The document discusses classifying microbes based on their metabolic requirements and laboratory techniques used for culturing bacteria. It covers Robert Koch's pioneering work developing strategies for cultivating bacteria. It describes the four phases of bacterial growth in laboratory conditions. Key techniques discussed include obtaining pure cultures using streak plating on semi-solid agar media, and maintaining and storing stock cultures.

1. CLASSIFYING MICROBES METABOLIC REQUIREMENTS & LABORATORY TECHNIQUES

2. Principles of Prokaryotic Growth Robert Koch (1843-1910) Developed the strategies for cultivating bacteria Defined growth requirements Media formulations First to use agar for semisolid media

3. Bacterial replication Binary fission Doubling time varies by species and conditions Growth can be calculated N t = N 0 x 2 n N t = total cells in a given time N 0 = starting population of cells n = number of cell divisions Assume t = 20 min (3 per hour) 3 x 4 hours = 12 doublings If N 0 = 10 then 10 x 2 12 = 40,960 bacteria

4. Bacterial Growth in Nature Biofilms Bacteria in nature tend to attach to solid surfaces These bacteria encase themselves in polysaccharide coatings to form communities Collectively, these communities are referred to as biofilms

5. Biofilm communities are highly organized Intracellular communication Channels Cellular movement within the biofilm (usually mediated by pili)

6. Biofilm Activities Ear infections Dental decay Bioremediation Sewage treatment Toxic waste sites Heavy metals Intracellular “warfare” (i.e., competition)

7. Obtaining a Pure Culture Isolation of pure cultures is mandatory for studying bacteria This is usually accomplished using semi-solid media composed with agar Agar is a polysaccharide obtained from marine algae It melts at about 95° C and remains liquid to 45° C

8. Media are made with agar and usually sterilized in an autoclave Media are cooled to 60° C, then dispensed into Petri dishes or tubes After cooling, the agar solidifies, providing a semi-solid surface

9. The streak plate method for obtaining a pure culture Agar media in Petri dish Collect a broth culture with a sterile loop Streak the plate to deposit individual bacteria at sites on the plate Incubate for 24-48 hr Single bacterium grows to millions, forming a colony on the plate

11. Maintaining a Stock Culture Once a colony is obtained it is considered pure This colony can be picked and inoculated into another tube or plate ( subculturing ) to provide a stock of the purified culture for short-term use (weeks) Long-term storage (years) This purified culture can also be grown in broth and lyophilized (freeze-dried) It can also be diluted 1:2 in glycerol and frozen at -70° C

12. Bacterial Growth in Laboratory Conditions The Growth Curve Bacteria exhibit distinct kinetic profiles of growth in the laboratory in closed cultures (systems) These profiles generally are the same, although time-course between species can be different

14. Bacterial Population Growth Curve 4 phases: Lag phase Bacteria absorb nutrients, synthesize enzymes and prepare for cell division Do not increase in # during this time Logarithmic growth phase/log phase/ exponential growth phase Multiply rapidly where the # doubles with each generation time Brief unless dividing culture is maintained by constant addition of nutrients and frequent removal of waste products Stationary phase Nutrients are used up and concentration of waste product build up Rate of division slows Culture is at its greatest population density Death phase Microbe die due to toxic waste build up

15. Continuous (Open) Cultures Removal of toxic metabolites Replenishment of medium Conducted in culture tanks called fermenters Internal sensors Computer controlled

16. 4.5 Environmental Factors that Influence Microbial Growth

17. Nutritional Factors that Influence Microbial Growth Heterotrophs Carbon source is organic carbon Medically important bacteria Autotrophs Carbon source is inorganic carbon Perform carbon fixation , the conversion of inorganic carbon into organic carbon Photoautotrophs use photosynthesis CO 2 + H 2 O ➔ Glucose + O 2

18. Cultivating Prokaryotes in the Laboratory Complex Media Contain a variety of biomolecule precursors Concentrations of precursors can vary between media Often, the source of the precursors are extracts , which are water-soluble substances Examples Nutrient agar Tryptic soy agar Blood agar Nutrient Agar, Mannitol Salt Agar, EMB Agar

19. Mannitol Salt Agar - contain: 1. high salt 2. mannitol (phenol red) if able to ferment mannitol, acidic byproduct is used turning yellow

20. Defined Media Known amounts of chemicals and biomolecules are formulated into the medium More expensive than complex media Selective Media Generally used for the propagation of particular bacteria MacConkey agar selects for Gram - enterics Bismuth sulfite agar cultures Salmonella and Proteus species to the exclusion of other bacteria Defined Media Known amounts of chemicals and biomolecules are formulated into the medium More expensive than complex media Selective Media Generally used for the propagation of particular bacteria MacConkey agar selects for Gram - enterics Bismuth sulfite agar cultures Salmonella and Proteus species to the exclusion of other bacteria

