Microbiology Lab Exam #2
???? Exercise 5-1: Oxidation-Fermentation Test
• The Oxidation-Fermentation (O-F) Test is designed to differentiate bacteria on the basis of fermentation or oxidative metabolism of carbohydrates.
• In this medium, oxidative organisms oxidize the carbohydrate to CO2, H2O, and energy by way of glycolysis, the oxidation of pyruvate, the Krebs cycle, and finally the electron transport chain with oxygen (the final electron acceptor) being reduced to H2O.
• Similarly, fermentative organisms convert the carbohydrate to pyruvate, but because oxygen is not available as an electron acceptor, the pyruvate and other organic intermediates are reduced to organic acids, gas, or alcohol.
• Consequently, fermenters acidify O-F medium to a much greater extent than do oxidizers. • One tube is sealed with a layer of sterile mineral oil to promote anaerobic growth and fermentation. The mineral oil creates an environment unsuitable for oxidation because it prevents the diffusion of oxygen from the air into the medium.
• The other tube is left unsealed to allow aerobic growth and oxidation.
• With Oil ✂ NO O2 ✂ Fermenters
• NO Oil ✂ O2 ✂ Oxidizers or Aerotolerant Fermenters
Don't forget about the age old question of How to distinguish sex from gender?
• Bromthymol Blue
- Green ✂ no pH change
- Yellow ✂ Acid produced If you want to learn more check out What is root cause analysis?
- Blue ✂ Basic product produced (increases pH).
Table of Results
Sealed Unsealed Interpretation Symbol
Green or Blue Any amount of
Yellow Oxidation O
throughout Yellow throughout Oxidation and Fermentation OR Fermentation
Green or Blue Green or Blue No Sugar Metabolism; organism is nonsacharolytic. N
We also discuss several other topics like What are esteem needs?
???? Exercise 5-2: Phenol Red Broth
• Phenol Red Broth is a differential test medium prepared as a base to which a carbohydrate is added. We also discuss several other topics like What is the slope of the total product curve?
• Phenol red is yellow when acidic, pink to magenta when basic, and red when neutral. • An inverted Durham tube is added to each tube to trap a portion of any gas produced. • Acid production from fermentation of the carbohydrate lowers the pH below the neutral range of the indicator and turns the medium yellow.
• Deamination of peptone amino acids produces ammonia (NH3), which raises the pH (basic) and turns the broth pink.
• Gas production is indicated by a bubble or pocket in the Durham tube where the broth as been displaced.
Phenol Red Table of Results
Result Interpretation Symbol Organism
Yellow broth, bubble in
Fermentation of glucose with acid and gas end
Yellow broth, no bubble Ferment sugar with acid (no gas) A/-
Red broth, no bubble No Fermentation -/- P. aeroginosa
Pink broth, no bubble Degradation of peptone; alkaline end products. K
We also discuss several other topics like What is accelerated depreciation?
???? Exercise 5-11: Starch Hydrolysis, Exercise 5-13 Casein Hydrolysis, Exercise 5-16 Lipid Hydrolysis
• Starch Agar Plate
- Exoenzyme ✂ Glucosidase ✂ Starch ✂ Glucose ✂ Starch
- E. coli + B. cereus
- Add drops of iodine to reveal clear areas surrounding the growth to detect the presence or absence of starch in the vicinity.
- Zone of clearing = a positive result ++++
- B. cereus is positive (amylase + glucosidase present)
- E. coli is negative.
• Casein Agar Plate (Milk Agar) Don't forget about the age old question of What is gender performativity?
- Exoenzyme ✂ Casease ✂ Casein (protein in milk) ✂ Amino Acids ✂ Milk Agar - E. coli + P. aeroginosa
- Casease is an enzyme that some bacteria produce to hydrolyze the milk protein casein, the molecule that gives milk its white color.
- The presence of casease can be detected easily with the test medium Milk Agar. - Clearing in the agar ✂ casease is present – P. aeroginosa.
- No clearing ✂ casease is absent – E. coli.
• Spirit Blue Agar (Lipase Test)
- Exoenzyme ✂ Lipase ✂ Lipids ✂ Molecules for Krebs Cycle ✂ Spirit Blue Plate - E. coli + S. epidermidis
- Bacterial enzymes that hydrolyze lipids are called lipases.
