UCF Microbiology Lab Midterm

Selective/Differential media

PEA, DES, EMB, blood agar

PEA

Phenyl ethyl alcohol agar

inhibits growth of gram -

selects for gram +

EMB

selects for gram -

differentiates lactose +/- even further

Lactose + show color change

Further differentiates Lactose + fermenters based on the amounts of acid produced

EMB- Mixed acid fermenters

produce more acid

dark blue-black colonies

center is the size of the colony

E. Coli produces metallic green sheen

EMB- Butanediol fermenters

produce less acid

pale pink to lavender centers

bull's-eye colonies

Blood Agar

differentiates based on hemolysis

Beta- Best

Alpha- Almost

Gamma- Garbage

Biochemical Tests

tests used to determine physiological characteristics

in terms of bacterial enzymes and chemistry of bio oxidation

Starch Agar

enzyme- amylase

reagent- iodine

positive- absence of blue/black color

negative- presence of blue/black color

Milk Agar

enzyme- caseinase

caseinase hydrolyzes casein into amino acid products

positive- clearing around colonies

negative- absence of clearing

Lipase Plate

enzyme- lipase

lipase hydrolyzes fat to form glycerol and fatty acids

production of fatty acids lowers the pH

positive- presence of dark blue precipitate

negative- absence of dark blue precipitate

Sugar Fermentation

used to determine if microorganism can ferment particular sugars

Sugars: glucose, lactose, mannitol

pH indicator- phenol red

contains durham tube

Alkaline reaction can occur due to the utilization of the peptone

Results of Sugar Fermentation

yellow=acid

yellow + gas= acid, gas

red to dark red= negative or alkaline

Methyl Red

HCOOH yields CO2+H2

tests for mixed acid fermenter

pH indicator- methyl red

lowers pH less than 5.1

positive- stays red

E. Coli- MR+

Voges-Proskauer

HCOOH yields acetyl methyl carbinol (acetoin) yields 2,3 butanediol

tests for 2,3 butanediol fermenter

positive- brick red color

Enterobacter- VP+

VP- Barrit's Reagents

VPI- alpha-napthol (used to intensify color)

VPII- KOH

Catalase

2H2O2 yields 2H2O+O2

tests for catalase

catalase converts hydrogen peroxide to water and oxygen

add H2O2 to test for presence of catalase

Oxidase

Oxidase oxidizes aromatic amines

reagent-dimethyl-p-phenylenediamine+HCL

positive- presence of dark blue-black color

negative- absence of dark blue-black color

Nitrate

tests for ability to reduce nitrate

NO3- +2e- +2h+ yields NO2+H2O

NO2 yields N2 +NH3

Tryptophan (Indole)

tests for enzyme tryptophanase

trytophanse converts tryptophan to indole and pyruvic acid

positive- presence of red color

negative- absence of red color

Tryptophan (Indole)- Kovac's reagents

p-dimethylaminobenzaldehyde+amyl or butyl alchol+ HCL

Urea

Urea yields 2NH3+CO2

pH indicator- phenol red

pH above 8.1

positive- presence of red color

negative- absence of red color

Proteus- Urease+

Hydrogen Sulfide Production (H2S)

tests for enzyme cystein desulfurase

cystein yields H2S+amino acrylic acid yields imino acid yields pyruvic acid+NH3

cystein desulfurase removes the sulfur side chain from cystein to produce H2S, which when in the presence of iron salts forms a black precipitate

positive- presence of black precipitate

negative- absence of black precipitate

Proteus- H2S+

SIM

Tests for Sulfur, Indole, Motility

H2S positive- black precipitate

Indole positive- Kovac's reagent addition stays red

Motility positive- turbidity

Simmons Citrate

tests for ability to use citrate as sole carbon source

positive- growth and deep Prussian blue color

negative- absence of growth and deep Prussian blue color

Phenylalanine (PPA)

PP in the pool turns green

tests for presence of phenylalanase

reagent- ferric chloride

ferric chloride turns green in presence of PPA

Phenylalanine yields phenylpyruvic acid (PPA)+NH3

positive- presence of green color

negative- absence of green color

Litmus Milk

tests for lactose fermentation, reduction of litmus, presence of caseinase, and deamination of amino acids to produce NH3

pH indicator- litmus and powderd milk

Litmus Milk- Acid Reaction

pink liquid due to drop in pH from the fermentation of lactose

Litmus Milk- Acid Curd Reaction

pink solid due to acid production and coagulation of proteins causing the solid formation

Litmus Milk- Reduction Reaction

litmus is reduced and is caused to be colorless and the tube appears white since only the milk reamains

Litmus Milk- Alkaline Reaction

blue liquid that is usually caused when protein breakdown produces amino acids that are deaminated and release ammonia

Litmus Milk Peptonization/Proteolysis Reaction

clearing of medium (brown or amber) caused by enzyme caseinase, which breaks down the white protein casein in milk

Gelatin

looking for enzyme gelatinase

positive- liquid after being put on ice (gelatin hydrolyzed)

negative- solid after being put on ice (gelatin not hydrolyzed)

What is the indicator in EMB?

eosin and methylene blue

What type of media will E. Coli produce a metallic green sheen?