21. Differential Media Media that can distinguish two or more groups of bacteria in a mixed culture

22. Providing Appropriate Atmospheric Conditions Increased CO 2 Capnophiles (15% CO 2 ) Haemophilus Neisseria Microaerophilic - low levels of oxygen Anaerobe - killed by prolonged exposure to oxygen

23. METABOLISM

24. The Central Metabolic Pathways

25. METABOLISM Sum of all chemical reactions within a living organism. Divided into: CATABOLISM Chemical reaction that PRODUCE ENERGY Breakdown of complex organic compounds into simpler ones ANABOLISM Building of complex organic molecules from simpler ones. Energy requiring reactions Formation of proteins from amino acids

26. Role of ATP in Coupling Reactions A metabolic pathway is a sequence of enzymatically catalyzed chemical reactions in a cell. Metabolic pathways are determined by enzymes, which are encoded by genes.

27. Energy Production: Oxidation-Reduction Reactions Oxidation = removal of e - Reduction = gain of e - Fig 5.9 Redox reaction = oxidation reaction paired with reduction reaction.

28. Oxidation-Reduction cont. In biological systems, the electrons are often associated with hydrogen atoms. Biological oxidations are often dehydrogenations. Fig 5.10

29. The Generation of ATP Phosphorylation : Substrate level phosphorylation : transfer of a high-energy PO 4 – to ADP. Oxidative phosphorylation : transfer of electrons from one compound to another is used to generate ATP by chemiosmosis .

30. Metabolic Pathways of Energy Production: COH Catabolism Cellular respiration Aerobic respiration Anaerobic respiration Fermentation The three steps of aerobic respiration Glycolysis (oxidation of _____ to ______) Krebs cycle (oxidation of acetyl CoA to ___) Oxidative phosphorylation (e - transport chain)

31. Respiration Cellular respiration Series of oxidation-reduction reaction in a membrane that generates ATP Respiration can be: Aerobic respiration – in the presence of oxygen, final electron acceptor is oxygen KREB CYCLE AND ELECTRON TRANSPORT CHAIN Anaerobic respiration – absence of oxygen, FERMENTATION PROCESS Aerobic respiration uses reducing agents produced by glycolysis and TCA cycle to produce ATP A phosphate group is covalently linked to ADP to form ATP, termed oxidative phosphorylation A two-step mechanism is used to generate ATP Electron transport chain that builds a proton (H + ) gradient across a membrane Proton motive force (PMF) that powers an enzyme, ATP synthase , to link phosphate to ADP

32. In prokaryotes, respiration happens in the cytoplasmic membrane surrounding the cell In eukaryotes, respiration happens in the inner membrane of the mitochondria Both systems are nearly identical Respiration (cont.)

33. GLYCOLYSIS 1 ST stage of carbohydrate catabolism Also known as Embden-Meyerhof pathway Splitting of sugar, oxidized, releasing energy, and their atoms are rearranged to produce pyruvic acid Series of 10 chemical reactions catalyzed by different enzymes.

34. Glycolysis Multi – step breakdown of glucose into pyruvate Generates small amount of ATP (how many?) small amount of reducing power – (?) Alternative pathways: Pentose phosphate and Entner-Doudoroff

35. The Steps of Glycolysis

37. GLYCOLYSIS

38. Krebs Cycle Other names? Transition step generates acetyl-CoA from pyruvate (decarboxylation) Acetyl group of acetyl- CoA enters TCA cycle Generates ATP and reducing power Generates precursor metabolites

39. Krebs Cycle

41. Electron Transport Chain generates the PMF NADH and FADH 2 are electron donors They give up their electrons to a series of protein complexes that use the energy to translocate H + across the membrane The accumulation of H + on one side of a membrane forms an electrochemical gradient Respiration (cont.)

42. Electron Transport Chain Formed by series of electron carriers ( cytochromes ) located in ___________ Oxidation/Reduction reactions . Electron carriers (reducing power) from glycolysis and TCA cycle transfer their electrons to the electron transport chain Generates proton gradient or proton motive force (pmf) In chemiosmosis , pmf generates energy via oxidative phosphorylation

43. Electron Transport and the Chemiosmotic Generation of ATP

44. Overview of Respiration and Fermentation

46. Net production from 1 glucose molecule is 38 ATP molecules 2 ATP from glycolysis 2 ATP from TCA cycle 34 ATP from aerobic respiration Respiration (cont.)

