ENVR 421 Laboratory #3: identification of pathogenic bacteria in water Introduction The purpose of this laboratory exercise is to familiarize you with biochemical identification assays for pathogenic bacteria that occur in water. We will be using the Gram stain and biochemical testing to identify four important bacteria that can occur in water: Shigella, V. cholerae, Klebsiella, and E. coli. Many biochemical tests are based on the ability of bacteria to use different carbon sources as energy for growth. By incorporating these carbon sources into solid and liquid media and observing the patterns of how different bacteria do or do not use them, we can differentiate genii and species of bacteria from each other. Shigella, Klebsiella, and E. coli belong to the family Enterobacteriaceae, a group of Gram-negative, rod-shaped, oxidase-negative bacteria. Identification of Shigella dysenteriae Purpose Shigella dysenteriae is an important cause of bacterial dysentery in developing countries, transmitted via the fecal-oral route. Biochemical testing to distinguish this bacterium from toxin-producing E. coli is important for diagnosing cases of severe diarrhea. Principle Members of the genus Shigella are distinguished from E. coli and Klebsiella by their inability to ferment lactose. Unlike Klebsiella, they do not use citrate as a carbon source or produce urease enzyme. Unlike E. coli, they produce no gas when they ferment glucose, and do not produce indole. Using a triple sugar iron agar slant, you will be able to see the differences in glucose and lactose fermentation between E. coli and Shigella. Materials: • TSA plate with colonies of S. flexneri • sterile sticks • MacConkey plates • Kliger Iron agar slant • Citrate slant• Urease slant • MR tube • Indole tube • VP tube • petri dish with paper in the bottom • oxidase test reagent • Glass slides • Bunsen burner • Gram stain reagents Protocol First day: inoculation and staining 1. Perform a Gram stain 2. look at the stain under the microscope and observe the appearance of the bacteria 3. Streak for isolation on MacConkey agar and TCBS agar 4. Perform an oxidase test 5. Inoculate the KIA slant 6. Inoculate the urease slant 7. Inoculate the citrate slant 8. Inoculate the indole tube 9. Inoculate the MR tube 10. Inoculate the VP tube 11. Incubate all tubes and plates at 35°C Second day: Reading. 1. Examine the colonies on MacConkey and record results 2. Examine the KIA, citrate, urease, and ONPG tubes and record results 3. Perform and read the indole test 4. Perform and read the VP test Third day: Reading. 5. Perform and read the MR testIdentification of Escherichia coli and Klebsiella pneumoniae Purpose Members of the genus Escherichia and Klebsiella are members of the Enterobacteriaceae and are also considered members of the fecal coliform group. They can grow at elevated temperatures (44.5°C). Escherichia coli is more feces-specific, but Klebsiella occurs in feces and it is important to distinguish the two. Principle E. coli and Klebsiella both have the ability to ferment lactose, and are biochemically similar. The main biochemical test that distinguishes the two is the urease test; E. coli do not produce urease and are negative in this test, but Klebsiella are positive. Traditionally there have been four biochemical tests to separate E. coli from other lactose-fermenting Enterobacteriaceae: • Indole test - detects indole production from tryptophane; E. coli is positive (+); many other coliforms are negative. • Methyl Red test - detects acid production in the medium; intended to distinguish between type of fermentation (mixed acid versus butylene glycol). E. coli is (+) and some other coliforms are (-). • Voges-Proskauer test - detects acetoin, an intermediate in the butylene glycol pathway. Acetoin is oxidized to diacetyl under alkaline conditions in the presence of air, and when reacted with creatine, it forms a pink color. E. coli is (-) and some other coliforms are (+). • Citrate utilization as sole carbon source. E. coli is (-) and many other coliforms are (+). Therefore, in this series of four tests, called the IMViC tests, E. coli is typically ++--. Other coliforms give different reaction patterns. However, the IMViC reactions have been found to be imperfect in speciating E. coli, and subsequently, other approaches to E. coli and coliform speciation have been developed. Materials: • TSA plate with colonies of K. pneumoniae • sterile sticks • MacConkey plates • Kliger Iron agar slant • Citrate slant• Urease slant • MR tube • Indole tube • VP tube • petri dish with paper in the bottom • oxidase test reagent Protocol First day: inoculation and staining 1. Perform a Gram stain 2. look at the stain under the microscope and observe the appearance of the bacteria 3. Streak for isolation on MacConkey agar and TCBS agar 4. Perform an oxidase test 5. Inoculate the KIA slant 6. Inoculate the urease slant 7. Inoculate the citrate slant 8. Inoculate the indole tube 9. Inoculate the MR tube 10. Inoculate the VP tube 11. Incubate all tubes and plates at 35°C Second day: Reading. 1. Examine the colonies on MacConkey and record results 2. Examine the KIA, citrate, urease, and ONPG tubes and record results 3. Perform and read the indole test 4. Perform and read the VP test Third day: Reading. 5. Perform and read the MR testIdentification of Vibrio chlorae Purpose Diagnosis of cholera is often made based on the symptoms and epidemiology of illness. However, isolation is important for identification of sporadic cases and identification of environmental sources of cholera. Principle Vibrio cholerae belongs to the family Vibrionaceae. Vibrios are distinguished by the fact that they are oxidase positive, salt tolerant, and ferment sucrose. They are marine organisms, capable of growth in the presence of 1% NaCl. The selective agar usually used for the isolation of Vibrio is thiosulfate-citrate-bile salts agar (TCBS). Vibrio forms bright yellow colonies on this agar when it ferments sucrose. Materials: • TSA plate with colonies of V. cholerae • sterile sticks • TCBS plates • oxidase reagents • 1% NaCl broth Protocol First day: inoculation 1. Select an isolated colony from the TSA plate using a sterile