BISC209/S11: Lab8

Cultured Bacterial Isolates Characterization continued

Complete Quorum Sensing & Mutualistic and Antagonistic Interactions Tests set up last week

Take photos of your plates.

Complete Antibiotic Production & Sensitivity Testing
Week 3

• Examine the plates and look for evidence of inhibition of growth of "test" organisms near the putative antibiotic producer's midline streak. This will appear as a measurable zone of inhibition (section of a circle of no growth). You should measure the radius of the area most affected and convert to diameter in millimeters of inhibition. The size is indicative of the diffusion potential of the antibiotic and/or an indication of how sensitive the test organism is to the mechanism of inhibition. Compare your results to other tested isolates in your lab. Think about why an antibiotic might work differently on a Gram positive vs. a Gram negative organism or between two bacteria that are both Gram positive.
• Take a photo of your plates. If you found no inhibition of growth, does that mean that your potential antibiotic producer does not secrete any antimicrobial compounds? Why or why not?

SIM TESTS DEVELOPMENT AND ANALYSIS

MOTILITY
Motility detection is possible due to the semisolid nature (low concentration of agar) of the SIM medium. Growth radiating out from the central stab inoculation line indicates that the test organism is motile. The motility test should be assessed first. Motile organisms will exhibit growth radiating from the stab inoculation line. Non motile organisms will exhibit growth only along the stab inoculation line.

TEST FOR TRYPTOPHASE and SULFUR REDUCTION
The ingredients in SIM (sulfate/ indole/ motility) medium enable detection of two other metabolic capabilities that some bacteria have and others lack: digestion of tryptophan by the enzyme tryptophanase to indole and/or sulfur reduction with the production of hydrogen sulfide. SIM medium contains nutrients, iron, and sodium thiosulfate.

The indole test is used for detecting tryptophanase. Casein peptone is rich in tryptophan, which is attacked by certain microorganisms resulting in the production of indole. Bacteria possessing the enzyme tryptophanase cleave tryptophan, producing three end products. One of these end products is indole, produced in aerobic conditions; another is skatole, produced in anaerobic conditions. Amyl alcohol in Kovacs reagent acts as a solvent for indole, which then reacts with p-dimethylaminobenzaldehyde to produce a red rosindole dye. Organisms which do not produce tryptophanase produce no color change in SIM medium when Kovacs is added while bacteria positive for tryptophanase produce a red color when Kovacs reagent is added.

PROTOCOL for DEVELOPING AN INDOLE TEST
To detect indole production due to the enzyme tryptophanase, add three or four drops of Kovacs’ reagent and observe the fluid for development of a ring of red color(positive reaction)at the top of the tube.

DETECTING A POSITIVE H₂S test:
The hydrogen sulfide test relies on the use of sodium thiosulfate and ferrous ammonium sulfate as indicators of hydrogen sulfide production. Ferrous ammonium sulfate reacts with H₂S gas to produce ferrous sulfide, a black precipitate.

When hydrogen sulfide gas is produced, a precipitation reaction will occur with the ferrous ammonium sulfate. An insoluble black precipitate is seen as a positive result. You will develop/analyze your SIM results in Lab 8.

SIM agar:
Approximate Formula* Per Liter
Pancreatic Digest of Casein - 20.0 g
Peptic Digest of Animal Tissue - 6.1 g
Ferrous Ammonium Sulfate - 0.2 g
Sodium Thiosulfate - 0.2 g
Agar - 3.5 g

Kovac's reagent: (per liter)
p-Dimethylaminobenzaldehyde 50,0g
Amyl Alcohol 750.0 ml
Hydrochloric acid 250.0 ml

Special Stains:

Detecting Endospores
All Gram positive bacilli or any bacteria that showed a spore shaped, unstained area in the cells when Gram stained should be stained for endospores. All isolates growing from your dried soil extract on Glyerol Yeast Extract Agar (GYEA) medium should be stained for endospores.
Directions for the Schaeffer-Fulton Endospore stain is found in the Protocols section of the wiki.
Stains : Simple, Gram, Endospore, Capsule

Confirmatory Tests for Motility
Directions for the Hanging Drop motility test and Flagella stain can be found in the Motility section of Protocols. All bacteria that were positive or ambiguous for motility in SIM medium should be looked at by Hanging Drop technique. Any "swarmers" (bacteria that spread all over the plate when cultured on solid medium)should be looked at by Hanging Drop, too. If the hanging drop test is positive and you have time after you have performed any other confirmation tests or special stains, you could try the Flagella stain.

Detecting Capsules by Negative Stain
Highly mucoid (sticky and wet) colonies should be tested for the presence of a capsule using the capsule stain protocol.

If your Gram stain results were ambiguous or not what you expected from growth on PEA and EMB you used, you should probably repeat those Gram stains.

Assignment

Graphical abstract: See models in research reports found in recent issues of the journal Cell

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11
Lab 12