- Because lipases are secreted by the organism to digest lipids outside the cell, they are called extracellular enzymes or exoenzymes.
- Clearing in the agar ✂ lipase is present ✂ S. epidermidis.
- No clearing ✂ lipase is absent ✂ E. coli.
???? Exercise 5-3: Methyl Red and Voges-Proskauer Tests
• Methyl Red and Voges-Proskauer (MV-VP) Broth is a combination medium used for both Methyl Red (MR) and Voges-Proskauer (VP) tests.
• The Methyl Red test is designed to detect organisms capable of performing a mixed acid fermentation, which overcomes the phosphate buffer in the medium and lowers the pH. The acids produced by these organisms tend to be stable.
• Mixed acid fermentation is verified by the addition of methyl red indicator dye following incubation. Methyl red is red at pH 4.4 and yellow at pH 6.2. Between these two pH values, it is various shades of orange.
• Red color is the only true indication of a positive result. Orange is negative or inconclusive. Yellow is negative.
• The Voges-Proskauer test was designed for organisms that are able to ferment glucose, but quickly convert their acid products to acetoin and 2,3-butanedoil.
• A positive VP result is red. No color change (or development of copper color) after the addition of the reagents is negative.
• The Methyl Red and Voges-Proskauer tests are components of the IMViC battery of tests (Indole, Methyl Red, Voges-Proskauer, and Citrate).
• IMViC ✂ Methyl Red ✂ Mixed Acid Fermentation ✂ Stable Acids (Lower pH) ✂ Methyl Red pH indicator.
• IMViC ✂ Voges-Proskauer ✂ Butanediol Fermentation ✂ Unstable Acid ✂ Acetoin + Butanediol ✂ A Reagent + B Reagent + Acetoin ✂ Red Color
• Add 3 drops of methyl red dye indicator for MR test.
• Add 15 drops Reagent A, mix, 5 drops Reagent B, mix ✂ for VP test.
Methyl Red Test Results
Result Interpretation Symbol
Red Mixed Acid Fermentation +
No Color Change No Mixed Acid Fermentation -
Voges-Proskauer Test Results
Result Interpretation Symbol
Red 2,3-butanediol fermentation (acetoin produced) +
No Color Change NO 2,3-butanediol fermentation (acetoin is NOT produced) -
???? Exercise 5-6: Nitrate Reduction
• Anaerobic respiration involves the reduction of (transfer of electrons to) an inorganic molecule other than oxygen. Nitrate reduction is one such example.
• Many Gram-negative bacteria contain the enzyme nitrate reductase and perform a single-step reduction of nitrate to nitrite (NO3✂NO2).
• Other bacteria in a multistep process known as denitrification, are capable of enzymatically converting nitrate to molecular nitrogen (N2). Some products of nitrate reduction are shown in the figure.
• Nitrate Broth contains an inverted Durham tube to trap a portion of any gas produced.
• The color reactions obtained in Nitrate Broth take place as a result of reactions between metabolic products and reagents added after incubation.
• Before a broth can be tested for nitrate reductase activity (nitrate reduction to nitrite), it must be examined for evidence of denitrification. If the Durham tube contains gas, the test is complete. Denitrification has taken place.
• If there is no visual evidence of denitrification, nitrate reagent A and B are added to the medium to test for nitrate reduction to nitrite. If present, nitrite will form nitrous acid (HNO2) in the medium. Nitrous acid reacts with the added reagents to produce a red, water-soluble compound. ** Therefore, formation of red color after the addition of reagents indicates that the organism reduced nitrate to nitrite.
• If no color change takes place with the addition of reagents, the nitrate either was not reduced or was reduced to one of the other nitrogenous compounds shown in the figure. Because it is visually impossible to tell the difference between these two occurrences, another test must be performed.
• A small amount of powdered zinc is added to the broth to catalyze the reduction of any nitrate to nitrite.
• If nitrate is present at the time zinc is added, it will be converted immediately to nitrite, and the medium will turn red. In this instance, the red color indicates that nitrate was not reduced by the organism.
• No color change after the addition of zinc indicates that the organism reduced the nitrate to NH3, NO, N2O, or some other nongaseous nitrogenous compound.