47. Anaerobic Respiration Inorganic molecule is final electron acceptor, e.g.: NO 3 - SO 4 2- ATP yield lower than in aerobic respiration because only part of Krebs cycle operates under anaerobic conditions.

48. Fermentation In respiration, a terminal electron acceptor is used to consume donated electrons Oxygen is commonly used as the acceptor by aerobic microorganisms Fermentation is used by organisms that cannot perform respiration to consume electrons Oxygen is not available Obligate anaerobe (lacks genes for the proteins of the electron transport chain) Fermentation products are particularly useful for identifying enteric bacteria

49. Fermentation Any spoilage of food by microorganisms (general use) Any process that produces alcoholic beverages or acidic dairy products (general use) Any large-scale microbial process occurring with or without air (common definition used in industry) Scientific definition: Uses an organic molecule as the final electron acceptor Does not use the Krebs cycle or ETC Energy yield low

50. The Relationship of Fermentation to Glycolysis

53. Catabolism of Organic Compounds Other Than Glucose Polysaccharides and disaccharides Broken down into simple sugars These simple sugars can enter glycolysis

54. Lipid Catabolism Lipids are broken down by lipases Glycerol can enter glycolysis Fatty acids can enter the TCA cycle

55. Protein Catabolism Proteins are broken down by proteases Amino acids can be recycled

56. Carbohydrate Catabolism 36 ATPs are produced in eukaryotes Pathway By Substrate-Level Phosphorylation By Oxidative Phosphorylation From NADH From FADH Glycolysis 2 6 0 Intermediate step 0 6 Krebs cycle 2 18 4 Total 4 30 4

57. Catabolism of Other Compounds Polysaccharides and disaccharides Amylases for digestion of ___________ (very common) Cellulase for digestion of cellulose (only bacteria and fungi have this enzyme) Disaccharidases

58. Protein Catabolism Protein Amino acids Extracellular proteases Krebs cycle Deamination, decarboxylation, dehydrogenation, desulfurylation Organic acid Decarboxylation

59. Chemolithotrophs Use reduced inorganic compounds as a source of energy Hydrogen sulfide (H 2 S) Ammonia (NH 3 ) These compounds are electron sources that are used in oxidative phosphorylation to produce ATP

60. Photosynthesis Photosynthetic bacteria capture photons from radiant energy for ATP synthesis Pigments, such as chlorophyl , capture light energy and funnels it to an electron transport chain

61. The ETC is used to generate a proton gradient that produces ATP and converts inorganic carbon into organic carbon ( carbon fixation ) 6CO 2 + 6H 2 O ➔ C 6 H 12 O 6 + 6O 2

62. SYMBIOTIC RELATIONSHIPS INVOLVING MICROBES

63. Symbiosis Symbiotic relationship Living together or close association of 2 dissimilar organisms (usually 2 different species) Organisms that live together in such a relationships are referred as symbionts Some relationships are beneficial to both symbiont, others are beneficial to only 1 symbiont while others are harmful to one symbiont.. Different symbiotic relationship that occur in microbes are: Neutralism Commensalism Mutualism Parasitism Syngergism (synergistic infection)

64. 1. NEUTRALISM A symbiotic relationship in which neither symbionts is affected by the relationship. Reflects a situation in which different microbes occupy the same ecologic niche, but have no effect on each other

65. 2. COMMENSALISM Relationship that is beneficial to one symbiont and of no consequence to the other. Ex. Microflora in/on our body are considered commensals Obvious benefits to the microbes as they are provided nutrients and “housing” and has no effect on the host. Host Living organism that harbors another living organism.

66. 3. MUTUALISM Relationship that is beneficial to BOTH symbiont Example Escherichia coli , which obtains nutrients from food materials ingested by the host and produces vitamins (Vit. K) that are used by the host. Some members of our indigenous microflora prevent colonization by pathogens and overgrowth by opportunistic pathogens Protozoa that live in the intestine of termites. Termites eat wood, but they can not digest wood. Protozoa digest the wood and convert it to nutrients used up by the termites. Termites provide food and warm moist environment for the protozoa.

67. 4. PARASITISM Symbiotic relationship that is beneficial to one symbiont (the parasite) and detrimental to the other symbiont (the host) “ Smart” parasites do not cause disease, but rather take only the nutrients they need to exist. “ Dumb” parasites kill their host, they must either find a new host or die. Certain parasites always cause disease, and some cause the death of the host.

