22.5: Fermentation and Utilization Media-Durham Sugar Tubes, MRVP, Oxidase, Catalase, Citrate

Introduction

Bacteria can be differentiated based on their ability to respire (aerobically, anaerobically, or facultative), or their ability to produce ATP via fermentation. Respiration produces ATP via oxidative phosphorylation, which occurs along an Electron Transport Chain (ETC). In aerobic respiration, \(\ce{O2}\) is the final electron acceptor. In anaerobic respiration, molecules other than \(\ce{O2}\) are the final electron acceptors. Fermentation also happens after glycolysis, however, ATP is produced by substrate level phosphorylation without an ETC or utilizing \(\ce{O2}\). Much less ATP is made, but it is sufficient for many bacteria. Acid is also produced during fermentation. The acid produced depends on the sugar that was consumed. Fermentation also may produce gas, \(\ce{CO2}\). Think of wine vs. champagne!

These tests help identify bacteria based on the sugars they utilize, the acids, the produce, their ability to utilize \(\ce{O2}\), etc.

Phenol Red Broths (aka Durham Tube Sugar Fermentations): Dextrose, Lactose, Sucrose

Purpose: To distinguish carbohydrate fermenters from non-fermenters, to detect and distinguish utilization of specific carbohydrates by the products formed.

Media used: 0.5% to 1% carbohydrate broth- Dextrose (glucose), Lactose, or Sucrose, Peptone, with Phenol Red and an inverted Durham tube for detection of gas.

Reagents and/or indicators: Phenol Red

Mechanism/reaction: Carbohydrate fermentation results in acid and sometimes gas production causing a pH change and possibly gas being trapped in the Durham tube.

Directions: Inoculate tubes and incubate at 35ºC for 24 – 48 hours.

Figure \(\PageIndex{1}\): Sugartubes: non-fermentive(L), acid production(center), acid and gas production(R). J.Gallai CC BY-NC SA

Interpretation: Observe for color change and gas production.

• (+)= color change from re to yellow, pH < 7.0
• (-)= no color change, pH = or > 7.0 (Note: Color may change to a darker red than an uninoculated tube. This darker color indicates alkaline metabolic products due to the utilization of the peptone instead of the sugar.)
• Gas production (+)= bubble trapped in the inverted Durham tube
• No gas production (-)= no bubble trapped in the inverted Durham tube

Record results as:

1. AG = acid with gas production
2. A = acid, no gas
3. (-) = negative for acid and gas

Methyl Red Test: Mixed Acid Fermentation

Purpose: To determine mixed acid fermentation (lactic, acetic, formic, etc). Part of the IMViC tests--(Indole, Methyl Red, Voges-Proskauer, Citrate)

Media: MRVP broth--buffered peptone glucose broth used for both MR and VP tests.

Reagents/indicators: Methyl Red- red in pH under 4.4, yellow in pH over 6.2, and orange in between

Mechanism/reactions: If the organism uses the mixed acid fermentation pathway and produces large amounts of organic acids from glucose, the acids will overcome buffers in the medium and the culture will be acidic.

Directions: Broth is inoculated and incubated for 48 hours – 5 days. After incubation, add 5 drops of Methyl Red indicator, do not shake the tube, read the results immediately.

Figure \(\PageIndex{2}\): Methyl Red test; negative(L), positive(R). J.Gallai CC BY-NC SA

Interpretation:

• (+) = bright red color immediately upon the addition of methyl red (pH < 4.4)
• (-) = yellow color (pH > 6.2)
• Weak (+) = orange color

Voges Proskauer Test: Butanediol Fermentation

Purpose: To detect the production of acetoin (acetylmethyl carbinol) or 2,3 butainediol(acetoin is the precursor) from glucose broth. Part of the IMViC tests.

Media: MRVP broth-- buffered glucose peptone broth used for both MR and VP test.

Reagent/indicators: Barritt’s reagents A- Napthol and B- Potassium Hydroxide (KOH)

Mechanism/reactions:

Glucose + diacetyl + \(\ce{KOH}\) + \(\ce{O2}\) + arginine → pink color (Acetoin is oxidized to diacetyl in the presence of \(\ce{KOH}\))

Directions: Inoculate broth and incubate 48 hours. After incubation add 20 drops of Barritt’sReagent A (napthol) and 20 drops Barritt’s Reagent B (\(\ce{KOH}\)). Shake well at frequent intervals and allow reaction to develop up to 1 – 2 hours if necessary.

