2 Optimum Temperature Requirements Most human pathogens are ???
3 Physical Requirements ˚C- most bacteria destroyed by enzyme denaturation.50-65 ˚C slow growth15-50 ˚C Danger of food spoilage5 -15 ˚C survival, and slow growth0-5 ˚C slow or no growth<0 ˚C no growth, but may survive.
8 AerotoleranceObligate Aerobe – Uses oxygen as final Hydrogen acceptor. Has Superoxide dismutase and catalase. Pseudomonas aeruginosaFacultative Anaerobe – Can use O2, but other hydrogen acceptors i.e., nitrates. They are also capable of fermentation. Efficiency decreases without oxygen. Has Superoxide dismutase and catalase. E. coli, Staphylococcus sp.Obligate Anaerobe – unable to use oxygen for energy. Most are harmed by it. No enzymes to detoxify oxygen forms. Clostridium sp.
9 Chemical Requirements Anaerobe jarChemicals make CO2 and H2H2 reacts with O2 + paladium --> H2OO2 consumed.Typical anaerobes:Clostridium, Bacteroides
10 Aerotolerance continued Aerotolerant Anaerobe – Cannot use oxygen i.e., Fermentative, but tolerate oxygen – possess Superoxide dismutase. Lactic acid producers.Microaerophiles- Require oxygen but too much causes the production of toxic oxygen compounds. No enzymes. Lower levels of oxygen required for grow.Capnophile – Some aerobic organisms require lower levels of oxygen and higher levels of CO2.Neisseria meningiditis or gonorrhea
11 Special Techniques Anaerobic growth Extra CO2 -capnophilic -Neisseria Selective media- selects for specific group.Differential media-allows you to tell one group from another.
12 Toxic Forms of Oxygen Normal Atmospheric Molecular Oxygen O2 Most stable form of oxygen, but quite reactive.Singlet oxygen – O single oxygen atom, higher energy state, much more reactive than O2. Found in phagocytesSuperoxide Free Radical O2- Produced during aerobic respiration must be detoxified by superoxide dismutase.Peroxide anion O22- very reactive and toxic. Removed by catalase.
13 Chemical Requirements Carbon -Photosynthesis - Basis for all organic compoundsNitrogenFrom nitrates, ammoinum salts, amino acids, and nitrogen fixationNeeded for protein and nucleic acid synthesisPhosphorousFrom phosphate salts.For DNA, RNA, ATP, etcSulfurFrom sulfate ions, hydrogen sulfide and sulfur containing amino acidsFor protein synthesisTrace ElementsMg, Fe, Mn, Zn, Cu etc for metabolism.
14 Culture Medium Medium Sterile - no living organisms present. Simple - definedInoculum- microbes that start the cultureCulture = medium + microbesAgar – polysaccharide derived from seaweed
15 Culture Medium Medium Agar - polysaccharide derived from seaweed Chemically definedComplex - serum, beef or yeast extract, peptones etcFastidious organisms have many nutritional requirements. Organic growth factors
16 Non-Selective Media Nutrient Agar Blood Agar LB agar (no antibiotics) Tryptic Soy Agar –TSA in petri dishesNutrient BrothSugar tubesTSA Agar (tube)TSA brothBrain Heart Infusion
17 Selective Media E. coli on EMB Mannitol Salt Agar Phenylethyl Alcohol Inhibitor - Methylene blueIndicator- EosinGives a dark purple colony with green metallic sheenMannitol Salt AgarPhenylethyl AlcoholMacConkey Agar
18 Enrichment MediaMedia to increase the numbers of desired species, but doesn’t purify them.Used in soil and fecal samples.Salmonella- Selenite broth.Bacto-Dubos - M. tuberculosisGrow overnight and then plate on elective medium.Increases chances of isolation when numbers are very low or contaminants very high.
19 Obtaining Pure Cultures Streak plate.Each new direction diltues organisms until you get individual ones.CFUSerial dilution.Dilute in broth until you get 1 organism. 1:10 --> 1:100 --> 1:000
21 Preserving Cultures Large stock tubes - 4 to 6 months @ 4˚C Freeze in medium with glycerol -20˚C- 1 yearStore -70˚C - 5 to 10 yearsStore liquid nitrogen - indefinitely.Lyophilization- freeze drying - indefinitely.
22 Frozen Culture TrickCan’t thaw and freeze very many times- kills organisms.Aseptically add mls of warm medium to top of the frozen vial.Swish with pipette tip and remove liquid.This thaws only a small top portion keeping the remainder frozen.Can do this 10 times per vial.
23 Bacterial Growth Binary Fission one cell makes two Cell first grows in size.Replicates its DNA (genome)Then divides.Time it takes between divisions is the Generation Time.
24 Dynamics of Bacterial Growth Generation Times GT.E. coli - 20 minutesBacillus subtilis hoursBorrelia burgdorferi hoursMycobacterium tuberculosis- 1 dayTreponema pallidum - 35 hours ( in rabbits)Mycobacterium leprae - > 30 days
25 Phases of Growth Lag - no growth. Exponential Growth- rapid growth- GT.Stationary Phase- equal numbers dying and dividing.Logarithmic Death - rapid death due to :Lack of nutrientsHostile media conditions (acid, toxic by-products)