Lab 11 Goals and Objectives:

Exercise 39: Oxidation and Fermentation TestsRead results: some tubes will require additional reagents

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)

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

Durham Sugar Tube Fermentation (Glucose, Lactose, Mannitol)

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

Control: Escherichia coli

Negative

Acid plus gas

Acid

MR-VP Medium: Methyl Red TestContains: 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

+ _

Control E.coli

+ _ + _

MR-VP Medium: Voges-Proskauer TestContains: 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 pathwayResults: Red = positive for acetoin and thus for 2,3-butanediol

productionYellow/Orange = no acetoin, negative for 2,3-butanediol

production

Control Enterobacter cloacae

Nitrate Reduction BrothContains: 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

Clear with/wo gas = positive for complete reduction of nitrate to nitrogen gas (or nongaseous N2O or NH4)

Red only after zinc = negative for nitrate reduction, negative for nitrate reductases

Controls: Pseudomonas aeruginosa and E. coli

Anaerobic respiration - Nitrate reduction

NO3- NO2

- NH4

NO N2O N2nitrate nitrite

ammonium

nitric oxide Nitrous oxide molecular nitrogen

Electron acceptor Products

NO3– NO2

–, N2 + H2O

SO4– H2S + H2O

CO32 – CH4 + H2O

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

Nitrate to nitriteadd zinc to negative tubes

No reductionComplete reduction

1. A + B + nitrite = red

2. Zinc converts nitrate to nitrite

Simmon’s Citrate AgarContains: 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

+ _

CitrateOxaloacetate Pyruvate + ammonium saltFermentation Alkaline pH

Control: Enterobacter cloacae

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 -

Bac

illu

s su

btil

is

Pse

udom

onas

aer

ugin

osa

Catalase + Catalase -

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)

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

Fig. 40.1

Control

UnknownsSeparate Plates!

broth

broth

• Each pair needs:

3 Starch plates3 Skim milk plates3 Spirit blue plates5 Urea broths (replaces urea slant) 5 Phenylalanine slants5 Tryptone broths

• One set of controls per pair using broth cultures:

• Bacillus subtilis• Staphylococcus aureus• Escherichia coli• Proteus vulgaris

Title Identification of an Unknown Bacterial Culture Laboratory Report

• Introduction ( Why the experiment is important? )– State a hypothesis (an “if then” statement, may require multiple sentences) that is clear and appropriately addresses

the purpose of this laboratory exercise.

• Materials and Methods (How each of the assay was performed?)– Describe each essential assay separately in a separate paragraph. – Media used to grow the organism and list any reagents or indicators – Do not include the enzymatic reactions here: save those for the Discussion

• Results (What are the results observed?)– Include only the key assays, not every test that was performed. In cases where multiple assays were employed

to determine one characteristic, only one type of assay need be presented.

• Discussion ( Discussion of the assays and interpretation of the results)– Discuss only the assays for which you presented results in the table in the – List these enzymes, substrates, products, and color change

• Conclusions (Interpretation of your collective results)– Analyze the results and determine the identification of the unknown based on the collective results – follow the

dichotomous key. – Approve or disapprove the hypothesis

• Literature Cited– List here all source material cited in the laboratory report. Use proper CSE format for scientific publications including

listing the sources in this section in alphabetical order by the first authors last name