Lab Exercise 9 - Cultural and Physiological Characteristics of Bacteria II

quick tests (yield results within minutes)

catalse

oxidase

tube media that test for other metabolic processes

nitrate broth

gelatin

urea broth

phenylalanine

SIm

IMViC

respiration

a cycle after glycolysis in which the final electron acceptor is an inorganic molecule

if the final electron acceptor is oxygen → aerobic respiration

if the final electron acceptor is nitrate or sulfate, etc → anaerobic respiration

catalase test

a test that detects catalase, which converts 2H₂O₂ → 2H₂O + O₂

catalase is important in breaking down reactive oxygen species produced during aerobic respiration

What is a positive result of the catalase test?

after 3% H₂O₂ is added to a colony → O₂ product appears as bubbling

function of cytochrome C oxidase

removes electrons from (oxidizes) cytochrome C and transfers them to oxygen (reduces) or oxidizes a chromogenic reducing agent

chromogenic reducing agents, e.g. dimethyl-p-phenylenediamine, develop color when oxidized

oxidase test

a test that detects cytochrome C oxidase, which transfers electrons to oxygen (the final e- acceptor)

indicator in the oxidase test

dimethyl-p-phenylenediamine hydrochloride

an aromatic amine

What is a positive result of the oxidase test?

cytochrome c oxidase is present → reagent oxidizes → turns dark blue/black

nitrate broth

a test that detects nitrate reductase, which reduces nitrate to nitrite (NO₃ → NO₂)

What are the reagents in nitrate broth?

Nitrate I: sulfanilic acid

Nitrate II: dimethyl-alpha-naphthylamine

What is a positive result in nitrate broth?

option 1: nitrate reductase reduces NO₃ to NO₂ → nitrate I and nitrate II react with NO₂ → brick red color

option 2: NO₃ denitrifies all the way down to N₂ → remains clear even after addition of zinc

brick red appears after addition of nitrate I and II, before Zn; or doesn’t appear at all

What is a negative result in nitrate broth?

nitrate reductase is not present, NO₃ remains after addition of nitrate I and II

Zn catalyzes reaction of NO₃ → NO₂

brick red color appears after addition of Zn, not before

urea broth

a test that detects urease, which degreates urea → 2 ammonia (NH₃) and CO₂

What is the indicator in urea broth?

phenol red, a pH indicator

What is a positive result in urea broth?

urease is present → degrades urea → pH increases >8.1 → cerise color

What is a negative result in urea broth?

urease is not present

pH remains neutral → color remains red

pH can decrease → color changes to yellow

What does the gelatin medium test?

presence of gelatinase, which catalyzes hydrolysis of gelatin → amino acids

What is needed to determine results of a gelatin test?

ice bath, because gelatin solidifies in low temperatures

What is a positive result for a gelatin test?

gelatinase is present → hydrolyzes gelatin

no gelatin → no solidification after ice bath

see last tube in picture

What is a negative result for a gelatin test?

no gelatinase → no gelatin hydrolysis

gelatin remains → resolidification after ice bath

see second tube in picture

What does a phenylalanine slant test for?

detects production of phenylalanine deaminase

process: phenylalanine → phenylpyruvic acid → ammonia

(Phe → PPA + NH₃)

What is the reagent in a phenylalanine slant?

FeCl₃ (ferric chloride), 5-10 drops

What is a positive result for a phenylalanine slant?

phenylalanine deaminase present → PPA present → FeCl₃ reacts and appears deep green

(PP in the pool turns green)

SIM medium

a medium that tests for sulfide production, indole, and motility

reagents in SIM media

iron (to detect sulfide production)

Kovac’s reagent, 3-5 drops (detects indole production)

What is the result for SIM if an organism produces H₂S?

FeS production → black precipitate

What is the result for SIm if an organism produces indole?

cerise ring at the top of broth

What is the result for SIM if an organism is motile?

turbidity (cloudiness)

fermentation

a pathway after glycolysis in which the final electron acceptor is an organic molecule

does not produce as much energy as respiration

can also refer to breakdown of polysaccharides → monomers that can be fermented

MR-VP broth

one broth to perform two separate tests: MR and VP

MR test

methyl red

tests for mixed acid fermenters

What is the reagent in MR-VP broth for MR test?

methyl red, 3-4 drops

What is a positive result of MR in MR-VP broth?

MR detects high acid production (pH lowered to 4.4 or less), produces red color

What is a negative result of MR in MR-VP broth?

low acid concentration (pH higher than 4.4) → no retention of red color from MR

VP test

Vogues-Proskauer

tests for 2,3-butanediol fermenters

pathway of 2,3-butanediol fermentation

glucose → acetoin intermediate → 2,3-butanediol

acetoin can react with VP reagents

What are the reagents in VP for MR-VP broth?

Barritt’s reagents

VP I: alpha naphthol, 10 drops; intensifies red color

VP II: KOH, 10 drops; produces alkaline conditions that favor acetoin oxidation

What is a positive result for VP test?

acetoin oxidation occurs → diacetal and O₂ are made → O₂ presence produces a red color

tryptone broth

a test that detects tryptophanase, which hydrolyzes tryptophan → indol, pyruvate, and NH₃

What is the reagent added to tryptone broth?

Kovac’s reagent (DMABA and HCl dissolved in amyl or butyl alcohol); 3-5 drops

nonpolar, extracts indole to the top

reacts with indole to produce cerise color

What is a positive result in tryptone broth?

tryptophanase → indole production → cerise ring at top

What does Simmons citrate slant test?

detects utilization of citrate as the sole carbon source for growth

What is the indicator in Simmons citrate sland?

bromothymol blue

What is a positive result on Simmons citrate slant?

growth, NOT blue reaction

however, blue reaction often accompanies growth, can be helped to determine if growth occurred

“if it’s blue, it grew”

IMViC

a set of four tests that differentiate E. coli from Enterobacter aerogenes

What four tests are included in IMViC?

Indole

Methyl red

Vogues-Proskauer

Citrate

What are the results of IMViC?

E. coli tests positive for indole and MR, tests negative for VP and citrate

E. aerogenes tests negative for indole and MR, tests positive for VP and citrate