BSC 242 Lab Test 2

Energy metabolism tests

• heterotrophs obtain energy through respiration or fermentation

• convert chemical energy in organic molecules to high yield-energy bonds of ATP

• Respiration—> glycolysis, TCA (Krebs), ETC (oxidation phosphorylation

• Fermentation—> glucose is electron donor and organic products are electron acceptors. Acids,CO2, organic solvents

O-F Test

• differentiate between fermentation or oxidation of carbohydrates (glucose)

• use Bromothymol blue to indicate pH change

• initial way to tell between fermentative Enterobacteriaceae and Oxidative Pseudomonas and nonreactive Alcaligenes

O-F Results

Sealed: green/blue and unsealed: any yellow = Oxidation

Sealed: yellow and unsealed: yellow = O-F or F

Sealed: Slight yellow on top and unsealed: slight yellow on top = O-F or F

Sealed: blue/green and unsealed: green/blue = no sugar metab

Phenol Red Broth

• fermentation of specific carbs

• differentiate members of enterobacteriaceae and distinguish from other Gram - rods

• has pancreatic digest of casein, NaCl, phenol red, Durhamn tube, carb

• phenol changes color based on pH

• look for fermentation, fermentation with has production, deamination of peptone amino

Phenol Red Broth Results

Yellow + bubble = fermentation, gas, acid

Yellow, no bubble = fermentation, acid

Red, no bubble = no fermentation

Pink, no bubble = deamination of peptone (basic)

Starch Hydrolysis

• are too large to cross membrane

• Linear (amylase) and branched (amylopectin)

• extracellular enzymes (amylase and oligo-1-6-glucosidase) hydrolyze into small subunits that can cross into cell

• add iodine to plate, binds with starch that hasn’t been hydrolyzed

Starch Hydrolysis Results

• clearing around growth = amylase present (+)

• no clearing = no amylase (-)

Casein Hydrolysis

• casease is an enzyme used to hydrolyze casein, causing clearing

• casein is milk protein and source of amino acids for organisms that can hydrolyze it and transport it into cell

• gives milk white color

• used to test for bacteria in milk products

Casein Hydrolysis Results

• clearing = casease present (+)

• no clearing = no casease (-)

Lipase Test

• hydrolyze fats (lipids)

• tributyrin plate—> an oil that is a simple triglyceride found in natural oils

• extracellular enzyme

• clearing

• detect and enumerate lipolytic bacteria

• differentiate members of staphylococcus, enterobacteriaceae, clostridium, neisseria

Lipase Test Results

• clearing = lipase present (+)

• no clearing = no lipade (-)

Biochemical Tests

• differentiate between organisms based on enzymatic reactions and metabolic pathways

IMVIC Tests

• indole, methyl red, citrate, voges-pa

Indole Test Using SIM Medium

• combination of differential medium (allow multiple biochemical determinations)

• sulfur reduction, indole production, motility

• differentiation of enterobacteriaceae, salmonella, shigella

Indole

• produced with pyruvate and ammonia during hydrolysis of tryptophan by the enzyme tryptophanase

Tryptophan

• in casein and animal protein

SIM

• low agar concentration and inoculated with a needle

• growth that radiates and gives hazy look indicates motility

• black precipitation after incubation indicates sulfur reduction

• red layer after adding Kovac’s reagent indicates tryptophan metabolism to indole -

Methyl Red and Vogues Proskauer test

• MR-VP test series, used in microbiology to figure out how bacteria ferment glucose—specifically, which metabolic pathway they use.

Methyl Red Test

• tests for mixed acid fermentation

• added after incubation to test pH

• red = pH of 4.4 (positive), mixed acid fermentation

• yellow= pH of >6.2 (negative), no mixed acid fermentation

VP Test

• tests for conversion of glucose fermentation products to acetoin and 2,3-butanediol

• reagents react with acetoin to form diacetyl, which reac ts with guididine nuclei from peptone to produce red color (+)

Utilization Media

• defined media designed to differentiate bacteria based on ability to grow when essential nutrients (C or N) aren’t present

Citrate Test

• can bacteria use citrate instead of glucose

• slant, IMViC test

• citrate—> componenet of Krebs cycle

• sodium citrate is only C source, ammonium ph

Nitrate Reduction

• anaerobic

• nitrate reductase—> reduces nitrate to nitrite

• many gram - including enterobacteriaceae

• denitrification—> takes nitrate to N2

• potassium nitrate, beef extract, peptonem durham tube

Urease Test

• can an organism break down urea using urease

• urea—> product of decarboxylation of amino acids

• hydrolyzed to ammonia and CO2 by enzyme urease

• many enterics have urease, but Proteus, Morganella, Providencia are rapid

• Urea broth—> limited nutrients, strong buffer for rapid, slow, negative

• Phenol red is indication—> turns pink to reveal alkalization due to production of ammonia and CO2

Catalase Test

• detect presence of catalse

• catalase—> an enzyme used to protect cell from hydrogen peroxide produced in ETC

• differentiates catalase + staphylococcus and micrococcus from catalase - streptococcus, enterococcus, lactococcus

• bubbles = +

• no bubbles = -

Oxidase test

• tests for presence of cytochrome C oxidase

• tested by its ability to catalyze reduction of cytochrome C by chromogenic reducing agent (develop color as oxidized)

• initial ID of oxidase + Neisseria, differentiation of oxidase - enterobacteriaceae from oxidase + pseudomonadaceae

• dark purple = +

Enterotube

• allows for many biological tests to be done at the same time and read in 24 hours

• used for enteric bacteria