Biochemical Tests

Fermentation Glucose acts as an electron donor to one or more of its organic products End products include alcohols, organic acids, and CO2 Today we use two tests Phenol red broth Triple sugar iron (TSI)

Phenol Red Broth Contains: Carbohydrate peptone Dextrose/glucose Lactose Sucrose peptone Phenol red (pH indicator) Below 6.8 = yellow 6.8-7.4 = red Above 7.4 = pink Durham tube – inverted to catch gas bubbles

TSI Similar to phenol red broth, but has a 3 sugars in one slant Only contains a small amount of glucose (0.1%), so if the organism only ferments glucose, it will quickly run out and will resort to breaking down protein, which will increase pH and cause red coloration Also tells if organism reduces sulfur Black precipitate Streak the surface and stab it (get to oxygen poor area)

Starch Hydrolysis Starch can be broken down into glucose if the proper enzymes are produced by bacteria Iodine turns from yellow to black in the presence of starch So if the bacteria produces starch hydrolyzing enzymes, there will be no starch present and iodine will not produce black color

Casein Hydrolysis Casein = milk protein Casease (enzyme) breaks down casein into amino acids to be taken up by bacteria Casease-positive bacteria will produce a clear zone

Gelatin Hydrolysis Gelatin is another protein Gelatinase (enzyme) breaks down gelatin into amino acids to be taken up by bacteria Gelatin liquifies when it’s broken down

Lipid Hydrolysis Enzyme lipase breaks down lipids Spirit blue dye gets pulled toward bacteria when lipids are taken up

Indole/H2S SIM determines Sulfur reduction (black precipitate) Indole production from trypophan (red color with Kovac’s reagent) Motility (radiating growth)

Methyl Red-Vogues Proskauer (MR-VP) Methyl red test determines mixed-acid fermentation Addition of methyl red reagent gives red color for positive bacteria Vogues Proskauer test determines if fermenting bacteria utilize the butylene glycol pathway Addition of vogues proskauer reagent gives red color for positive bacteria Because both result in red color, next week we need to pour ½ of each broth into a fresh culture tube

Citrate Utilization Determines if bacteria use citrate as carbon source Contains bromthymol blue dye Green at pH 6.9 Blue at pH 7.6 Produces alkaline products when citrate is used So it turns BLUE