ENVR 421 Laboratory #9: identification of pathogenic bacteria in foods

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1 ENVR 421 Laboratory #9: identification of pathogenic bacteria in foods Introduction The purpose of this laboratory exercise is to familiarize you with biochemical identification assays for pathogenic bacteria that occur in foods. We will be using the same methods applied for identification of waterborne bacteria to identify organisms found in chicken, oysters, and ground beef. In addition to trying to identify your own isolates from these foods, we will test positive controls. The controls available will be cholerae, E. coli, Aeromonas hydrophila, and Pseudomonas aeruginosa. In addition, isolates will be tested using the Enterotube, a commercial biochemical test. The Enterotube allows you to include some additional tests. Identification of bacteria from chicken Purpose Salmonella from chicken. You will test a positive control culture along with any isolates you get from the chicken analyzed. Principle Members of the genus Salmonella are distinguished from E. coli and Klebsiella by their inability to ferment lactose. You will use the standard tests for members of the Enterobacteriaceae, including oxidase, KIA, indole, citrate, MRVP, and urease. Materials: TSA plates with isolates sterile sticks Kliger Iron agar slant Citrate slant Urease slant MRVP tube Indole tube petri dish with paper in the bottom oxidase test reagent Enterotube

2 Protocol First day: inoculation 1. Perform an oxidase test 2. Inoculate the KIA slant 3. Inoculate the urease slant 4. Inoculate the citrate slant 5. Inoculate the indole tube 6. Inoculate the MRVP tube 7. Inoculate the enterotube 8. Incubate all tubes and plates at 35 C Second day: Reading. 1. Examine the KIA, citrate, urease, and ONPG tubes and record results 2. Perform and read the indole test 3. Perform and read the VP test 4. Read the enterotube Third day: Reading. 5. Perform and read the MR test

3 Test or substrate 1. Glucose (TSI) 2. Lysine decarboxylase (LIA) 3. H 2 S (TSI and LIA) Table 1. Biochemical and serological reactions of Salmonella Positive Result Negative yellow butt red butt + purple butt yellow butt + blackening no blackening + 4. Urease purple-red color no color change - 5. Lysine decarboxylase broth 6. Phenol red dulcitol broth purple color yellow color + yellow color and/or gas no gas; no color change 7. KCN broth growth no growth - 8. Malonate broth blue color no color change - (c) 9. Indole test violet color at surface yellow color at surface Polyvalent flagellar test agglutination no agglutination Polyvalent somatic test 12. Phenol red lactose broth 13. Phenol red sucrose broth 14. Voges-Proskauer test agglutination no agglutination + yellow color and/or gas no gas; no color change yellow color and/or gas no gas; no color change - pink-to-red color no color change Methyl red test diffuse red color diffuse yellow color Simmons citrate growth; blue color no growth; no color change v a +, 90% or more positive in 1 or 2 days; -, 90% or more negative in 1 or 2 days; v, variable. Salmonella species reaction (a) + (b) - (c) b Majority of S. arizonae cultures are negative. c Majority of S. arizonae cultures are positive.

4 Identification of bacteria from ground beef Purpose Escherichia coli is the most important bacterial pathogen in ground beef, as well as an indicator of fecal contamination. 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 (+). Materials: TSA plate with colonies sterile sticks Kliger Iron agar slant Citrate slant Urease slant MRVP tube Indole tube petri dish with paper in the bottom oxidase test reagent Enterotube

5 Protocol First day: inoculation 1. Perform an oxidase test 2. Inoculate the KIA slant 3. Inoculate the urease slant 4. Inoculate the citrate slant 5. Inoculate the indole tube 6. Inoculate the MRVP tube 7. Inoculate the enterotube 8. Incubate all tubes and plates at 35 C Second day: Reading. 1. Examine the KIA, citrate, and urease and record results 2. Perform and read the indole test 3. Perform and read the VP test 4. Read the enterotube Third day: Reading. 5. Perform and read the MR test

