Microbiology Lab Exam

• Lysozyme


  • Found in many body fluids (tears and saliva)
  • Functions by breaking the beta(1-4) glycosidic bond between the NAM and NAG sugars in the peptidoglycan, thereby weakening the cell wall
  • Once the wall is compromised, osmotic pressure causes the cell membrane to burst, killing the cell
  • In some gram-positive organisms, modification of the cell wall by teichoic acids lends resistance to lysozyme degradation
• Catalase test


  • Differential test to determine if an organism has the enzyme catalase
  • This enzyme can be found in aerobes, microaerophiles, and facultative anaerobes
  • The ability to make these protective enzymes accounts for an organism's ability to live in aerobic environments
  • Oxygen is toxic to obligate anaerobes because they lack the enzymes catalase and superoxide dismutase, which makes superoxide radicals into a much less damaging hydrogen peroxide, to break down toxic oxygen intermediates that tend to evolve under aerobic conditions
• Catalase reaction


  2H2O2 ---> 2H2O + O2 (hydrogen peroxide ---> water + oxygen)
• Oxygen is toxic to obligate anaerobes because these microbes lack enzymes such as catalase and superoxide dismutase to effectively break down toxic oxygen intermediates that tend to evolve under aerobic conditions
• Some bacteria use catalase to thwart immune defenses by breaking down the hydrogen peroxide that certain phagocytic white blood cells (macrophages) use to kill bacteria
• Catalase positive


  Bubbles will form when hydrogen peroxide is added to a bacterium that is catalase positive, no bubbles mean catalase negative
• Citrate


  A six-carbon weak organic acid
• Citrate utilization


  1. If yes bacterium makes citrate permease, allows transport of citrate into cell

  2. Then citrate lyase converts the imported citrate into oxaloacetate and acetate

  3. The oxaloacetate is decarboxylated (CO2 is removed) by oxaloacetate decarboxylase to form pyruvate

  4. Resulting pyruvate may then be fermented to a variety of intermediates
• Citrate is not a good carbon source, not a lot of growth even if positive
• When growing organism produces NH3 and NH4OH from nitrogen source in medium, because these are bases, they alkalinize the agar (raising the pH)
• Citrate positive


  • Medium turns blue because of the pH indicator in the broth (bromothymol blue)
  • Blue means the bacterium is actively growing and positive citrate test
• If growth is seen with no color change, usually indicates too short of an incubation time
• Urea


  • Produced as a byproduct of amino acid catabolism and is the primary nitrogenous waste in the urine of many land animals (including mammals)
  • High levels in urine but not only in urine, also blood, sweat and in the stomach
• Urea hydrolysis


  • Differential test to determine if a bacterium produces urease
  • Because urease generates an alkaline byproduct (ammonia), the enzyme can serve as a virulence factor for some pathogens by counteracting acid in the microbe's environment
• Urea hydrolysis reaction


  (NH2)2CO + H2O CO2 + 2NH3 ammonia being detected alkaline product raises the pH gives color change, positive result
• Positive urea hydrolysis


  • 24 hours of incubation and tube turns completely pink = positive fast reaction (a lot of urease production)
  • 24 hours of incubation and not completely pink = positive slow reaction (not much urease production)
  • ANY PINK AT ALL IS A POSITIVE REACTION
• Negative urea hydrolysis


  Yellow means acidified pH down
• SIM medium


  • Multiple Test Medium
  • S: Sulfur reduction – sulfur-containing compounds (ex. SO4^-2) can be reduced anaerobically producing a black precipitate (SO4^-2 H2S + Fe black precipitate)
  • I: Indole production – tryptophanase breaks amino acid tryptophan into pyruvate, NH3, and indole
  • M: Motility – can bacterium produce flagella and swim from stab line
• Triple Sugar Iron Agar

  • Multiple test medium (differential for gram-negative rods): does bacterium ferment? Which sugars? Does the bacterium produce H2S
  • Places cells in aerobic and anaerobic environments
  • Contains 3 sugars: glucose, lactose, and sucrose
  • Also contains animal proteins as carbon and nitrogen sources and sodium thiosulfate as an oxidized sulfur source
  • Phenol red serves as the pH indicator; it appears yellow at pH less than 6.8 and reddish above pH 7.4
  • Ferrous sulfate allows to determine if the tested bacteria reduce sulfur to make H2S; if it does it will produce a black precipitate
• Obligate aerobe on Triple Sugar Iron Agar