Result Interpretation Symbol Organisms
Gas Bubble Denitrification – production of nitrogen gas (NO3
✂ NO2 ✂ N2)
Red Color After the
Nitrate Reduction to Nitrite (NO3 ✂ NO2) + E. coli
Addition of the Reagents
A and B
No Color After the
Incomplete test; requires the addition of zinc
Addition of the Reagents
A and B
No Color Change After
Nitrate reduction to nongaseous nitrogenous
compounds (NO3 ✂ NO2 ✂ nongaseous
Red Color After Zinc No nitrate reduction - A. faecalis
???? Exercise 5-7: Citrate Test
• The citrate utilization test is used to determine the ability of an organism to use citrate as its sole source of carbon.
• IMViC test
• Simmons Citrate Medium provides the means for a few bacterial species that possess the enzyme citrate-permease to transport citrate into the cell and metabolize it by way of the fermentative pathway.
• Bacteria that do not possess citrate-permease will not survive on the medium. • Bromthymol blue dye, which is green at pH 6.9 and blue at pH 7.6, is added as an indicator. • Citrate-positive bacteria produce ammonia (NH3) and ammonium hydroxide (NH4OH), both of
which alkalinize the medium and turn it blue. Thus, blue color is a positive citrate test result. • Growth on the slant, even in the absence of color change, indicates that citrate is being utilized and is considered a positive test result.
• Citrate Permease – allows an organism to bring citrate into the cell.
• Blue – Positive result for citrate utilization – Increase in pH (basic) – able to ferment citrate
Result Interpretation Symbol
Blue (even a small amount) Citrate is utilized +
No color change; growth Citrate is utilized +
No color change (stays green); no growth Citrate is NOT utilized -
???? Exercise 5-12: Urea Hydrolysis
• Urea is a product of decarboxylation of certain amino acids.
• It can be hydrolyzed to ammonia and carbon dioxide by bacteria containing the enzyme urease.
• The Urea Hydrolysis Test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease.
• Phenol red, which is yellow or orange below pH 8.4 and red or pink above, is included to expose any increase in pH.
• Pink color in the medium in less than 24 hours indicates a rapid urease-positive organism. • Orange or yellow is negative.
Pink Rapid urea hydrolysis; strong urease production
Orange or Yellow No urea hydrolysis; organism does not produce urease or cannot live in broth.
???? Exercise 5-18: SIM Medium (Indole Test)
• IMViC test
• Indole production from tryptophan
• Indole production in the medium is made possible by the presence of tryptophan (contained in casein and animal protein).
• Bacteria possessing the enzyme tryptophanase can hydrolyze tryptophan to pyruvate, ammonia, and indole.
• The hydrolysis of tryptophan in SIM medium can be detected by the addition of Kovacs’ reagent after a period of incubation.
• The formation of the red color in the reagent layer indicates a positive reaction and the presence of tryptophanase.
• No red color is indole-negative.
Red in the alcohol layer of Kovacs’s
Tryptophan is broken down into indole and pyruvate.
Reagent color is unchanged Tryptophan is NOT broken down into indol and pyruvate.
???? Exercise 5-4: Catalase Test
• Superoxide dismutase catalyzes conversion of superoxide radicals to hydrogen peroxide. • Catalase converts hydrogen peroxide into water and gaseous oxygen.
• Bacteria that produce catalase can be detected easily using typical store-grade hydrogen peroxide.
• When hydrogen peroxide is added to a catalase-positive culture, oxygen gas bubbles form immediately.
• If no gas bubbles appear, the organism is catalase-negative.
• This test is used to identify organisms that produce the enzyme catalase.
Bubbles Catalase is present
No Bubbles Catalase is absent
???? Exercise 5-5: Oxidase Test
• The oxidase test is designed to identify the presence of cytochrome c oxidase.
Dark Blue within 20 seconds Cytochrome c oxidase is present
No color change Cytochrome c oxidase is absent
???? Exercise 9-5: Enterotube II
• The Enterotube II is a multiple test system designed to identify enteric bacteria based on: glucose, adonitol, lactose, arabinose, sorbitol, and dulcitol fermentation, lysine and ornithine decarxylation, sulfur reduction, indole production, acetoin production from glucose fermentation, phenylalanine deamination, urea hydrolysis, and citrate utilization.