Lab #7 Microbial growth and metabolism So far what we know: Colony morphology and cell morphology (rod vs cocci) Motility Oxygen requirement Gram stain, endospore, and acid fast reaction This information not enough to identify a particular organism In order to identify an organism specifically (species) biochemical and metabolic tests need to be performed Microorganisms are very diverse in their ability to: 1) Ferment/ utilize particular substrates as food 2) Produce specific enzymes that allow them to metabolize Microbial growth and metabolism Metabolism = sum of processes occurring within the cell that lead to the production of energy (ATP) Requires enzymes proteins that carry out the chemical processes within the cell Anabolism synthesis of new bonds and larger molecules (requires energy) Catabolism breakdown of bonds and macromolecules (releases energy) Anabolism and catabolism occurs via redox reactions Overview of metabolism: Enzymes Substrate Product(s) Microbial growth and metabolism Major nutritional needs: Energy source Carbon source Nitrogen source Phosphorous, sulfur Water (hydrogen, oxygen) Vitamins Minerals Physical needs: Temperature Oxygen Osmotic pressure (salt concentration) pH How do microorganisms get nutrients in their natural habitat? Environment How do microorganisms get nutrients in the lab? Growth media Growth Media Pg Classified based on composition (ingredients) and function Solid vs. Liquid Solid media contains Agar Synthetic vs. Non-synthetic Synthetic = artificial; exact chemical composition (formula) is known Non-synthetic = contains natural (unpurified) mixtures; exact chemical composition is not known (digested proteins, beef, peptone, animal/plant extract) Growth Media Pg General purpose vs. Minimal General = allows many different organisms to grow Minimal = contains very specific nutrients will allow specific organisms to grow Enriched contains complex ingredients (blood, yeast, serum, brain infusion) Reducing chemicals added to remove oxygen (thio, cysteine) Buffered chemicals added to stabilize pH of media (two phosphate salts KH 2 PO 4 and K 2 HPO 4 ) Growth Media Selective vs. differential Selective = allows some organisms to grow but prevents others (salt, pH, dyes, and toxins) Differential = different species will grow but will look different (sugars, pH indicators) Mannitol Salt Agar: Selective for Staphylococcus species Differential for S. aureus able to ferment sugar mannitol (yellow color) Growth Media Media must be sterilized before use Autoclave Steam at high pressure 121 o C and 15 psi for 15 minutes achieves sterilization Radiation Filtration heat liable chemicals (antibiotics, dyes) Biochemical Testing Pg How do you know a reaction has occurred? 1) Patterns and location of growth Aerobic vs. anaerobic 2) Turbidity (cloudiness) Increase growth Decrease hydrolysis/ breakdown substance in media 3) Physical condition of the medium Liquefaction hydrolysis/ breakdown of substances in media Loss of fluidity (coagulation) liquid media becomes solid Gas production bubbles can be detected Biochemical Testing How do you know a reaction has occurred? 4) Colorimetric changes A result of pH change detected by pH indicator Non-pH changes a reagent is used to detect the production of a particular substance Table on pg. 65 pH Indicators pH Indicator Very acidic AcidicNeutralBasic Phenol red- pH 8.0 = magenta/ hot pink Litmus-pinkpurpleblue Bromocresol Purple -yellowburgundyviolet Methyl red pH 6.3 = yellow - Memorize pH indicators and color reactions! Biochemical Testing Things to keep in mind for biochemical tests (pg. 66): Controls Must include a control for comparison & to validate the test Everything will be the same except one factor (m/o) A positive result will look different from the control Proper recording of results Positive result Reaction has occurred (color change, gas production, turbidity, etc.) Indicate using a + sign Negative result No reaction has occurred (looks the same as control) indicate using a - sign Sugar Fermentation Pg Bacteria have the ability to breakdown certain carbohydrates to produce energy (ATP) Fermentation (pathway) Carbohydrates = sugars (made up of C, H, and O) Fermentation does not require oxygen (anaerobic) Fermentation of sugars results in the production of: Acid or an alcohol Gas (CO 2 ) in some cases Overview of fermentation: Sugar Pyruvic acid acids/alcohol + ATP (maybe CO 2 gas) Sugar Fermentation Fermentation can be detected using a media that includes a sugar and a pH indicator Positive sugar fermentation reaction: A = acid production (yellow color change) A/G = acid production (yellow) and gas in Durham tube SA = slow reaction (orange color change) Negative sugar fermentation reaction: B = base production (pink/magenta color change) (-) = No change (looks exactly like the control) Sugar Fermentation Experiment - DEMO Types of sugars: Glucose Lactose Mannitol Sucrose Record results on pg. 74 Water Analysis Project and Lab Report Pg. 131 138 Test for presence of coliform (E. coli) G- rod bacteria found in the GI tract Perform three tests (Pg. 136): Presumptive Confirmed Completed Today start the presumptive test Obtain 3 water samples A, B, and C inoculate 1mL of each sample into a separate lactose broth tube Inoculate 1mL of the (+) and (-) controls into separate Lactose broth tubes Make sure all 5 tubes are properly labeled Incubate the tubes Read results in hrs (Wed Fri open lab hours) Water Analysis Project and Lab Report Presumptive test results (table on pg. 133) Positive: Acid and gas production = a/g (color change to yellow and gas bubble in Durham tube) On Wed-Fri open lab: Use positive tubes to streak an EMB (Eosin Methylene Blue) plate Confirmed test One plate for each positive result! Confirmed test results Positive: Metallic green colonies Obtain a single colony from the positive streak plate and inoculate it into a lactose broth Completed test Completed test results Positive: Acid and gas produced Indicates the presence of coliform bacteria fecal contamination! Todays Inoculations Nitrate reduction test (#15) use loop to inoculate 3 Nitrate broths: 1) B. subtilis 2) Alcaligenes faecalis 3) E. coli 4) Control tube no inoculation Production of decarboxylase (#17) (total 12 tubes + 1 control) Use loop to inoculate Lysine broths with: 1) Enetrobacter aerogenes 2) Enterobacter cloacea 3) Klebsiella pneumoniae 4) Morganella morganii 5) Control tube no inoculation Repeat using Arginine broths and Ornithine broths Add 1mL of sterile mineral oil on top of EACH broth culture AFTER inoculation Todays Inoculations SIM Reations (#19) 1 tube, 3 tests Inoculate 4 SIM deeps using a needle: 1) Proteus vulgaris 2) E. coli 3) Citrobacter freundii 4) Morganella morganii 5) Control no inoculation MR-VP Reactions (#20) 1 tube, 2 tests Inoculate 3 MR-VP broths using a loop: 1) Enterobacter aerogenes 2) E. coli 3) K. pneumoniae 4) Control no inoculation Make sure each tube is properly labeled!! Name of media Inoculated bacteria Your name Date Place all your tubes in your basket and fasten them with a rubber band Place basket in the incubator Todays Inoculations