  This would have a pink slant and no color change in butt
• Mannitol Salt Agar

  • Selective and differential growth medium
  • Selective: contains 7.5% NaCl, Halotolerant/Halophile can grow, most often to select for Staphylococcus species
  • Differential: contains mannitol (sugar alcohol) (fermentable carb), pH indicator detects acid production, can the bacteria ferment? (plate will turn yellow)
• Bile Esculin Azide Agar

  • Selective and Differential
  • Isolation and identification of enterococcus species
  • Selective: contains bile (oxgall) which inhibit GP other than Group D strep, azide inhibits the growth of GN (bile-resistant)
  • Differential: enterococci hydrolyze esculin in the presence of bile in the presence of bile – Esculetin + Fe(III) dk. Brown precipitate
• Bacteriophage

  • Replicate only within bacterial cells
  • Infect the cells causing them to undergo lysis and form the clear areas (plaques)
  • Following the replication inside its bacterial host the phage lyses the cell from within, releasing ~100 new phages into the environment. When a phage encounters a new bacterial cell, this replication cycle repeats
• Plaque


  • These appear instead of colonies and they are physical evidence of phage replication
  • Formed by the progeny of a single phage that has replicated and lysed the bacterial cells in that area
  • Like colony forming units, plaque forming units (pfu) can be counted to determine the number of phage particles in a suspension
  • Plaque: killed bacteria in an area; Colony: growth of bacteria in an area
• Isolation of Antibiotic Producer: the Streptomyces

  • Soil is rich with microorganisms
  • Antibiotics: small, natural or synthetic molecules that inhibit bacterial growth, many are made by soil microorganisms
  • Plate contains a glycerol yeast extract that has the drug CHX that inhibits any fungal growth from the soil. We are SELECTING for soil bacteria
  • Streptomyces species: GP, filamentous form endospores produce goemins that give dirt its characteristic odor
  • When isolating the streptomyces look for these distinct features: dry looking, wrinkle, small, color pigmentation (chalky white), often grow down into agar
• Kirby-Bauer Test

  • Disk Diffusion (Kirby-Bauer method): Antimicrobial susceptibility test
  • Useful for assessing a pure culture's susceptibility to various antimicrobial drugs
  • During incubation the bacteria grows as a confluent lawn
  • The drugs infused on the paper discs of antibiotics seep into the agar
  • ZOI (zone of inhibition ( a clear zone visible around some of the disks)
  • These clearings are areas where the antimicrobial drug concentration was high enough to inhibit bacterial growth
  • If ZOI is not observed around a disk, then the bacterium is by default considered resistant to the drug
  • The mere presence of ZOI does NOT mean susceptibility, there has to be an amount of ZOI present to show this (measured in mm)
  • Mueller-Hinton agar pH is between 7.2 and 7.4, anything different can affect the drug activity
  • Mueller-Hinton agar is poured to a depth of 4mm because the depth can affect the drug diffusion
• Penicillin

  Prohibits cell wall formation in GP
• Polymyxin B


  Binds to liquid polysaccharide and disrupts membrane (GN)
• Tetracycline


  Must get into cell and inhibits ribosome function/protein production (GN and GP, broad spectrum)
• Vancomycin

  Inhibits cell wall synthesis kills GP only
• Epidemiology


  The study of the causes, occurrence, transmission, distribution, and prevention of infectious and noninfectious diseases in populations
• Incidence rate

  A measure of the frequency of new disease cases in a population within a specified time period
• Period Prevalence

  This measure helps us understand the burden of disease in a population during a specified period
• Epidemic


  Sudden unexpected increase in number of disease cases over time
• Endemic

  There are an expected number of cases we can count on over time
• Pandemic

  An epidemic disease covering a large geographical area
• Antiseptic

  Chemical substance used on living tissue, that reduces CFUs on surface
• Blood Agar

  • Nutritionally complex: TSA + 5% sheeps blood
  • Used to grow fastidious bacteria and distinguish based on patterns of hemolysis
  • Differentiation of bacteria, gram-positive cocci such as streptococcus, staphylococcus, and enterococcus species based on the degree to which they conduct hemolysis
• Hemolysis

  Bacterial toxin that disrupts cell membranes (lyses Red Blood Cells)
• Patterns of Hemolysis


  • Beta hemolysis: complete lysis of Red Blood Cells, clearing around growth
  • Alpha hemolysis: incomplete lysis of Red Blood Cells, medium has greenish brown appearance, involves the oxidation of hemoglobin inside Red Blood Cells to methemoglobin
  • Gamma Hemolysis: no hemolysis, growth is still there, Red Blood Cells not affected