EMB

What is the reagent used in starch agar?

iodine

Which enzyme hydrolyzes starch to simple sugars?

amylase

Which enzyme hydrolyzes casein into amino acid products?

caseinase

Which enzyme hydrolyzes fat to form glycerol and fatty acids?

lipase

What is the pH indicator used in sugar fermentation?

phenol red

What does methyl red test for?

mixed acid fermenters

Which enzyme converts hydrogen peroxide to water and oxygen?

catalase

What is the reagent used in catalase?

H2O2

What is the reagent used in oxidase?

dimethyl-p-phenylenediamine and HCL

Which enzyme oxidizes aromatic amines to form colored products?

oxidase

What does the enzyme tryptophanase do?

converts tryptophan to pyruvic acid, indole, and NH3

What is the pH indicator used in Urea?

phenol red

What does cystein desulfarase do?

H2S+ amino acrylic acid yields imino acid yields pyruvic acid+HCL

What is the indicator in Simmons Citrate?

bromothymol blue

What is the reagent used in phenylalanine?

ferric chloride

What does phenylalanine yield?

phenyl pyruvic acid and NH3

What are the pH indicators used in litmus milk?

litmus and powdered milk

Known positive results: E.Coli

MR+

Known positive results: Enterobacter

VP+

Known positive results: Proteus

Urease+

H2S+

Positive Result: PEA

growth

Positive Result: DES

growth

lactose fermenters- red

Positive Result: EMB

growth

only lactose + fermenters show color change:

mixed acid fermenter- dark blue-black center

butanediol fermenter- pale pink to lavender center

Positive Result: Starch Agar

absence of blue/black color

Positive Result: Milk Agar

presence of clearing

Positive Result: Lipase Plate

dark blue precipitate

Positive Result: Sugar Fermentation

yellow= acid

yellow+gas= acid and gas

red to dark red= negative or alkaline

Positive Result: Methyl Red

stays red

Positive Result: VP

presence of brick red color

Positive Result: Catalase

production of bubbles

Positive Result: Nitrate Reductase

add N1 and N2 and it turns red

or

after addition of zinc it is colorless

Positive Result: Nitrite Reductase

after addition of zinc it is colorless

Positive Result: Tryptophanse

presence of red color

Positive Result: Urease

presence of red color

Positive Result: Cystein Desulfurase

presence of black precipitate

Positive Result: Simmons Citrate

growth

presence of Prussian blue color

Positive Result: Phenylalanase

presence of green color

Positive Result: Gelatinase

liquid after being put on ice

Positive Result: Motility

turbidty

presence of red color away from inoculation line

Reagents used in nitrate

N1- sulfanillic acid

N2- dimethyl-alpha-napthylamine

What is the growth indicator in motility media?

tetrazolium chloride

What is the primary dye used in Gram stain?

crystal violet

What is the mordant (bonding agent) used in Gram stain?

iodine

What is the decolorizer used in Gram stain?

95% ethanol

What is the secondary dye used in Gram stain?

safranin

What is the primary dye used in acid-fast stain? Type?

Ziel's Carbol Fuchsin, lipid-soluble dye

What is the mordant used in acid-fast stain?

steam

What is the decolorizer used in acid-fast stain?

acid alcohol

What is the secondary dye used in acid-fast stain?

methylene blue

What is the primary dye used in spore stain? Type?

Malachite Green, water soluble (Fat insoluble)

What is the mordant used in spore stain?

Steam

What is the decolorizer used in spore stain?

Water

What is the secondary dye used in spore stain?

Safranin

Simple Microscope

single lens

Compound Microscope

two lenses

Ocular

eyepiece lens (usually 10x)

Objective

nosepiece lenses (commonly 4x, 10x, 45x, 100x)

Mag(total)

mag(objective) X mag(ocular)

Parts of the Microscope

framework

stage

lens system

Framework

arm and base

structural parts of the microscope which support the basic frame

Stage

holds the slide

the mechanical stage clamps the slide and moves the slide around the stage

Lens System

oculars, objectives, condenser

Oculars

eyepiece lenses (usually 10x)

Objectives

lenses attached to rotatable nosepiece

usually 4x, 10x, 45x in low power,high-dry objectives

usually 100x in oil immersion lens

What doesn't need to be changed when changing the objective lenses?

parfocalized microscope focusing adjustments need not to be made when changing objective lenses

What do oil immersion lens use oil for?

oil immersion lens uses oil with approximately the same refractive index as glass to prevent light loss due to diffraction which would occur if light traveled from one refractive index to another