IDENTIFICATION AND CONFIRMATION OF BACTERIA TOOLS AND MATERIALS • Test bacterial suspension • Media MCA (Mac Conkey Agar), VJA (Vogel Johnson Agar), CETA (Cetrimide Agar) and XLDA (Xylose Lysine Desoxychollate Agar) • IMVIC Media (Indole, Methyl Red, Voges Proskauer, Simmons Citrate) + TSIA (Triple Sugar Iron Agar) • Indole reagent, Methyl Red, Voges Proskauer, and Gram Staining Reagent, • Round and straight Ose needle, Bunsen, immersion oil, distilled water, 370C incubator, and Microscope EXPERIMENT PROCEDURE BIOCHEMICAL TESTS 1. Inoculate the bacterial suspension on MCA, VJA, CETA, and XLDA media. Incubate in an incubator at 370C for 1 week. Observe every day the growth and color of the colonies that occur on each medium. Observation result : • MCA: positive for E. coli • VJA: positive for Staphylococcus aureus, if there are black colonies with or without yellow circles, if there are red colonies • CETA: positive for Pseudomonas aeruginosa, if there are bluish green colonies • XLDA: positive for Salmonella, if there are red colonies 2. Also inoculate the bacterial suspension on IMVIC + TSIA media. Indole, Methyl Red and Voges Proskauer are in the form of liquid media, while Simons Citrate is in the form of agar slants. TSIA is a combination of slanted agar and upright agar. Incubate in an incubator at 370C for 1 week and observe every day. Observation results for the IMVIC + TSIA test: Indol To the 24 hour old culture add 3 - 4 drops of Kovacs reagent. Shake, if a red color appears on the surface layer, it means Indol is positive. Examples of bacteria whose Indol reaction is positive: E. coli. Methyl Red To the 48 hour old culture, add 1 – 2 drops of Methyl Red reagent, shake. The occurrence of red color means a positive reaction. Examples of bacteria whose Methyl Red reaction is positive: Salmonella, Shigella, E.coli. Voges Proskauer To the 48 hour old culture add: 0.5 mL of alpha naphthol solution and 1 mL of 16% KOH solution. Shake vigorously, leave for 10 minutes at room temperature.If a rosa or red color appears on the surface/is evenly distributed throughout the culture, it means the reaction is positive. Examples of bacteria whose VP reaction is positive: Klebsiella, Enterobacter, Serratia. Simmons Citrate If Simmons Citrate is positive, the color will change from green to blue. Examples of bacteria that are positive for citrate: Klebsiella and Enterobacter. TSIA (Triple Sugar Iron Agar) Observe changes that occur in TSIA agar cultures that are 48 hours old or more. Glucose: observe the erect part, it is positive if the color changes from red to yellow. Lactose and saccharose: observe on the agar slant, positive if the medium turns yellow. Gas formation: gas bubbles appear in the upright part.if there is a black color in the middle H2S gas : positive, is the boundary between the upright and inclined parts. 3. Draw conclusions about what microbes were inoculated by discussing the results obtained! GRAM COLORING 1. Make a dry smear of the test bacteria 2. The dye is discarded. Pour the lugol to remove the remaining crystal violet (the crystal violet is rinsed by the lugol). Then cover the preparation with Lugol's solution, leave it for approximately 30 seconds. 3. Lugol's solution is discarded. The preparation was rinsed with 96% alcohol, drop by dropuntil the last drop of alcohol rinse remains clear. 4. The preparations were washed with distilled water and stained with Fukhsin dye 30 seconds. 5. Rinse with distilled water and let dry. 6. Observe under a microscope with an objective lens magnification of 100 x use immersion oil. 7. Note the color, arrangement and shape of the bacteria obtained.