6 Identification of organisms from oysters Purpose The most important bacterial pathogens of oysters are members of the genus Vibrio. This may include cholera, but is more likely to be other vibrios, including parahaemolyticus and vulnificus. The method we used may also capture other salt tolerant marine organisms, including members of the genus Aeromonas. To observe the differences, you will also test a culture of Aeromonas. Principle Vibrio 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 NaCl. The use of TCBS and TSA with NaCl for streaking the contents of your alkaline peptone broth tubes allows the isolation of salt tolerant marine organisms from oyster meat. Vibrios and Aeromonas are oxidase positive. In the KIA test, parahaemolyticus and vulnificus will not ferment sucrose. They can be differentiated from each other and the Aeromonas by the VP test and their amino acid utilization patterns. Materials: TSA plate with colonies sterile sticks 5% NaCl broth Kliger Iron agar slant Citrate slant Urease slant MRVP tube Indole tube petri dish with paper in the bottom oxidase test reagent enterotube

7 Protocol First day: inoculation 1. Perform an oxidase test 2. Inoculate the NaCl broth 9. Perform an oxidase test 10. Inoculate the KIA slant 11. Inoculate the urease slant 12. Inoculate the citrate slant 13. Inoculate the indole tube 14. Inoculate the MRVP tube 15. Inoculate the enterotube 16. Incubate all tubes and plates at 35 C Second day: Reading. 6. Examine the KIA, citrate, urease, and ONPG tubes and record results 7. Examine the NaCl broth for growth (turbidity) 8. Perform and read the indole test 9. Perform and read the VP test 10. Read the enterotube Third day: Reading. 11. Perform and read the MR test

8 TABLE 1. Biochemical characteristics of human pathogenic Vibrionaceae commonly encountered in seafood* alginolyti cus choler ae fluvia lis furnis sii hollis ae metsch nikovii mimic us parah aemolyti cus vulnifi cus A. hydro - philia ** TCBS agar Y Y Y Y NG Y G G G Y G mcpc agar NG P NG NG NG NG NG NG Y NG NG CC agar NG P NG NG NG NG NG NG Y NG NG AGS KA Ka KK KK Ka KK KA KA KA KK nd Oxidase Arginine dihydrolase Ornithine decarboxylase Lysine decarboxylase V + Grow th in (w/v) : 0% NaCl 3% NaCl 6% NaCl 8% NaCl 10% NaCl Growth at 42 C Acid from: V + V V nd V V + Sucrose V D- Cellobi ose + V + + Lactose + V Arabin ose V P. shigel - loides **

9 Sensi - tivity to: D- Manno se D- Mannit ol V V + ONPG Voges- Proska uer 10 µg O/ µg O/129 Gelatin ase + V + + R S R R nd S S R S R S S S S S nd S S S S R S Urease V * Adapted from Elliot et al. (31) ** Aeromonas hydrophila, Plesiomonas shigelloides Abbreviations: TCBS, thiosulfate-citrate-bile salts-sucrose; mcpc, modified cellobiosepolymyxin B-colistin; AGS, arginine-glucose slant; Y = yellow NG = no or poor growth S = susceptible nd = not done G = green V = variable among strains R = resistant P = purple, V = variable KK = Slant alkaline / Butt alkaline KA = Slant alkaline /Butt acidic, Ka = Slant alkaline/ Butt slightly acidic

10

11 Protocols for biochemical tests The oxidase test 1. Select a colony with a stick and smear it on the paper in the petri dish 2. Drip a few drops of oxidase reagent on the smear. A positive result is a purple color within 10s (it cannot be interpreted accurately after 10s) KIA slant 1. Pass an inoculation needle through a Bunsen burner and let cool 30s. 2. Using the needle, select a colony from the TSA plate 3. Inoculate the KIA slant by stabbing the needle straight into the agar slant. 4. Draw the needle out and streak it across the surface of the slant in a zigzag pattern Urease and citrate slants 1. Select a colony using a sterile stick 2. Inoculate the urease slant by streaking across the surface 3. Inoculate the citrate slant in the same manner using a new stick Liquid media in tubes 1. Select a colony with a sterile stick and dip into the indole tube 2. Repeat with a new stick for the MR tube 3. Repeat with a new stick for the VP tube 4. Repeat with a new stick for the ONPG tub Reading the Indole test 1. Add 5 drops of Kovac s reagent to the tube and watch for color change (immediate) Reading the VP test 1. pipette 1 ml of liquid out of the MRVP tube and place in a clean tube 2. Put the original back in the incubator for another 24 hours 3. Add 6 drops of α-napthol to the new tube 4. Add 2 drops of potassium hydroxide to the new tube 5. Shake tube gently for 30s 6. wait 10 min and read result

12 Reading the MR test (after 48 HOURS incubation) 1. Add 5 drops of methyl red reagent to the tube and watch for color change (immediate)

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