68. OPPORTUNISTIC PATHOGENS Many indigenous microflora are opportunistic pathogen, awaiting the opportunity to cause disease. Immunosuppressed individuals are especially susceptible to it. Opportunists can also cause disease in otherwise healthy persons if they gain access to the blood, urinary bladder, lungs, or other organs and tissues of those individuals. Conditions that may enable opportunists to cause disease include: Burns Laceration Surgical laceration Disease that debilitate(weaken) the host Interfere with host defense mechanism.

69. 5. SYNERGISM (SYNERGISTIC INFECTION) 2 or more microbes that team up to produce a disease that neither could cause by itself. – SYNERGISTIC RELATIONSHIP SYNERGISTIC INFECTION Diseases that are caused by polymicrobial infection or mixed infection. EX. Fusobacteria and spirochetes, which together cause the disease “trench mouth”

70. NORMAL HUMAN MICROBIAL FLORA

71. INDIGENOUS MICROFLORA Also known as normal flora All microbes that reside on and within that person. Our bodies composed of 10 trillions of cells and about 10x that many microbes that live on and within our bodies A fetus has no indigenous microflora. During and after delivery, a newborn is exposed to these microbes (mother, air, food and everything that touched the baby)

72. Conditions for proper growth (moisture, pH, temperature, nutrients) vary throughout the body Thus, the types of resident flora differ from one anatomic site to another. Blood, lymph, spinal fluid and most internal tissues and organs are free of microbes.

73. TRANSIENT MICROFLORA Take up temporary residence on and within humans since we are constantly exposed to microbes These microbes are attracted to moist, warm body areas.

74. Temporary because: Washed from external areas by bathing Not be able to compete with the resident microflora Fail to survive in acidic/alkaline environment May be killed by substances produced by resident microbes Flushed away by bodily excretions or secretions (urine, feces, tears, perspiration) Superinfection An overgrowth or population explosion of an organism that is usually present in low numbers.

75. Microflora of the Skin Primarily bacteria and fungi The number and species depend on the environmental conditon (moist, temperature, pH and etc) Moist, warm environment in hairy areas where there are many sweat and oil glands, such as under the arms and in groins, stimulate the growth of Staphylococci, diptheroids, aerobic spore-forming bacilli, nonpathogenic mycobacteria, gram negative enteric bacilli ( E.coli ), Candida albican and Cryptococcus .

76. Dry,calloused areas of the skin have few bacteria, whereas moist folds between the toes and fingers support many bacteria and fungi. Frequent washing of soap and water removes most of the potentially harmful transient microbes harbored in sweat, oil, and other secretions from moist body parts. Responsibilities: Particularly careful to keep patient’s skin and clothing as free of transient microbes as possible To help prevent personal infections To avoid transferring pathogens to patients. Most infections following burns, wounds and surgery from the growth of resident or transient skin microflora in these susceptible areas.

77. Microflora of the Mouth Mouth and throat contain many or varied population of microbes Areas provide moist, warm mucus membranes that furnish excellent conditions for microbial growth. Bacteria thrive well I particles of food and in the debris of dead epithelial cells around the teeth. Shelters anaerobic and aerobic microbes due to oral cavity’s peculiar anatomy. Anaerobic microbes thrive in gum margins, in cervices between the teeth, and in deep folds on the surface of the tonsils.

78. Most microbes found in mouth are harmless and beneficial like diptheroids, lactobacilli and micrococci. Streptococci and staphylococci are potentially opportunistic pathogens and are frequently associated with disease. Some people carry virulent microbes in their nasal passages or throat but do not have the disease associated with them Ex. Diptheroids, meningitis, pneumonia and TB. These people are healthy CARRIERS who are resistant to these pathogens but can transmit them to susceptible person. Poor dental hygiene allows the growth of microbes that cause dental caries, gingivitis, and periodontitis. Bacteria includes Actinomyces, Lactobacillus, Streptococcus, Neisseria and Veillonella.

79. Many alpha-hemolytic streptococci are indigenous inhabitants of the mouth and oropharynx. When large numbers of this group is present, antibiotic therapy should be given to destroy these pathogens that may cause “strep throat and its complication (scarlet fever, rheumatic fever, and glomerulonephritis)

80. Microflora of the Respiratory tract Lower respiratory tract, below the larynx is sterile (free of microbes) Mucus membranes and lungs have defense mechanisms that efficiently removes the invaders. Membranes of URT, include the naso and oropharynx, provide a suitable environment for growth of manny species of Streptococcus, Staphylococcus, Neisseria, Corynebacterium , yeast, and other microbes. These microbes are opportunist that cause disease to susceptible host. Presence of staphylococci. Streptococci, Pseudomonas sp , or yeast found in the sputum specimens would indicate either: An infectious disease of the lungs Specimen contamination by indigenous microflora of the URT

81. 7 CAPABILITIES OF A PATHOGEN