Dividing into 2 planes

Square Rectangle

no charge

Sudan Black stains granules of poly-B-OH-butyric acid

water insoluble

India Ink

Stains background, not cell

Nigrosin: (-) charge repels membrane of cell

India Ink: insoluble dye, does not penetrate cell

Dyes same cell all the same color

Can tell morhpology and size of cell

Common Dyes: methylene blue, and crystal violet

Causes cell to stain differently based on properties of cell

Differential 

Based on characteristic of cell wall

Crystal Violet, Mordant:Iodine, Decolorizer:Ethanol, Counterstain:Saffranin

Differential stain testing for high wax content of cells

Steam allows dye to penetrate cell

Primary Stain: Carbol Fuschn (red)

Steam

Decolorizer: Acid Alcohol (- test is colorless)

Counterstain: Methylene Blue (- test is blue)

Acid Fast Organisms

(Acid Fast Positive)

Myrobacterium- if mature cell, because high wax content

if young cell, appear as gram+ tapered rod, sometimes fragment

Mycrobacterium: Tuberculosis and Leprae

Nocardia

Spore Staining 

Structural Stain

Must be steamed to allow dye (malachite green)to enter spore

Endospores appear as green center with pink sporangium

Free spores as small green oval bodies

Primary: Malachite Green

Steam

Decolorizer: Water

Counterstain: Safranin

Bacillus- Aerobic, gram + rod

Clostridium- Anaerobic, gram + rod

Sporsarcinae- Cocci

Pour= liquid agar--> pour plate

Broth= liquid media

Deep=media solidified in upright position

Slant= solidified at angled position

Fermentation Broth= broth with durham tube added

Exact chemical composition known

Examples:

Selective Media- favors growth of one microorganism over another; inhibiting or providing nutrients

Differential Media- media distinguishing between different types of organisms based on apperance of growth or color changes

selects for growth of gram + microorganisms

PEA is inhibitory to the growth of gram (-) organisms

Selects for gram (-) microorganisms

Differentiates for lactose fermentors

Lactose fermentors produce acid, acid absorbs neutral red dye

Appearing Red

Non-fermentors do nor appear Red

Selects for gram (-) organisms

Differentiates lactose +/-

Lactose + shows color change

Lactose - no color change

EMB 

Lactose + results, based on acid

Can further differentiate based on amounts of acid

Mixed Acid Fermentors produce more acid

colonies have dark blue-black centers

E.Coli- Metallic Green Sheen

Butanediol Fermentors produce less acid

colonies have pale pink to lavender centers

Differentiates organisms based on blood reaction

Gamma Hemolysis- None

Alpha Hemolysis- Partial hemolysis, partial clearing around colony; sometimes appear green

Beta Hemolysis- Complete blood hemolysis

Used to determine physiological characteristics of microorganism

Bacterial Enzymes and biooxidation

Tests for presence of Amylase--> hydrolyzes starch into simple sugar

Iodine added to plate and appears blue/black when interacting with starch

If amylase present, starch not present blue/black color not observed

Tests for presence of Caseinase

Hydrolyze Caseinase into amino acid product

Breakdown of caseinase causes agar to lose white coloring

Determining if microorganism can ferment particular sugars

Test Tubes contain: sugar (glucose, lactose, mannitol)

pH indicator (phenol red)

Durham tube

Fermentation= acid increase changing color from red to yellow

Durham tube= testing if gas produced during fermentation

Alkaline Reaction= darkening of red pH indicator color

Yellow= acid

Yellow + gas= Acid, gas

Red to Dark red= Neg. or Alkaline

HCOOH--> CO2 + H2

Tests for Mixed acid Fermentor

Mixed Acid Fermentor produce large amounts of acid

Acid drops below 5.1

When methyl red added, stays red

E. Coli = MR+

HCOOH--> Acetyl methyl carbinol (AMC)--> 2,3 butanediol

Tests for 2,3 butanediol fermentor

2,3 butanediol fermentor produce less acid than mixed

AMC= acetoin is tested

VP1= Barrit's reagent, intensify possible red color

VP2= KOH, reacts with AMC to produce brick red color

Enterobacteria= VP+

2H2O2--> 2H2O and O2

Hydrogen peroxide toxic (produced in oxygen utilization), Catalase converts H2O2 into water and oxygen

