Test Gram stain

Purpose Determine morphology and arrangement to determine the ability of an organism to ferment a specific carbohydrate with or without the production of gas.

Result Gram positive, long pinkish rods

(bacilli) with purple dots

positive Media turns yellow (fermentation has occurred) and gas produced Negative Media remains red (no fermentation). -Positive Bubbling (O2 gas is liberated from the H2O2) Negative No bubbling -Positive A zone of clearing appears adjacent to the streak line. Negative No clearing; only a blue/black area surrounding the streak line Positive Red layer forms on surface of the media Negative Yellow layer forms on the surface of the media Positive Intense pink/red color Negative No color change

Carbohydrate Fermentation

Catalase Test

to identify the presence or absence of catalase via the use of hydrogen peroxide
to determine the ability of an organism to hydrolyze (break down) starch

Starch hydrolysis test

Indole Test

Used to determine the ability of an organism to split indole from the amino acid tryptophan using the enzyme tryptophanase. Used to determine the ability of an organism to split urea to form ammonia (an alkaline end product) by the action of the enzyme urease. Media also contains the pH indicator phenol red, which turns an intense pink at alkaline pH. Used to determine if an organism is capable of using citrate as the sole source of carbon with production of the enzyme citratase Used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) or nitrogen gas (N2) by the production of the enzyme nitratase.

Urease Test

Citrate Utilization

Nitrate Reductase Test

Positive Alkaline pH causes media to change from green to Prussian blue Negative No color change Positive Red color; nitrate reduced to nitrite; test is complete Negative No color change; do confirmation test by adding a small pinch of zinc powder Positive Organisms change color to a dark red/black Negative No color change If bacteria have mixed acid fermentation lifestyle, the broth remained red. If they did not, then the broth turned yellow, signifying that no acidic products were made

Oxidase Test

Used to determine the presence of oxidase enzyme. Used to determine the ability of an organism to produce mixed acid end products from glucose fermentations.

Methyl Red Test

VogesProskauer to distinguish bacteria based upon their Test butanediol fermentation abilities Gelatin Liquefaction Used to determine the ability of an organism to produce enzyme gelatinase, which liquefies gelatin. Gelatinase breaks down large proteins into smaller components, which can then enter the organism and be metabolized. Differentiate facultative anaerobes between motile and non-motile Used to determine oxygen requirements. The media contains glucose, cystine, and sodium thioglycollate to lower the oxidationreduction potential.

Motility Deep

Positive bright orange red red color develops Negative Brown color develops Positive Gelatin is liquefied Negative Gelatin is solid (Note continue incubation of negative tubes for another 4 to 5 days to see if gelatinase is produced Motile (growth spread throughout) indicates

Oxygen test

Aerobe Growth at the top of the media Facultative Growth throughout the media Anaerobe Growth at the bottom of the media

Protease production The protease producing isolates were inoculated in 250 ml Erlenmeyer flasks containing 50 ml saline skim-milk broth and incubated at 37 C and 150 rpm [9]. After incubation for 48 h, the cultures were centrifuged at 4500g for 10 min at 4 C, and the cell free supernatants used as crude enzyme for extracellular protease assay.

2.3. Protease assay Protease activity was determined using casein as a substrate. The assay mixture contained 1.1 ml of 1% (w/v) casein in 0.1 M Tris-HCl buffer (pH 8.0) and 0.1 ml of enzyme solution. After incubation at 37 C for 30 min, the reaction was terminated by adding 1.8 ml of 5% (w/v) trichloroacetic acid. Then each test tube was centrifuged at 4000g for 20 min and the absorbance of the supernatant was determined at 280 nm. One international unit (IU) of protease activity is the amount of enzyme which liberates 1 mol of tyrosin

Culture and Growth Conditions for Protease Production For protease production, the isolated bacterium was grown in a medium (pH=8.0) consisting of 0.5 % (w/w) glucose, 0.75% (w/w) peptone, 0.5 % (w/w) MgSO4, 0.5% (w/w) KH2PO4 and 0.01% (w/w) FeSO4, in orbital shaking incubator at 60 C and 140 rpm for 72 h. After incubation, the culture broth was centrifuged at 4 C and 3600 rpm for 30 min. The cell precipitate was discarded to obtain the supernatant, which was used as crude protease preparation for further purification [11]. Assay of Protease Activity Enzyme activity was measured according to the method described by Kunitz et al. [12]. The sample contained enzyme solution and 0.5% (w/v) casein in 0.1 M potassium phosphate buffer (pH=8.0) and was incubated at 60 C for 10 min. This reaction was terminated by the addition of 5% (w/v) trichloroacetic acid (TCA) solution and then centrifuged to remove the resulting precipitate. Protease activity was estimated using a tyrosine standard curve. One unit of alkaline protease activity (U) was taken as the amount of enzyme liberati ng 1g of tyrosine/min under the assay conditions.