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Bacteria identification :Media

What you have to know about the media

• What are the sources of C,H,N,O,P,S?

• What type of media is it?

• What are the indicators?

• What are the selective agents?

• They allow the growth of what bacteria?

• What are the possible reactions?

Ex. MacConkey Agar

• Peptone - 17 g

• Proteose peptone - 3 g

• Lactose - 10 g

• Bile salts - 1.5 g

• Sodium chloride - 5 g

• Neutral red - 0.03 g

• Crystal Violet - 0.001 g

• Agar - 13.5 g

• Sources of C,H,N,O,P,S?

• Type of media?

• Indicators?

• Selective agents?

• Allow growth of what bacteria?

• Possible reactions?

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Identification :Complex Carbon Sources

Complex Carbon Utilization

• Too large to be transported inside

• Requires exocellular enzymes for the external degradation into smaller units

– Polysaccharides (starch)

– Lipids (triglycerides, etc…)

– Proteins (casein)

– Polynucleotide chain (DNA)

Complex Carbon Sources: Starch

• Media used: Starch Agar

• Detected Enzyme: α-amylase

– cleaves α-1,4 bound between glucose monomers

• Identification: Iodine (halo = starch digestion)

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Complex Carbon Sources: Starch

Before iodine addition After iodine addition

Complex Carbon Source: Protein

• Media used: Milk agar

• Detected Enzyme: Caseinase (protease)

– cleaves peptide bounds joining amino acids in the casein protein

• -Identification: clear area (halo) under and surrounding growth

Complex Carbon Source: Protein

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Complex Carbon Sources: Fatty Acids

• Media used: Spirit Blue

• Detected Enzyme: Lipase

– can degrades complex fats (triglycerides) into individual fatty acids

• Identification: Spirit Blue (clear area (halo) under and surrounding growth)

Complex Carbon Sources: Fatty Acids

Complex Carbon Sources: DNA

• Media Used: DNA agar

• Detected Enzyme: DNase

– catalyzes the hydrolytic cleavage of phosphodiester bounds in the DNA backbone

• Identification: Precipitates of polymerized DNA are opaque, clearing represents digestion of DNA

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Complex Carbon Sources: DNA

Identification: Metabolic Tests

• Phenol red broth

– Allows determination of carbon source preferred and metabolism (Oxidation or fermentation)

– Contains simple carbon sources:

• Peptone (protein amino acids)

• Desired sugar added

– Contains a pH indicator

• Phenol red– Yellow - acid pH

– Orange - neutral pH

– Red - alkaline pH

Phenol Red Broths - Interpretation

A. Yellow (acid) + gas = Fermentation of sugar

B. Yellow (acid) no gas = Fermentaion of sugar

C. Orange (neutral) no gas = Oxidation of sugar

D. Red (alkaline) no gas = Oxidation of proteins

E. Uninoculated

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TSI — Three Sugars and Iron

• Three sugars

– Glucose (limiting)

– Sucrose

– Lactose

• Proteins

– Cysteine

• Indicator

– Phenol red

IMViC Tests

• Indole, Methyl Red, Voges-Prosakaur, Citrate(IMViC) :

– These four tests include an important series ofdeterminations which are collectively called theIMViC reaction series

– The IMViC reaction series allows thediscrimination of bacteria of theEnterobacteriaceae family

IMViC Test Methyl Red-Voges Proskauer

• Methyl Red Test :– Fermentation with accumulation of acids:

• Glucose pyruvate lactic and/or acetic acid + CO2

• Voges Proskauer Test – Fermentation with accumulation of butanediol– Glucose pyruvate acetoine 2 butanediol + CO2

- +

- +

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Methyl Red Test

• Test for acid accumulation

– Carbon Sources: Glucose and proteins

– Indicator -methyl red; Added after growth

• MR +: red (pH < 5.2)

• MR - : Yellow (pH > 5.2)Neutral Acid

Voges-Proskauer Test

VP + = redVP - = Yellow

Usual results of MR/VP:MR+/VP-; MR-/VP+ MR-/VP-

Reagents VP:

butanediol + -naphthol + KOH + O2 acetoin

Neutral Acid

- +

Acid

produced

No

acetoin

Neutral Acetoin

IMViC: Indole Test

• Principal

– Some microorganisms can metabolize tryptophane by the tryptophanase

TryptophaneTryptophanase

Indole + acide Pyurvic + NH3

Kovac’s reagent

Red color

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IMViC Test : Citrate Utilization

• Unique carbon source

– Citrate

• Indicator

– Bromthymol blue

• Citrate utilization generates alkaline end products

– Changes from green to blue

Positive

Klebsiella, Enterobacter

Negative

E. coli

Urea Utilization• Enzyme tested

– Urease

• pH Indicator

– Phenol red (turns pink)

C O + 2 H2O CO2 + H2O + 2 NH3 (NH4)2CO3

H2N

H2N

Ureaammonium

carbonate

(alkaline)Amino acids

PositiveNegative

Urea Utilization – Phenol Red

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Ornithine Decarboxylase Assay

