Module 4 Notes:How organisms obtain carbon – autotroph vs. heterotroph (most are heterotrophs)- Aerobic respiration - Carbohydrate fermentation - Proteolytic activity - Decomposition of proteins (proteases break down larger proteins)Aerobic Respiration: release of energy from glucose or another organic substrate in the presence of oxygen - Glucose + Oxygen = CO2 + H2O + Energy- 2 ATP  Glycolysis  4 ATP + 2 Pyruvate  Krebs  e- (NADH & FADH2)  oxidative phosphorylation/ETC  creates gradient as e- pumped out  protons back in to naturalize  forms ATP  ~38 ATPo O2 picks up extra e- forming H2OFermentation: conversion of carb such as sugar into – acid (lactic, acetic, etc.), neutral (ethyl alcohol), gas (CO2, hydrogen, etc.)- W/o O2- Glycolysis  fermentation - Depending on enzyme/organism, different end products - Not all organisms can ferment - In presence of O2, fermentation will not occur- How to determine:o Special brother contains the carb, nutrients and Phenol Red; if pH changes there will be a color change; if gas is, produced gas will be trapped in aerobe tube; both can occur together Anaerobic Respiration:- Converts the chemical energy into an electrochemical gradient through use of the ETC- True anaerobes will never be fermenters Obligate Anaerobes cannot live in presence of O2 b/c reactive O2 will kill; in order to grow in lab you must remove O2All organisms that live in presence of O2 must have some form of 2 enzymes: SOD and catalaseGastight jar containing GasPAK removes all O2 from jar; indicator strip will tell you whether isworking or not (if there’s O2 in the jar or not)Thioglycolate Broth: takes up/removes O2 from medium; is a O2 free suspension, small oxic (oxygen) zone at top of suspension in tube, high oxygen content directly above suspension - Obligate aerobes: must grow at top- Obligate anaerobes: must grow at bottom- Facultative Anaerobes: can grow anywhere, grow much faster at top because can more efficient ATP production in presence of O2; ones at bottom must ferment- Aero-tolerant anaerobes: O2 does not effect them; grow equally throughout; O2 does not help or hurt them - Microaerophile: grows slightly below top; don’t have catalase Release of proteases: how do microbes eat? Excretion of digestive enzymes  digestive enzymes degrade substrate  digested food absorbed by hyphae - Proteolytic activity; positive result – liquefied gelatin, negative result – solid matrix, no protease activity- Decomposition of proteins: odiferous gas, hydrogen sulfide (rotten egg smell), is commonly given off by the action of proteolytic bacteria on peptides; can use this to detect proteolytic bacteria; Peptone Iron Agar (contains iron and protein)  if bacteria produces Hydrogen Sulfide, it will react with the iron in the agar  iron sulfide becomes black precipitate in the media - Relevance: we have to characterize bacteria by means of their phenotypic traits; this is how technicians will identify what bacteria is making your pts sick Module 4 Worksheet:1. Indicate bacterial growth in the following conditions with + or –Aerobic Anaerobic a. Obligate aerobe sample + ++ -b. Facultative anaerobe sample +++ +c. Obligate anaerobe sample - +++2. How are obligate anaerobes grown in suspension or in agar plates are cultured in the laboratory? a. Liquid culture: Thioglycolate broth, which removes oxygen from the suspension b. Agar plates: are put into Aneorobic jar/GasPAK which will remove the oxygen 3. Bromcresol purple sugar fermentation tubes are used to determine if a given bacterium ferments a specific sugar (e.g., glucose or lactose). How do you recognize the formation of a. Acid: the change in the pH will change the color of the suspension (from purple toyellow with pH drop) b. Gas: if gas is produced there will be air bubbles formed in the tube4. Discuss the role of sugar fermentation products in the etiology of dental caries (as indicated in the lecture (be brief).o When there is no oxygen present for the microbes present on teeth to produce energy through aerobic respiration, they will go through fermentation to break down glucose which produces lactic acid which erodes that calcium in tooth enamel (plaque itself is source of food plus food you eat)5. Explain 2 reasons why drinking soda pop increases risk of dental caries?a. It is adding sugar to the mouth which will feed the bacteria present in the mouth b. The carbonation is acidic 6. Explain the procedure (culture medium and color reaction) employed to determine bacterial production of hydrogen sulfide (H2S).o The medium is called Peptone Iron Agar; the hydrogen sulfide will react with the iron and this will from a black precipitate in the suspension 7. What kind of bacterial enzyme causes gelatin liquefaction? Is this an intracellular or extracellular enzymes?o Protease/gelatinase, extracellular (since it does it’s job outside of the cell)8. What is a Gas-Pak and how is it used?o It is an anaerobic jar that removes all of the oxygen inside of it and add specific catalyst, not a vacuum; there is an indicator strip to tell whether there is actually O2 present inside or not; allows obligate anaerobes to grow9. Do different bacteria vary in their ability to ferment sugars, produce H2S, and liquefy gelatin? How do we use this information to identify bacteria?o Yes, do to the different enzymes and such present, based on what they can ferment/produce/liquefy allows us to identify what they are 10. Describe a flow chart.o Starts broad and then slowly branches off getting more specific Module 5 Notes:Gram Stain: most important and most common used technique when looking at bacteria1. Stains bacteria that are normally colorless2. Allows you to see morphology (shape)3. Allows you to differentiate between gram positive and negative- Always first step in identification- Always use fresh culture - Give idea how to treat pt (ex. If pink no penicillin)- Understanding the staining process is important (same as lecture); if you don’t add alcohol everything will be purple; without iodine all would be pink Gram (-): have two membranes (inner and outer), thin layer of PD, has LPS (lipopolysaccharides), pink, penicillin will not fxn Gram (+): thick layers of peptidoglycan, lipostoic; dark purple/blueModule 5 Worksheet:1. 4 key steps of staininga. crystal violet stains – stains purple/violet (both),