Bio235_Exe.15 Fermentation

Bio235_Exe.15 Fermentation_Fall19.pptx

Mar 6, 2023Download as PPTX, PDF0 likes9 views

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

Exercise 15:
Carbohydrate Catabolism (part2)
Fermentation

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.

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).

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)

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.

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.

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)

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

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

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

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

Citrate test
• Record your results on Page 106.

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Bio235_Exe.15 Fermentation_Fall19.pptx

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.

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Bio235_Exe.15 Fermentation_Fall19.pptx

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