• Detects ornithine decarboxylase

– Catalyzes the decarboxylation of ornithine

– Produces diamine putrescine and carbon dioxide

(causes alkaline change)

• Indicator: Brom Cresol purple

– Purple when alkaline or neutral

– Yellow when acid

Ornithine Decarboxylase Assay

• Left: Alkaline with and without ornithine

• Center: Alkaline with ornithine, acidic without

ornithine

• Right: Acidic with and without ornithine

Phenylalanine Slants

• Detects phenylalanine deaminase

• Phenylpyruvic acid reacts with ferric chloride

to produce a green colour

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Phenylalanine Slants

A: Positive for phenylpyruvic acid

B: Negative for phenylpyruvic acid

A B

Lysine Agar Slants

• Detects lysine decarboxylase

• Primarily used to detect bacteria in the

Enterobacteriaceae group (for example,

salmonella)

• Indicator: Brom cresol purple

Lysine Agar Slants :Brom Cresol Purple

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Lysine Agar Slants

• Purple butt : lysine

decarboxylase positive

• Purple slant: lysine

deamination negative

• Yellow butt: glucose

fermentation

• Red slant: lysine

deamination positive

• Black precipitate:

sulfur reduction

SIM — H2S, Indole and Motility

• Semi-solid medium

– Allows to visualize motility

• Cystein metabolism

CysteineH2S; H2S+ FeSO4 Black precipitate

• Tryptophan metabolism

(A) Tryptophan Indole + NH4 + Pyruvate

(B) Indole + Kovac reagent Red

Non inoculated Non-motile

H2S and motile

Indole+ -

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Anaerobic Respiration :Nitrate Reductase

2 H+

2 H+

3 H+ + 3 OH -

3 H2O

2 H+

NO2- + H2O (N = +3) nitrite

NO3- + 2 H+ (N = +5) nitrate

2 e-

2 e-

2 e-

Fp

Fe-S

2 e-

Q

Cyt

b

NADH + H+

FADH2

Nitrate

reductase

Interior

Exterior

Final e- acceptor

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

- NO, N2O,

NH2OH,

NH3, N2

nitrate nitrite

Step 1: Test for nitriteNO2

- + sulfanilic acid and alpha naphthylamine HNO2

Nitrate is not reducedNo Nitrite

Yellow

Nitrate is reducedProduction of Nitrite

Red

Nitrate is reduced to nitriteNitrite is reduced

No Nitrite Yellow

Anaerobic Respiration :Nitrate Reductase (con’t)

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

- NO, N2O,

NH2OH,

NH3, N2

nitrate nitrite

Step 2: Test for the presence of nitrateNO3

- + Zn (s) NO2-

Nitrate is presentReduction to Nitrite

Red

Nitrate is absentNitrite was reduced

Yellow

Anaerobic Respiration :Nitrate Reductase (con’t)

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Oxidase Test : Aerobic RespirationElectron Transport Chain

3 H2O

H+

2 H+

2 H+

3 H+ + 3 OH-

2 H+

H2O

3 H+ + 1/2 O2

2 e-

2 e-

2 e-

Fp

Fe-S

2 e-

Q

Cyt b

Cyt o

NADH + H+

FADH2

interior

exterior

Oxidase Test : Aerobic Respiration

• Cytochrome oxidase catalyzes the reduction of a final electron acceptor, oxygen

• An artifcial e- donor, phenylenediamine, is used to reduce the cytochrome oxidase

• If the enzyme is present, the colorless reagent (reducedstate) will turn blue (oxidized state)

phenylenediamine

Differential Tests for the Identification of Gram Positive Cocci

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Blood Hemolysis

• Media used: Blood agar

• Detected Enzyme: hemolysins

• Identification:

– α-hemolysis: greenish hue, partial breakdown of

red blood cells

– β-hemolysis: clearing, breaks down red blood cells

and hemoglobin completely

– γ-hemolysis: no hemolysins

Blood Hemolysis

β

α

γ

Catalase

• Enzyme found in most organisms living in the presence of oxygen

• Reduces peroxide, which can be damaging to a cell (free radical)

• First step in the discrimination between:

– Micrococcaceae (catalase positive)

– Streptocaccaceae (catalase negative)

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Catalase

2H2O2 2H2O + O2 catalase

Product of respiration

Damaging for DNA

Aerobic metabolism requires catalase

bubbles

(O2)

Add 3% H2O2 to

bacterial growth

We add this.

Does bacteria make this?Detect bubbles.

Other Gram Positice Cocci Identification Tests

• Bile-Esculin

• Bacitracin, optochin, and Novobiocin sensitivity

• Mannitol + Salts Agar

• Tellurite Agar or Baird Parker Agar

• Pyr Test

Multi Test: Enteropluri-test

• Tube of multiple metabolic tests

– Uses a constant inoculum

– Quick

– Reading can be automated

– Preparations and inconsistencies are normalized