1. Biochemical Tests

2. 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)

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

4. 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)

5. 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

6. 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

7. 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

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

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

10. 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

11. 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