Microbiology Lab Exam 3

IMViC

• What does IMViC stands for, and what does it test?

• Examples of Fermentation products and commercial use.

• IMViC identify enteric bacteria, and it comprises of four separate tests: indoles, methyl red, Voges-Proskauer, citrate

• Organisms: Escherichia, Acetobacter → acetic acid → vinegar

• Organisms: Lactobacillus, Streptococcus → lactic acid → cheese

• Organisms: Clostridium → isopropanol → rubbing alcohol 

• Organisms: Saccharomyces → ethanol → wine, beer

SIM

• What does SIM stand for?

• What does indole test?

• What does the motility test?

• How to perform the test?

• What does the medium contain?

• What are the reactions?

• What reagent is used?

• What were the lab results?

• SIM is an acronym for sulfide, indole, and motility

• Indole test is used for differentiating the Enterobacteriaceae

• Motility test is useful for testing a wide variety of organisms

• Use a needle, stab and pull along the same streak line. Add 3 drops of Kovac’s reagent after incubation

• SIM contains iron salt, sodium thiosulfate, peptone (amino acid - tryptophan)

• Sulfide positive: when thiosulfate is reduced, hydrogen sulfide (H₂S) produced and react with iron salt to produced ferric sulfide (black precipitate).

• Organisms that produce enzyme tryptophanase hydrolyze (breaks down) tryptophan and releasing indole (organic compound). 

• Kovac’s reagent is added after incubation, cherry red color indicates indole positive. pale yellow (no color change) indicates negative. 

• S. typhirium: Sulfide+, Indole-, Motility+

• E. coli: Sulfide-, Indole+, Motility+

• K. pneumoniae: Sulfide-, Indole-, Motility-

MRVP

• What does MRVP stand for?

• What is it used for?

• What does the medium contain?

• How to perform the test?

• What are the two pathways? What are the reactions

• How to interpret?

• What were the lab results?

• Methyl-Red and Voges-Proskauer

• Identification of gram-negatives

• MRVP contains peptone, glucose and buffers. All enteric bacteria metabolize glucose to pyruvate

• One incubated tube is split into two tubes to test MR and VP separately

  • VP procedures: 3 drops of VP A (agitate by tapping on the tube), then 3 drops of VP B (agitate gently). Wait for 30 minutes

  • MR procedures: 4 drops of methyl red

• Mixed acid fermentation or butylene glycol pathway. Mutually exclusive

  • MR tests for mixed acid fermenter, the stable end acid that lowers pH below 4.4 and color turns red. Remains yellow for negative. Orange means inconclusive

  • VP tests for butylene glycol pathway, neutral end products acetone and butanediol form red-complex with the reagent, color turns to reddish-brown color. Remains yellow for negative

  • E.coli is MR+

  • E. areogenes is VP+

Citrate

• What is it used for?

• What does the medium contain?

• How to perform the test?

• What is the reaction?

• What were the lab results?

• Test organisms ability to utilize citrate as carbon source, ammonium dihydrogen phosphate as nitrogen source. Differentiates Enterobacteriaceae.

• It contains citrate (carbon), phosphate (nitrogen), and pH indicator bromthymol blue

• Use loop and streak the surface of slant agar

• Bacteria growing on citrate use phosphate and convert it to ammonia. The alkaline condition increases to 7.5 turns blue indicates citrate positive. The color remains green at 6.9 means negative.   

• E. coli: Citrate-

• K. pneumoniae: Citrate+

Triple-Sugar Iron Agar

• What is it used for?

• What does the medium contain?

• How to perform the test?

• What are the reactions?

• What were the lab results?

• Tests for fermentation of certain carbohydrates and the ability to reduce compounds containing sulfur. As well as protein deamination. Purpose is to identify gram-negative enteric bacteria.

• The medium contains following:

  • Three types of sugars: glucose (low amounts), lactose, sucrose.

  • Two sources of sulfur and iron salt to detect hydrogen sulfide gas (black precipitate).

  • Nutrient base peptone to detect protein catabolism

  • pH indicator phenol red

• Use the needle and stab it into the butt, then streak the surface of the slant

• These are the possible reactions:

  • If the bacteria fermented glucose and other sugars. Both the butt and slant will be yellow.

  • Yellow butt and bright red slant indicates only glucose fermentation

  • Black precipitate in the butt indicate H₂S production

  • Bright red indicates alkaline reaction

  • Cracks, bubbles, lifted agar indicate gas was produced

• S. typhirium: K/A, H₂S, G

• E. coli: A/A, G

• Proteus. vulgaris: A/A, H₂S, G

• P. aeruginosa: K/K