Figure \(\PageIndex{3}\): Voges Proskauer Test https://www.asmscience.org/content/education/protocol/protocol.3204

Interpretation:

(+) = red layer at top in 10 minutes. (earliest detection), progressing downward.

() = no red color, disregard any copper or brownish-purple color.

Catalase Test

Purpose: Production of catalase.

Media: TSA

Reagents/indicators: 3% Hydrogen Peroxide (\(\ce{H2O2}\))

Mechanism/reactions: Catalase converts hydrogen peroxide, a by-product of oxidative respiration, to oxygen and water. Anaerobes and aerotolerant anaerobes lack this enzyme.

Directions: Apply several drops of 3% hydrogen peroxide to growth from a TSA plate.

Figure \(\PageIndex{4}\): Catalase test K.C.Burke CC BY-NC-SA

Interpretation: Vigorous bubbling due to the release of oxygen via catalase.

Oxidase Test

Purpose: The oxidase test identifies organisms that produce the enzyme cytochrome oxidase

Media: Use growth from a TSA plate or slant

Reagents/indicators: Oxidase dry slides

Mechanism/reactions: In organisms that use oxygen as the terminal electron acceptor in the electron transport chain, cytochrome oxidase transfers electrons to the oxygen. In the test, the reagent in the dry slide acts as the electron acceptor and changes from yellow to purple when it is oxidized.

Directions:

Using a sterile wooden stick (do not use an inoculating wire) pick a small amount of bacteria from a TSA plate or slant and touch an area on one section of the dry slide. Look for the color change to purple within about 30 sec. DRY SLIDES CAN BE USED FOR MANY TESTS (4 / SQUARE, 4 SQUARES PER SLIDE. USE UP EACH SLIDE BEFORE OPENING A NEW SLIDE PACKET).

Figure \(\PageIndex{5}\): Oxidase test K.C.Burke CC BY-NC-SA

Interpretation:

(+) = Color change to purple within 30 sec.

(-) = No color change, or a change after more than 30 sec.

Citrate Test

Purpose: To determine an organism’s ability to use citrate as the sole source of carbon. Part of the IMViC tests.

Media: Simmons Citrate Agar- contains sodium citrate as sole carbon source, mineral salts, and pH indicator Bromothymol blue

Reagents/indicators: Bromothymol Blue is a pH indicator. Yellow at less than pH 6.0, green between pH 6.0-7.6, Prussian blue at pH greater than 7.6.

Mechanism/reactions: Utilization of citrate leaves a sodium residue, increasing pH of the medium

Directions: Streak slant, cap loosely (this is an aerobic process), incubate 24 – 48 hours.

Figure \(\PageIndex{6}\): Citrate test; positive(L), negative(R). K.C.Burke CC BY-NC-SA

Interpretation:

• (+) = medium changes color from green to Prussian blue
• (-) = no change, medium remains green

Caution

Simmons Citrate can sometimes give a false positive result. If the media is blue, then check for growth on the slant to confirm a positive.

Materials

• Student stock organisms
• 1 tube Dextrose/GNR
• 1 tube Lactose/GNR
• 1 tube Sucrose/GNR
• 2 tubes MRVP broth/GNR (label one tube MR, the other tube VP)
• Hydrogen Peroxide, empty petri dishes, sterile wooden sticks
• Oxidase Dry Slides
• 1 tube Simmons Citrate/GNR

Procedures

1. Inoculate each GNR into each of the sugar tubes.
2. Inoculate each of the GNRs into an “MR” tube.
3. Inoculate each of the GNRs into a “VP” tube.
4. Perform the catalase test on all organisms.
5. Perform the oxidase text on the GNRs.
6. Inoculate each of the GNRs onto the slants of the Simmon’s Citrate tubes.

Results

Give the results for the inoculated bacteria.

Bacterium	Sugars (A/G) D L S	MR/VP	Catalase	Oxidase	Citrate

Conclusion

• Examine your results and state which test or set of tests, if any, would be best to help identify (it would be a good idea to do this for all of your stock cultures!) the following-
  • E. coli
  • Pseudomonas
  • staphylococci
  • Lactococcus lactis