Lab 10: Media Demonstrating Carbohydrate Metabolism

Question 1

Name the 4 types of tests capable of demonstrating carbohydrate metabolism in bacteria

Answer 1

1) Citrate
2) Methyl red
3) Voges-Proskauer (VP)
4) ONPG

Question 2

1) What is citrate media capable of identifying?
2) What is the pH indicator in citrate media?

Answer 2

1) Identifying G- rod bacteria
2) Bromothymol blue

Question 3

1) What does growth on citrate media do to the CO2 level?
2) What does this change in CO2 level do to pH?
3) What does this change in pH do?

Answer 3

1) Increases CO2
2) Increases pH
3) Causes a vivid blue color (indiciates (+))

Question 4

1) What indicates a negative result to a citrate test?
2) What indicates a positive result to a citrate test?

Answer 4

1) Dark green = Negative (-) (E.Coli)
2) Vivid blue = Positive (+) (E.aerogenes)

Question 5

What is the sole carbon source?

Answer 5

Citrate

Question 6

1) What can methyl red identify? What does it look for?
2) What is methyl red media rich in, and what is its indicator?
3) How many drops of methyl red mix must be added?

Answer 6

1) It can identify G- rod bacteria; looks for mixed acid fermentation
2) Rich in protein and glucose, and doesn’t have an indicator.
3) 8 drops of methyl red mix

Question 7

1) What indicates a negative result of a methyl red test?
2) What indicates a positive result of a methyl red test?

Answer 7

1) Not red = Negative (-) (E. aerogenes)
2) Turns red = Positive (+) (E. Coli)

Question 8

1) What type of bacteria is a Voges-Proskauer (VP) test capable of identifying?
2) What media does this test use? How much?
3) What substance does this media detect?

Answer 8

1) It can identify G- rod bacteria
2) It uses the same media as a methyl red test; only needs 1-2ml
3) Detects an intermediate of alcoholic fermentation called acetylmethylcarbinol

Question 9

What are the two steps to a Voges-Proskauer (VP) test?

Answer 9

1) Add 10 drops of VP-A (alpha-naphthol); shake gently and wait 1-2 minutes
2) Add 5 drops of VP-B (40% KOH); incubate at 37 degrees for 1 hour. Look for color change.

Question 10

1) What indicates a negative result of a Voges-Proskauer (VP) test?
2) What indicates a positive result of a VP test?

Answer 10

1) No change = Negative (-) (E. Coli)
2) Dark red/ purple = Positive (+) (E. aerogenes)

Question 11

1) What question does an ONPG test answer about a Lac (-) culture?
2) What have some bacteria lost?

Answer 11

1) “Does a Lac (-) culture have B-galactosidase?”
2) Some bacteria have lost their lactose permease

Question 12

1) In an ONPG test, what is a part of the process in which LAC breaks down into Glu and Gal?
2) What type of lactose analog is used in an ONPG test?

Answer 12

1) B-galactosidase
2) One that can be taken up nonspecifically (so no lactose permease is needed)

Question 13

1) What results from the breakdown of ONPG?
2) What color is it and what does this indicate?

Answer 13

1) Nitrophenol and GAL
2) Nitrophenol causes a yellow color, indicating a positive (+) result

Question 14

1) What indicates a negative result of an ONPG test?
2) What indicates a positive result of an ONPG test?

Answer 14

1) Colorless = Negative (-) (E. Coli)
2) Bright yellow = Positive (+) (E. aerogenes)

Question 15

What are the three steps to performing an ONPG test?

Answer 15

1) Transfer 0.5ml culture into a sterile tube
2) Aseptically add 0.5 ml ONPG solution (or disc)
3) Incubate at 37 degrees and observe every 20 minutes for up to an hour

Question 16

Tests: SIM (indole), methyl red, VP, Citrate
1) In which test(s) does E. coli yield a positive result?
2) In which test(s) does E. aerogenes yield a positive result?

Answer 16

1) E. coli (+): SIM and methyl red
2) E. aerogenes (+): VP and citrate

Question 17

1) What does a phenol red sugars test test for?
2) What sugars are in the media?
3) What are the two indicator mechanisms?
4) The fermentation of any sugars in the media can result in the production of what two things?
5) What kind of fermentation produces gas?

Answer 17

1) If a bacteria will ferment any of the sugars in the media.
2) Glucose, lactose, mannitol, sucrose, and xylitol (Glu, Lac, Man, Suc, Xyl)
3) Phenol red pH indicator (pH<6.8: yellow; >7 red to purple) and Durham tubes to trap gas
4) Acid and/or gas
5) Alcohol fermentation

Question 18

How is a phenol red sugars test scored?

Answer 18

pH over gas production:
1) pH: A (acidic, yellow) or K (alkaline, red)
2) Gas presence: G+ (gas present) or G- (no gas)

Question 19

1) What type of glucose fermentation does E. Coli (EC) do?
2) What type of glucose fermentation does E. aerogenes (EA) do?

Answer 19

1) EC: mixed acid fermentation
2) EA: alcoholic fermentation

Question 20

Why is differentiation between E. coli and E. aerogenes important?

Answer 20

EC cannot live long in water, but EA can. MPN tests often test for lactose fermentation, which both EC and EA do, so they need to be differentiated between.

Question 21

1) What tests are able to aid in differentiating between EC and EA?
2) What test is the indole test a part of?

Answer 21

1) IMViC tests: Indole (I), Methyl Red (M), Voges-Proskauer (Vi), Citrate (C)
2) SIM test