This document describes several biochemical tests used to identify bacteria based on their metabolism of carbohydrates and other substrates. [1] Fermentation tests detect acid and gas production from carbohydrates like glucose, lactose, and sucrose. The MR-VP tests identify bacteria that produce acids or alcohols from glucose fermentation. The Methyl Red test checks for acid production and the Voges-Proskauer test detects alcohol production. [2] The Citrate test examines bacteria's ability to use citrate as an energy source, indicated by a color change from green to blue. These tests are performed on bacterial isolates using fermentation tubes, MR-VP medium, and Simmons citrate agar

1. Exercise 15:
Carbohydrate Catabolism (part2)
Fermentation

2. Fermentation tubes
• To detect acid and gas production
• Contain:
- peptone – source of nitrogen
- an acid/base indicator (phenol red),
o It is red in neutral condition,
o Will turn yellow in acidic condition
- inverted tube to trap gas (durham tube), -
- 0.5-1.0 % carbohydrate.

3. The MR-VP Tests
• MR-VP stands for the names of 2 biochemical tests:
MR – Methyl red VP – Voges-Proskauer
• Both of these tests are performed on MR-VP Medium (contains
glucose).

4. The Methyl Red Test
• This test determines if a bacterium is capable of mixed acid fermentation.
• Mixed acid fermentation results when glucose is fermented to produce acids, which
lowers the pH of the media.
• A MR test is done by adding a pH indicator (Methyl Red) to the test tube.
• Upon addition of the methyl red you may have 2 results:
– Large amounts of acid = medium remains red (pH below 4.4)
– Neutral products produced = medium turns
yellow (pH above 6.2)

5. The Voges-Proskauer Test
• This test identifies organisms that ferment sugars to produce alcohol end
products acetoin and butanediol.
• The VP test is done by adding Barritt’s reagents to determine if these
products were produced by the bacteria.
– Red color indicates a positive test result.

6. Methyl Red + Vogeus-Proskaeur +
Alcohol

8. The Citrate Test
• Citrate can be utilized by some bacteria as a source of energy but ONLY if
they can transport it into their cell.
• Nitrogen (from a source such as ammonium - NH4
+) is also a requirement
for growing bacteria.
• Citrate agar contains citrate (carbon source), ammonium phosphate
(nitrogen source), and bromthymol blue (pH indicator).
• Utilization of citrate and nitrogen release alkaline by-products which
raise the pH and result in a color change of the media from green to blue.

9. The Citrate Test

11. Materials
( per group of 2 -3 students)
• Glucose Fermentation tube ( 1 )
• Lactose Fermentation tube ( 1 )
• Sucrose Fermentation tube ( 1 )
• MRVP tubes (4)
• Simmons citrate agar slants (2)

12. Bacterial cultures (as assigned)
• Fermentation tubes:
– Group 1,5,9 Escherichia coli
– Group 2,6,10 Enterobacter aerogenes
– Group 3,7,11 Alcaligenes faecalis
– Group 4,8,12 Proteus vulgaris
• MRVP tests: (all groups)
– 2 MRVP tubes Escherichia coli
– 2 MRVP tubes Enterobacter aerogenes
• Citrate tests: ( all groups)
– 1 Simmons citrate tubes Escherichia coli
– 1 Simmons citrate tubes Enterobacter aerogenes

13. observation
• Fermentation Tubes:
– Yellow means positive
– Red means negative
– Bubbles means gas
production
- Record your results on page 105

14. MR (Methyl Red) test
– To one tube of each MRVP set (one E. coli and
One Enterobacter), add 5 drops of methyl red.
• Record your results on page 106

15. V-P Test
– To the other set of MRVP tubes,
add 0.6 ml (12 drops) of VP reagent I
and 0.2 ml (2-3 drops) of VP reagent II.
– Shake well and then leave the cap open
for 15 – 30 minutes.
– VP positive test will develop a pinkish red color.
• Record your results on Page 106

16. Citrate test
• Record your results on Page 106.