1. Lab #7

2. 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

3. 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)

4. 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

5. Growth Media – Pg. 59-61 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)

6. Growth Media – Pg. 59-61 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 )

7. 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)

8. 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)

9. Biochemical Testing – Pg. 64-66 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

10. 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

11. pH Indicators pH Indicator Very acidic AcidicNeutralBasic Phenol red- pH <6.8 = yellow pH 6.9 - 8.0 = red pH >8.0 = magenta/ hot pink Litmus-pinkpurpleblue Bromocresol Purple -yellowburgundyviolet Methyl red pH <4.4 = red pH 5 - 6 = orange pH >6.3 = yellow - Memorize pH indicators and color reactions!

12. 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

13. Sugar Fermentation – Pg. 71-74 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)

14. 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)

15. Sugar Fermentation Experiment - DEMO Types of sugars: Glucose Lactose Mannitol Sucrose Record results on pg. 74

16. 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 24-48 hrs (Wed – Fri open lab hours)

17. 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!

18. Today’s 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

19. Today’s 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

20. 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 Today’s Inoculations