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

API 20E Strip

• What does API 20E stand for?

• What is it used for?

• How does it work?

• How to set up tests for aerobic and anaerobic bacteria?

• It stands for Analytical Profile Index and has 20 tests

• Identify gram-negative bacteria, especially Enterobacteriaceae - E. coli, Salmonella and Shigella

• Results of the reaction correspond to numerical value and sum the numbers to arrive at 7-digit code.

• Aerobic reaction requires completely fill cupule and tube

• Anaerobic reaction under-fill the tube to leave room for oil

Kirby Bauer Method

• What does antimicrobial mean?

• What is zone of inhibition

• what does minimum inhibitory concentration (MIC) mean?

• In culture & sensitivity, what does the last part mean?

• What is Kirby-Bauer method?

• What are the procedures?

• How does antibiotic resistance occur?

• How does antibiotic function?

• Purpose of broad vs narrow spectrum?

• Agent that inhibits growth of microorganisms (bacteria, viruses, fungi, parasites)

• Clear area around the disk where bacteria unable to grow

• The concentration of antibiotic at the edge of the disk zone of inhibition.

• Sensitivity aspect test if bacteria are sensitive to specific antibiotics

• It tests the sensitivity of bacteria to antimicrobial

• Use Mueller-Hinton agar, completely streak the plate (streak, rotate plate 90* streak again, rotate 45* streak the third time. Evenly disperse the discs and incubate. Measure the zone and compared results with standard report

• Using antibiotic develop bacteria-free zone where antibiotic resistant bacteria thrive with no competition and spread throughout the community. Animals inject with antibiotic can also develop resistant. Spread via meats or crops with animal fertilizer.

• Target cell wall, cell membrane, protein synthesis, and nucleic acid synthesis

• Broad spectrum targets wide variety of bacteria and useful before pathogen is identified. Narrow spectrum target specific bacteria and cause less disruption to normal microbiota