Micro Lab Exam 2

Urea test

• Purpose: To determine whether bacteria produce urease (a protease).

• Content: Urea

• Detection method: pH indicator. When bacteria secrete urease, it hydrolyzes urea into ammonia (alkaline) and carbon dioxide changing the color of the media from yellow to pink

• Original test color/physical appearance: Yellow slant

• Positive result appearance: Media turned pink

• Positive result interpretation: Urease was produced, breaking down the urea to form ammonia. This raised the pH (alkaline), causing the phenol red indicator to turn pink

• Negative result appearance: Media stayed yellow

• Negative result interpretation: Urease was not produced. Therefore, urea was not hydrolyzed, no ammonia was produced, and pH did not increase

• Reagents: None. Media already contains pH indicator (phenol red)

Gelatin test

• Purpose: To determine whether bacteria produce gelatin hydrolase (a protease)

• Content: Protein gelatin

• Detection Method: When bacteria secrete gelatin hydrolase into the media, the gelatin liquefies and the deep will no longer appear solid

• Original test color/physical appearance: Clear/slightly amber solid medium (nutrient gelatin) in test tube

• Positive result appearance: Medium becomes liquid (even if only top layer is liquid)

• Positive result interpretation: Gelatin hydrolase was produced, hydrolyzing the gelatin and destroying its ability to solidify

• Negative result appearance: Medium remains solid

• Negative result interpretation: Gelatin hydrolase was not produced. Gelatin molecules remained intact, allowing medium to stay solid

• Reagents: None

SIM test

• Purpose: To determine if bacteria produce tryptophanase, are able to move and produce desulfhydrase

• Content: Kovac’s reagent turns reed in the presence of indole. Reduced concentration of agar for motility. Sulfur-containing amino acids (methionine and cysteine) that can be desulfurylated (by enzymes like cysteine desulfhydrase)

• Detection Method: Kovac’s reagent turns red in the presence of indole. Media turned black if hydrogen sulfide was produced and you will see a cloudy or fuzzy growth throughout the agar if the bacteria are motile

• Original test color/physical appearance: Semi-solid agar deep that is transparent and amber/straw-colored with a single stab line

• Positive result appearance:

  • Indole - Media turned red (red/pink ring at the top after adding reagent)

  • Hydrogen sulfide - Media turned black

  • Motility - Cloudy growth spreading away from the stab line

• Positive result interpretation: The organism can reduce sulfur to H₂S, (meaning it possesses cysteine desulfhydrase), possesses the enzyme tryptophanase to produce indole, and/or has flagella allowing it to swim through the agar

• Negative result appearance:

  • Indole - Reagent remains yellow/green at the top

  • Hydrogen sulfide - No black color (remains amber)

  • Motility - Growth is restricted only to the original stab line

• Negative result interpretation: The organism cannot reduce sulfur (does not possess cysteine desulfhydrase), does not produce the enzyme tryptophanase, and/or lacks the ability to move (non-motile)

• Reagents: Hydrogen sulfide and motility are read visually, but 4-5 drops of Kovac’s Reagent must be added to the top of the tube after incubation to detect the presence of indole

Phenylalanine test

• Purpose: To determine whether bacteria produce phenylalanine deaminase (enzyme). This allows it to remove the amino group from the amino acid phenylalanine to produce phenylpyruvic acid (PPA) and ammonia

• Content: Amino acid phenylalanine

• Detection Method: PPA (phenylpyruvic acid) can be detected by ferric chloride. The ferric ion reacts with PPA to produce a green color

• Original test color/physical appearance: Solid agar slant that is transparent and straw-colored/light yellow

• Positive result appearance: Slant color changes from yellow to green

• Positive result interpretation: Phenylalanine deaminase was produced, allowing it to deaminate phenylalanine into phenylpyruvic acid. This acid reacted with the added reagent to form a green-colored complex

• Negative result appearance: Slant color stays yellow

• Negative result interpretation: Phenylalanine deaminase was not produced, so no phenylpyruvic acid was formed to react with the reagent

• Reagents: Add 4-5 drops of 10% Ferric Chloride (FeCl₃) to the slant after incubation

Obligate aerobe

• Oxygen requirement: requires oxygen for growth (used in metabolism)

• Oxygen tolerance: High

• Growth pattern: Only at top (where oxygen is highest)

• Metabolism: Aerobic respiration

• Use oxygen in metabolism

  • + for catalase

  • + for cytochrome c oxidase

• Possesses enzymes needed to process toxic oxygen products

  • Makes catalase

  • Makes superoxide dismutase

Obligate anaerobe

• Oxygen requirement: Oxygen is toxic, cannot grow in its presence

• Oxygen tolerance: None

• Growth pattern: Only at bottom (away from oxygen)

• Metabolism: Anaerobic respiration or fermentation

• Lack the metabolic enzymes for using oxygen in respiration

  • - for catalase

  • - for oxidase

• Lack the enzymes for processing toxic oxygen

  • Does not make catalase

  • Does not make superoxide dismutase

Facultative anaerobe

• Oxygen requirement: Don’t require oxygen but use it when it is present, grows better with it (aerobic respiration), switches to fermentation/anaerobic respiration if not present

• Oxygen tolerance: High

• Growth pattern: All throughout tube but denser at top

• Metabolism: Aerobic respiration (O₂ present), anaerobic respiration and fermentation (O₂ not present)

• Possess the enzymes needed to use oxygen in respiration

  • + for catalase

  • Can be +, - for cytochrome c oxidase (usually negative)

• Possess the enzymes needed to process toxic oxygen products

  • Makes catalase

  • Makes superoxide dismutase

Aerotolerant anaerobe

• Oxygen requirement: Does not use oxygen for growth (fermentation) but can survive and grow to a limited extent in its presence (block reactive oxygen species)

• Oxygen tolerance: Moderate to high

• Growth pattern: Evenly throughout tube

• Metabolism: Fermentation

• Do not possess the enzymes needed to use oxygen in respiration

  • - for catalase

  • - for cytochrome c oxidase

• Possess some enzymes needed to process toxic oxygen products (have alternate mechanisms for breaking down peroxides and superoxide)

  • Does not make catalase

  • Makes superoxide dismutase

Microaerophile

• Oxygen requirement: Grows best with CO₂ and minimum oxygen. Harmed by normal atmospheric concentrations of oxygen. Require a small amount of it in metabolism

• Oxygen tolerance: Low (high levels are toxic)

• Growth pattern: Strip of growth not quite at top but close

• Metabolism: Aerobic respiration

• Possess the enzymes needed to use oxygen in respiration

  • + for catalase (often weak)

  • + for cytochrome c oxidase

• Possess the enzymes needed to process toxic oxygen products

  • Makes catalase (low levels)

  • Makes superoxide dismutase (low levels)