to O2 • Medium usually boiled during
  • 90. Culture methods • Manipulations usually carried out under a jet of O2-free N2 or N2/CO2 to exclude O2 • Roll-tube (Hungate) method often used instead of conventional plates for isolation and culture of strict anaerobes
  • 91. 1.Exclude oxygen by flushing the tube with the desired gas 2. Place 4.5ml of pre- reduced anaerobic agar medium into tube 3. Seal the tube with the butyl rubber stopper and screw cap 4.Autoclave the tube 5.Inoculate with a syringe 6.Prepare on roll tube spinner 7.Incubate in water bath
  • 92. Culture methods • Use of anaerobic cabinet/glove box allows conventional bacteriological techniques e.g. replica plating, antibiotic sensitivity testing etc. to be carried out anaerobically
  • 93. Chapter 5 Respiration • Overview; – Glucose to Carbon dioxide + Water +Energy – C6H12O6 + O2  6CO2 + 6H2O + 38 ATP – Glucose is highly reduced; contains energy – Oxygen receives the electrons to form energy • 4 separate reactions – Glycolysis, Transition Reaction, Krebs Cycle, Electron Transport, Chemiosomosis • Requires Oxygen
  • 94. Chapter 5 Steps in Respiration • Electron Donors – Organic Compounds (Glucose preferred) • Electron Carriers – NAD to NADH – FAD to FADH • Electron Acceptors-Terminal – O2 to H2O • Phosphorylation Reactions – ADP to ATP • Chemiosmosis Reactions
  • 95. Glycolysis- 10 steps • Glucose is Phosphorylated to form Fructose 1,6-diphosphate • Split to form 2 Glyceraldehyde 3- phosphate • Final Products are: – 2 Pyruvic Acid (C3H4O3) • Compare to original glucose - C6H12O6 – 2 NADH – 2 ATP
  • 96. Transition Reaction • Pyruvic Acid  Acetyl - Co A + CO2 + NADH • C2H4O2
  • 97. Kreb’s Cycle Acetyl CoA  Carbon Dioxide – C2H4O2 to CO2 – Energy produced/Acetyl CoA (x2 for /Glucose) • 3 NADH • 1 FADH • 1 ATP •Metabolic Wheel – Fats, amino acids, etc. enter or leave – Citrate is product of first reaction – Simmons Citrate Media
  • 98. Electron Transport Chain • NADH oxidized to NAD • FAD reduced to FADH • Cytochromes shuffle electrons finally to O2 – Cytochrome Oxidase important in G - ID • H2O formed and ATP • 3 ATP / 1 NADH • 2 ATP / 1 FADH
  • 99. Chapter 5
  • 100. Fermentation Products from Pyruvate • Homolactic = Lactic Acid – Yogurt, Lactobacillus • Alcohol + CO2 • Propionic Acid • Butyric Acid • Acetic Acid • Succinic Acid • Butylene to Acetoin – basis for VP Test (Vogues-Proskauer)
  • 101. Fermentation Products • Alcohol and Carbon Dioxide – Yeast mostly • Lactic Acid – Humans, muscles without oxygen – Bacteria (Lactobacillus-yogurt) • Butyric Acid – Rancid butter, Clostridium-gangrene • Acetoin – Butanediol fermentation in Klebsiella • Propionic Acid – Swiss Cheese
  • 102. Chapter 5
  • 103. Chapter 5