Microbial Growth

Optimum Temperature Requirements Most human pathogens are ???

Physical Requirements 65 -130 ˚C- most bacteria destroyed by enzyme denaturation. 50-65 ˚C slow growth 15-50 ˚C Danger of food spoilage 5 -15 ˚C survival, and slow growth 0-5 ˚C slow or no growth <0 ˚C no growth, but may survive.

Physical Requirements <6.0 acidophiles 6 - 8 neutrophiles >8.0 basophiles Most pathogens are neutrophiles.

Physical Requirements Osmotic Pressure Isotonic - 0.85% NaCl - 300 mOs Hypotonic- low salt or pure water Hypertonic - high salt - halophiles

Nutritional Classification Photoautotroph – Energy = sunlight, Carbon source = CO2 Photosynthesis Photoheterotroph - Energy source = sunlight, Carbon source = organic compounds. Chemoautotrophs - Energy source = Oxidation of inorganic compounds Nitrates, Iron, Sulfur. Carbon source = CO2 Chemoheterotroph Both Energy and Carbon sources are organic compounds.

Chemical Requirements

Aerotolerance Obligate Aerobe – Uses oxygen as final Hydrogen acceptor. Has Superoxide dismutase and catalase. Pseudomonas aeruginosa Facultative 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.

Chemical Requirements Anaerobe jar Chemicals make CO2 and H2 H2 reacts with O2 + paladium --> H2O O2 consumed. Typical anaerobes: Clostridium, Bacteroides

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

Special Techniques Anaerobic growth Extra CO2 -capnophilic -Neisseria Selective media- selects for specific group. Differential media-allows you to tell one group from another.

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 phagocytes Superoxide Free Radical O2- Produced during aerobic respiration must be detoxified by superoxide dismutase. Peroxide anion O22- very reactive and toxic. Removed by catalase.

Chemical Requirements Carbon - Photosynthesis - Basis for all organic compounds Nitrogen From nitrates, ammoinum salts, amino acids, and nitrogen fixation Needed for protein and nucleic acid synthesis Phosphorous From phosphate salts. For DNA, RNA, ATP, etc Sulfur From sulfate ions, hydrogen sulfide and sulfur containing amino acids For protein synthesis Trace Elements Mg, Fe, Mn, Zn, Cu etc for metabolism.

Culture Medium Medium Sterile - no living organisms present. Simple - defined Inoculum- microbes that start the culture Culture = medium + microbes Agar – polysaccharide derived from seaweed

Culture Medium Medium Agar - polysaccharide derived from seaweed Chemically defined Complex - serum, beef or yeast extract, peptones etc Fastidious organisms have many nutritional requirements. Organic growth factors

Non-Selective Media Nutrient Agar Blood Agar LB agar (no antibiotics) Tryptic Soy Agar –TSA in petri dishes Nutrient Broth Sugar tubes TSA Agar (tube) TSA broth Brain Heart Infusion

Selective Media E. coli on EMB Mannitol Salt Agar Phenylethyl Alcohol Inhibitor - Methylene blue Indicator- Eosin Gives a dark purple colony with green metallic sheen Mannitol Salt Agar Phenylethyl Alcohol MacConkey Agar

Enrichment Media Media to increase the numbers of desired species, but doesn’t purify them. Used in soil and fecal samples. Salmonella- Selenite broth. Bacto-Dubos - M. tuberculosis Grow overnight and then plate on elective medium. Increases chances of isolation when numbers are very low or contaminants very high.

Obtaining Pure Cultures Streak plate. Each new direction diltues organisms until you get individual ones. CFU Serial dilution. Dilute in broth until you get 1 organism. 1:10 --> 1:100 --> 1:000

Obtaining Pure Cultures

Preserving Cultures Large stock tubes - 4 to 6 months @ 4˚C Freeze in medium with glycerol -20˚C- 1 year Store -70˚C - 5 to 10 years Store liquid nitrogen - indefinitely. Lyophilization- freeze drying - indefinitely.

Frozen Culture Trick Can’t thaw and freeze very many times- kills organisms. Aseptically add 0.2-0.3 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.

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.

Dynamics of Bacterial Growth Generation Times GT. E. coli - 20 minutes Bacillus subtilis - 1-2 hours Borrelia burgdorferi - 8-10 hours Mycobacterium tuberculosis- 1 day Treponema pallidum - 35 hours ( in rabbits) Mycobacterium leprae - > 30 days

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 nutrients Hostile media conditions (acid, toxic by-products)

Methods to Measure Growth Direct Plate Counts Direct Counting Indirect (estimations) Turbidity Metabolic activity Dry Weight

Plate Counts Perform serial dilutions Plate out samples from dilutions

Plate Counts Can do spread plates for aerobes. Can do pour plates for facultative anaerobes or microaerophiles.

Direct Counts Petroff-Hausser chamber Dilute sample. Add volume of diluted medium to chamber. Count bacteria Calculate number in original sample.

Indirect Methods Metabolic Activity Dry Weight By product - CO2 More product / min = higher rate = more bacteria Dry Weight Collect by centrifugation Decant liquid Dry Weigh

Turbidity Use spectrophotometer. Measure transmission in sterile medium. Measure transmission in culture.