1. 3 starch plates 5 urea broths (replaces urea slant)
Lab 11 Goals and Objectives:
Exercise 39: Oxidation and Fermentation Tests
Read results: some tubes will require additional reagents
(share reagents across the bench)
Do controls first so you have something to compare to!
Exercise 40: Hydrolytic and Degradative Reactions
Set up according to Fig 40.1 except both of your two unknowns well separated on each type of plate and control on a different plate (one control plate per pair)
One set of controls per pair using broth cultures:
Bacillus subtilis Staphylococcus aureus
Escherichia coli Proteus vulgaris
Each pair needs:
3 starch plates 5 urea broths (replaces urea slant)
3 skim milk plates 5 phenylalanine slants
3 spirit blue plates 5 tryptone broths
***Save streak plates of unknowns for use next class***

2. Durham Sugar Tube Fermentation (Glucose, Lactose, Mannitol)
Inoculation method: loop transfer
Contains: single carbohydrate peptone broth with durham tube for gas collection, Phenol red pH indicator: alkaline pH = red, acidic pH = yellow
Discriminates the ability to ferment a single carbohydrate (glucose, lactose, or mannitol) into acid products (e.g. pyruvic acid) or acid plus gas
Results: Red = inert, negative for fermentation of specified
carbohydrate
Yellow = positive for fermentation of carbohydrate to acid products
Yellow with bubble = positive for fermentation of carbohydrate to acid + gas

3. Acid plus gas
Acid
Negative

4. _ + MR-VP Medium: Methyl Red Test Inoculation method: loop transfer
Contains: peptone, glucose, and buffer (buffer will neutralize weak acids so only strong stable acids will be detected by methyl red)
Additional reagents added: methyl red pH indicator: acid pH = red, neutral or alkaline pH = yellow
Distinguishes ability to catabolize glucose into stable mixed acids (lactic, acetic, and formic acids) in the mixed acid pathway
Results: Red = positive for mixed acid formation
Yellow = negative for mixed acid formation _ +

5. _ _ + + MR-VP Medium: Voges-Proskauer Test
Inoculation method: loop transfer
Contains: peptone and glucose
Additional reagents added: Barritt’s A (alpha napthol) and Barritt’s B (KOH) (will react with acetoin to produce a red product, alone produce a copper colored product)
Distinguishes the ability to catabolize glucose into the neutral end product butanediol (the oxidized product is acetoin) in the butylene glycol pathway
Results: Red = positive for acetoin and thus for 2,3-butanediol
production
Yellow/Orange = no acetoin, negative for 2,3-butanediol production _ _ + +

6. _ + Simmon’s Citrate Agar
Inoculation method: streak and stab slant with needle
Contains: citrate as sole carbon source, ammonium salts as sole
nitrogen source, bromthymol blue pH indicator: neutral pH = green, alkaline = prussian blue
Discriminates organisms that can produce citrase to metabolize citrate into oxaloacetate and pyruvate. These organisms are forced to utilize ammonium salts as the nitrogen source producing alkaline ammonia waste.
Results: Prussian blue slant and or butt = positive for
citrase production
Green = negative for citrase production _ +

7. Oxidase Test
Discriminates organisms that can produce cytochrome oxidase which catalyzes the transfer of electrons from reduced cytochrome c in the electron transport chain to molecular oxygen.
Test uses NNNN-tetramethyl-p-phenylenediamine (Oxidase Reagent) as an artificial electron acceptor: when oxidized it is colorless, when reduced it turns purple
*Look for color change on the bacteria, not on the cotton swab! (The reagent will turn light purple when exposed to oxygen in the air)

8. Catalase Test
Discriminates aerobic organisms that produce catalase to degrade hydrogen peroxide into water and oxygen _ +

9. 12 Possible Unknowns Gram Positive Gram Negative Gelatinase +
Catalase +
Catalase -
Pseudomonas aeruginosa
Bacillus subtilis

10. Nitrate Reduction Broth
Inoculation method: loop transfer
Contains: beef extract, peptone, KNO3 as nitrate source, durham tube for gas collection
Additional reagents added: sulfanilic acid (reagent A), dimethyl-alpha- naphthylamine (reagent B), (together form a complex with nitrite creating a red product), zinc (reduces nitrate to nitrite allowing reaction with reagent A and B)
Discriminates organisms that can produce nitrate reductases to utilize nitrate as a final electron acceptor resulting in the production of either nitrite (partial reduction)
or to NH4, N2O or N2 gas (complete reduction).
Results: Red with reagents A and B = positive for nitrate to nitrite reduction
Red only after zinc = negative for nitrate reduction, negative for nitrate reductases
Clear with/wo gas = positive for complete reduction of nitrate to nitrogen gas (or nongaseous N2O or NH4)

11. sulfanilic acid (reagent A) + dimethyl-alpha-naphthylamine (reagent B)
A + B + nitrite = red
Zinc converts nitrate to nitrite
sulfanilic acid (reagent A) + dimethyl-alpha-naphthylamine (reagent B)
add zinc to negative tubes
Nitrate to nitrite
Complete reduction
No reduction

12. Fig. 40.1
Unknowns
Separate Plates!
broth
Control
broth

13. 3 starch plates 5 urea broths (replaces urea slant)
Lab 11 Goals and Objectives:
Exercise 39: Oxidation and Fermentation Tests
Read results: some tubes will require additional reagents
(share reagents across the bench)
Do controls first so you have something to compare to!
Exercise 40: Hydrolytic and Degradative Reactions
Set up according to Fig 40.1 except both of your two unknowns well separated on each type of plate and control on a different plate (one control plate per pair)
One set of controls per pair using broth cultures:
Bacillus subtilis Staphylococcus aureus
Escherichia coli Proteus vulgaris
Each pair needs:
3 starch plates 5 urea broths (replaces urea slant)
3 skim milk plates 5 phenylalanine slants
3 spirit blue plates 5 tryptone broths
***Save streak plates of unknowns for use next class***