Lab 11 Goals and Objectives:

Exercise 39: Oxidation and Fermentation TestsRead 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 ReactionsSet 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 aureusEscherichia coli Proteus vulgaris

Each pair needs:3 starch plates 5 urea broths (replaces urea slant) 3 skim milk plates 5 phenylalanine slants3 spirit blue plates 5 tryptone broths

***Save streak plates of unknowns for use next class***

Durham Sugar Tube Fermentation (Glucose, Lactose, Mannitol)Inoculation method: loop transferContains: 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

Negative

Acid plus gas

Acid

MR-VP Medium: Methyl Red TestInoculation method: loop transferContains: 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 pathwayResults: Red = positive for mixed acid formation

Yellow = negative for mixed acid formation

+ _

+ _ + _

MR-VP Medium: Voges-Proskauer TestInoculation method: loop transferContains: peptone and glucoseAdditional 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

Simmon’s Citrate AgarInoculation method: streak and stab slant with needleContains: 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

+ _

Oxidase TestDiscriminates 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)

Catalase Test

Discriminates aerobic organisms that produce catalase to degrade hydrogen peroxide into water and oxygen

http://ftp.ccccd.edu/dcain/CCCCD%20Micro/Catalase.jpg

+ _

12 Possible Unknowns

Gram Positive Gram Negative

Gelatinase + Gelatinase - Gelatinase + Gelatinase -

Baci

llus s

ubtil

is

Pseu

dom

onas

aer

ugin

osa

Catalase + Catalase -

Nitrate Reduction BrothInoculation method: loop transferContains: beef extract, peptone, KNO3 as nitrate source, durham tube

for gas collectionAdditional 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)

sulfanilic acid (reagent A) + dimethyl-alpha-naphthylamine (reagent B)

Nitrate to nitriteadd zinc to negative tubes

No reductionComplete reduction

A + B + nitrite = red

Zinc converts nitrate to nitrite

Fig. 40.1

Control

UnknownsSeparate Plates!

broth

broth

Lab 11 Goals and Objectives:

Exercise 39: Oxidation and Fermentation TestsRead 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 ReactionsSet 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 aureusEscherichia coli Proteus vulgaris

Each pair needs:3 starch plates 5 urea broths (replaces urea slant) 3 skim milk plates 5 phenylalanine slants3 spirit blue plates 5 tryptone broths

***Save streak plates of unknowns for use next class***