Look for oxygen bubbles produced

Oxidase can oxidize aromatic amines to form colored products

Amine used to test is dimethyl-p-phenylenediamine hydrochloride

If dimethyl-p-phenylenediamine hydrochloride+oxidase= turns blue/ black color

NO3^-+ 2e + 2H^---> NO2 + H2O

NO2--> N2

Tests for ability to reduce nitrate

Reagents Nitrate I(Sulfanilic Acid

Nitrate II(dimethyl-alpha-naphthylamine)

NO2 present broth appears red--> Nitrate Positive

If not red, Zinc added to tube

Red after adding Zinc, Nitrate (-)

After Zinc added, if not Red

Nitrate (+)

Tryptophan--> Pyruvic Acid and Indole

Tests for tryptophanase

Indole tested by adding Kovac's Reagent

Kovac's Reagent= p-dimethylaminobenzaldehyde, amyl or butyl alcohol, and HCl

Kovacs appears in presence of indole product

Urea--> NH3 + CO2

Tests for Urease

Broth has urea and phenol red

As ammonia released pH rises, once above 8.1

Phenol Red appears Red 

Red coloring= Urease +

Proteus= Urease (+)

Cysteine--> H2S+ Amino Acrylic Acid--> Imino Acid--> Pyruvic Acid + NH3

Tests for cysteine defulfurase

Black precipitate= positive test for H2S 

Proteus= H2S positive

Tests for Sulfur, Indole, Motility

H2S Positive= Black precipitate

Indole Positive= Kovacs Reagent turning Red

Motility positive= growth away from inoculant

Tests for Ability to utalize citrate as sole carbon source

Microorganism can grow on media and turn it a deep Prussian Blue 

Growth+ Blue Coloring= Positive Citrate organism

Phenylalanine--> Phenylpyruvic Acid (PPA) +NH3

Tests for presence of phynlalanase

To test for PPA, ferric chloride added to media

Ferric Chloride in PPA turns deep green

Deep green= positive

Test for Lactose fermentation, reduction of litmus, presence of caseinase, and the deamination of amino acids to produce NH₃

pH indicator Litmus and powdered milk

Acid Reaction- Pink liquid due to drop in pH

Acid Curd- Pink solid acid production and coagulation of protein

Reduction- litmus reduced and turns white from milk

Alkaline Reaction- Blue liquid caused by protein breakdown

Proteolysis- Clearing of medium by caseinase

Kliger's Iron Agar (KIA)

read 18 hours after inoculation (ideally)

Tests for fermentation of glucose/lactose, and production of H2S or gas

Fermentation= pH indicator

Glucose fermented= bottom yellow

Lactose fermented= all yellow

Black precipitate if H2S positive

Gas determined by slant movement

determining if bacteria can use glucose in aerobic or anerobic condition

2 test tubes innoculated

1 covered in mineral oil

contains pH idicator Brom Thymol Blue

§  Open Tube: Yellow, Closed Tube: Uninoculated color = Incompletely

        Oxidative (O)

§  Open Tube: Yellow, Closed Tube: Yellow = Strictly Fermentation (F)

§  Open Tube: Uninoculated color, Closed Tube: Uninoculated color = Strictly Oxidative

§  Open Tube: Uninoculated color, Closed Tube: Yellow = Facultative

Indol, Methyl Red, Voge-Proskauer, Citrate

E.Coli = + + - -

E. aerogenes= - - + +

Test if bacteria are motile or not

Tetrazolium chloride, growth indicator

differential for lactose/ sucrose fermentation

lactose fermentig= yellow/green or yellow colonies

nonlactose= opaque red/pink/white colonies

Salmonella ususally produces black colony

Shigella a colorless colony

all lactose + colonies appear red

Selects for gram -, lactose - microorganism

some lactose + grow, but appear red

incubate in plasma tube

if plasma becomes clumpy/ solidifies= bacteria coagulase +

test only valid on gram + staphylococcus

selects for staphylococcus due to high salt conc.

medium is red (phenol red), but plate colonies yellow if mannitol +

contains mannitol and 7.5% NaCl, lacks phenol red

natural colony pigment

tests for exoenzyme DNase which hydrolyze DNA

Zones clear after addition of .1M HCl

Clearing= positive for DNase

selects for Gram -

Differential for lactose

lactose + = red colonies

Coliforms produce golden metallic golden sheen