Lab 15

• Enterobacteriaceae family consists of pathogenic and non-pathogenic bacteria that usually inhabit the guts of humans and other animals but can be found in almost all of the natural habitats
• Enterobacteriaceae


  • They are oxidase negative, glucose fermenters, and nitrate reducers
  • Non lactose-fermenters are usually pathogenic while the lactose-fermenters are not
• Biochemical tests are crucial to the identification, diagnosis, and treatment of infection for Enterobacteriaceae
• Fermentation reactions

  1. Oxidation of organic compounds such as sugars

  2. Strict aerobes oxidize sugars to carbon dioxide and water

  3. Strict anaerobes and facultative anaerobes perform fermentation reactions

  4. Facultative anaerobes can perform either respiration or fermentation
• Fermentation test media

  Contains a single carbohydrate for energy production, non-fermentable sources of nitrogen and other nutritional requirements, a pH indicator such as phenol red, and an inverted Durham tube that collects gas
• Positive fermentation reaction


  Change in the color of the indicator from red to yellow (below pH 7) due to the production of acid end products
• Methyl Red and Voges-Proskauer Tests (MR-VP)


  1. Used to differentiate certain intestinal bacteria called coliforms

  2. MR test measures if dextrose is fermented into various acid products

  3. VP test measures if dextrose is converted into neutral end products such as ethanol or butanediol
• Enzymes


  • Exoenzymes are used to degrade large polymers into smaller compounds
  • Endoenzymes catalyze intracellular reactions
• Catalase


  An enzyme that splits hydrogen peroxide into water and oxygen
• Oxidase


  Enzymes that catalyze the reduction of oxygen during respiration
• Citrate utilization


  Some bacteria in Enterobacteriaceae family are able to use citrate as their sole source of carbon
• Urease


  An enzyme that breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia, and water
• Nitrate reduction

  The enzyme nitrate reductase catalyzes the transfer of electrons from cytochrome b to nitrate, reducing it to nitrite
• SIM medium

  Tests for production of hydrogen Sulfide, Indole production and Motility
• Triple Sugar Iron Agar (TSIA)

  Tests for fermentation of glucose, lactose and sucrose, and production of hydrogen sulfide
• The purpose of this experiment is to identify coliforms that were isolated from the samples collected from each of the zones (pre- UV treatment plant, after UV treatment plant and the swash zone) of moonlight beach
• The unknown refers to the day 3 slant culture of the coliform isolated from Moonlight Beach water sample in Exercise 11
• Sugar fermentation reactions


  1. Inoculate glucose, lactose and sucrose broths with the unknown coliform, Escherichia coli (positive control), and Pseudomonas fluorescens (negative control)

  2. Incubate for 24-48 hours at 37°C

  3. Examine the tubes for growth, acid and gas production
• This will allow you to determine if the isolated bacteria using the Most Probable Number method is indeed Escherichia coli
• Sugars tested: Glucose, Lactose, Sucrose, and Mannitol
• Cultures: Escherichia coli (positive control), Pseudomonas fluorescens (negative control), Unknown coliform from water sample (Day 3 slant from Exercise 11)
• Controls: E. coli (positive for MR; negative for VP), E. aerogenes (positive for VP; negative for MR)
• Cultures for catalase production: Positive Control: E. coli, Negative Control: Streptococcus mutans, Unknown from water sample (Day 3 slant)
• Cultures for oxidase production: Positive Control: Pseudomonas fluorescens, Negative Control: E. coli, Unknown from water sample (Day 3 slant)
• Cultures for citrate utilization: E. coli (negative control), Enterobacter aerogenes (positive control), Unknown from water sample (Day 3 slant)
• Cultures for urease test: Escherichia coli, Citrobacter freundii, Unknown from water sample (Day 3 slant)
• Cultures for nitrate reduction: Escherichia coli (positive control), Streptococcus mutans (negative control), Unknown from water sample
• Cultures for SIM test: Escherichia coli, Pseudomonas fluorescens, Citrobacter freundii, Staphylococcus epidermidis, Unknown from isolated water sample
• Cultures for TSIA: Pseudomonas fluorescens, Escherichia coli, Citrobacter freundii, Unknown from water sample
• All biochemical (metabolic) reactions are driven by enzymes
• Each test determines the presence of an enzyme
• Different species of bacteria carry out a unique set of biochemical reactions that is analogous to a fingerprint
• By organizing the test results in a form of dichotomous key, bacterial can be identified
• A set of four tests, IMViC, is an example of commonly used method in quick differentiation of Enterobacteriacea
• An organism to be identified is inoculated into an appropriate test medium which contains the substrate for the enzyme
• Following the incubation, enzyme's activity is confirmed by a formation of the product or a disappearance of the substrate
• Indicator reagents (usually colorimetric) are added to determine the presence of the product or substrate in the media
• For each biochemical test, you need to know the proper name of the test, the enzyme name, the name and components of the media, how the organism is inoculated and processed, what positive and negative results look like, and positive and negative control organisms
• Fermentation involves the conversion of glucose to pyruvate, which can then be further converted to acid and/or gas depending on the organism
• The Methyl Red and Voges-Proskauer tests differentiate between mixed acid fermenters and butanediol fermenters