BIOCHEMICAL TEST

SULFUR INDOLE MOTILITY (SIM) TEST SIM medium is a bacterial growth medium that is designed to test for multiple characteristics in an effort to identify an organism. All tubes were incubated for 48 hours at 37C after inoculation. Kovcs reagent was added after incubation. SIM tubes test for 3 things: (1) hydrogen sulfide production (2) indole production, and (3) motility. Indole production: If an organism produces the enzyme tryptophanase, it will degrade tryptophan into indole, pyruvate, and ammonia. To test for the presence of tryptophanase, the agar deep is inoculated with a sterile needle and incubated for 24-48 hours. After incubation, a solution called Kovacs Reagent is added to the tube. Kovacs Reagent contains HCl, Butanol, and para-dimethyl-amino-benzaldehyde (pdaba). The acidified butanol extracts any indole that is produced and brings it to the surface of the agar, where it reacts with the pdaba, turning red.

Hydrogen sulfide production: Organisms which produce the enzyme thiosulfate reductase can reduce sulfur to hydrogen sulfide gas. In the SIM deep tubes, the source of sulfur is sodium thiosulfate. If hydrogen sulfide gas is produced, it reacts with ferrous sulfate, giving a black precipitate. Motility: Organisms which are motile will exhibit diffuse growth that spreads out from the initial stab line, whereas organisms which are not motile will only grow where the agar was initially stabbed. SIM PREPARATION: Dissolve 30g in 1L of distilled water Dispense 10mL in sterile tubes with screw Autoclave for 15 minutes at 121 C METHOD: Inoculate sample into SIM media with needle aseptically Leave another SIM media without sample, this will serve as the control ncubate for 4 48 hours at C Add 6-8 drops of Kovacs Reagent
***Ehrlichs reagent is more sensitive in detecting indole production in anerobes and non-fermenters.

INTERPRETATION: Indole Positive: Red coloration at the top layer H2S Positive: Black precipitate at the bottom layer Motile: Diffusion of stab line

NEGATIVE NONMOTILE NO H2S

POSITIVE

H2S

MOTILE

DeltaBiology (2012). Sulfur Indole Motility (SIM) Test. Retrieved from http://deltabiology.com/2011/sulfur-indole-motility-sim-test/

BIOCHEMICAL TEST
METHYL RED (MR) TEST - detects the ability of an organism to produce and maintain stable acid end products from glucose fermentation. Some bacteria produce large amounts of acids from glucose fermentation that they overcome the buffering action of the system. Methyl Red is a pH indicator, which remains red in color at a pH of 4.4 or less. METHYL READ VOGES PROSKAUER MEDIA PREPARATION: Dissolve 17g of media in 1L distilled water Dispense 10mL in sterile tubes with screw Autoclave for 15 inutes at C

METHOD: Inoculate sample into MRVP broth aseptically ncubate at C for 48 hours Add 5 drops of Methyl Red Reagent Shake sample INTERPRETATION: MR POSITIVE: Broth color changes to red

VOGES PROSKAUER (VP) TEST - detects butylene glycol producers. Acetyl-methyl carbinol (acetoin) is an intermediate in the production of butylene glycol. In this test two reagents, 40% KOH and alpha-naphthol are added to test broth after incubation and exposed to atmospheric oxygen. If acetoin is present, it is oxidized in the presence of air and KOH to diacetyl. Diacetyl then reacts with guanidine components of peptone, in the presence of lphanaphthol to produce red color. Role of alpha-naphthol is that of a catalyst and a color intensifier.

METHYL READ VOGES PROSKAUER MEDIA PREPARATION: Dissolve 17g of media in 1L distilled water Dispense 10mL in sterile tubes with screw Autoclave for 15 minutes at C METHOD: Inoculate sample into MR-VP Broth aseptically ncubate at C for at least 48 Hours Add 0.6mL of alpha-naphthol and shake Add 0.2 ml of 40% KOH and shake Allow tube to stand for 15minutes INTERPRETATION: POSITIVE: Appearance of red color change
Rao S. (2006). Department of Microbiology, JJMMC, Davangere. IMViC Reactions. Retrieved from http://www.microrao.com/micronotes/imvic.pdf

BIOCHEMICAL TEST
CITRATE TEST - detects the ability of an organism to utilize citrate as the sole source of carbon and energy. Bacteria are inoculated on a medium containing sodium citrate and a pH indicator bromothymol blue. The medium also contains inorganic ammonium salts, which is utilized as sole source of nitrogen. Utilization of citrate involves the enzyme citritase, which breaks down citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and CO2. Production of Na2CO3 as well as NH3 from utilization of sodium citrate and ammonium salt respectively results in alkaline pH. This results in change of ediu s color fro green to blue.

SIMMONS CITRATE MEDIA PREPARATION: Dissolve 24g in 1L distilled water Dispense 10mL in sterile tubes Autoclave for 15 minutes at C METHOD: Streak sample into media ncubate at C for 4 Hours INTERPRETATION: POSITIVE: Media changes color from green to blue

Rao S. (2006). Department of Microbiology, JJMMC, Davangere. IMViC Reactions. Retrieved from http://www.microrao.com/micronotes/imvic.pdf