LAB: Biochemical Tests

Question 1

Biochemical Tests

Answer 1

• Used to evaluate metabolic properties of specific bacteria
• Used in conjunction with staining and culture characterization to definitively identify bacteria

Question 2

Catalase Test (TSA plate)

Staphylococcus (Positive)

Answer 2

• The catalase enzyme catalyzes the breakdown of H2O2 (hydrogen peroxide) into H2O and O2
• If the bacteria has the catalase enzyme, bubbling will occur
• H2O2 —–> (catalase enzyme) H2O + O2 (bubbles)
• Negative result: no bubbling
• Positive result: bubbling

Question 3

Denitrification Test (Nitrate Broth Medium)

Pseudomonas (Positive)

Alcaligenes (Negative)

Answer 3

• The substance potassium nitrate is converted into nitrogen gas
• Nitrate reductase enzyme
  
  • break down the nitrate (NO3) in nitrate broth, there will be nitrogen gas (N2) bubbles produced
• Durham tube - collects gas generated by the culture
• NO3 —-> (nitrate reductase enzyme) N2 gas (bubbles)
• Negative Result: no bubbles in Durham tube
• Positive Result: bubbles in Durham tube

Question 4

Hydrogen Sulfide (H2S) Production (SIM media tube)

Proteus (Positive)

E. coli (negative)

Answer 4

• Sulfide Indole Motility (SIM) media contains amino acid cysteine as well as iron
• Cysteine desulfurase enzyme
  
  • catalyzes the breakdown of cysteine to produce H2S
  • H2S will combine with iron to produce iron sulfate
    
    • blackens the medium
• Cysteine —> (cysteine desulfurase) H2S
• H2S + Fe —-> Blackening
• Negative Result: no color change
• Positive Result: blackening of the medium

Question 5

Indole Production (SIM media tube)

E. coli (Positive)

Enterobacter (Negative)

Answer 5

• SIM also contains amino acid tryptophan
• Tryptophanase enzyme
  
  • catalyzes the breakdown of tryptophan to indole
  • indole will react with Kovac’s reagent to form a red color
• Tryptophan —-> (tryptophanase) indole
• indole + Kovac’s —-> red
• Negative Result: no color change
• Positive Result: red color

Question 6

Methyl Red Test (MR-VP Broth)

E. coli (Positive)

Enterobacter ​(Negative)

Answer 6

• Methyl red pH indicator
  
  • yellow-orange at slightly acidic levels (5-6)
  • red at more acidic levels (below 4.4)
• If bacteria has multiple enzymes that break down sugars, turns red
• If bacteria does not have multiple enzymes to break down sugar, remain yellow
• glucose —-> pyruvic acid only
• glucose —-> pyruvic, acetic, formic
• Negative result: yellow (neutral fermentor)
• Positive result: persistent red color (acid fermentor)

Question 7

Oxidation-Fermentation Test (O-F media)

E. coli (facultative anaerobe)

Pseudomonas (obligate aerobe)

Answer 7

• Determines oxygen requirements for the breakdown of sugar
• O-F media
  
  • green at neutral pH
  • yellow when pH drops below 6
• Aerobic and Anaerobic metabolism
• Obligate Aerobe: tube w/oil remains green; tube w/out oil turns yellow (pseudomonas)
• Facultative Anaerobe: both tubes turn yellow (E. coli)