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Bacteria identification :
Media
What you have to know about the
media
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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
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Sources of C,H,N,O,P,S?
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Type of media?
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Indicators?
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Selective agents?
Allow growth of what bacteria? •
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• Possible reactions?
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
Identification :
Complex Carbon Sources
Complex Carbon Utilization
• Too large to be transported inside
• Requires exocellular enzymes for the external
degradation into smaller units
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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)
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
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
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
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 of
determinations which are collectively called the
IMViC reaction series
– The IMViC reaction series allows the
discrimination
of
bacteria
of
the
Enterobacteriaceae 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
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
Reagents VP:
butanediol + -naphthol + KOH + O2  acetoin
VP + = red
VP - = Yellow
Usual results of MR/VP:
MR+/VP-; MR-/VP+ MR-/VPAcid
produced
No
acetoin
Neutral Acetoin
- +
Neutral
Acid
IMViC: Indole Test
• Principal
– Some microorganisms can metabolize
tryptophane by the tryptophanase
Tryptophane
Tryptophanase
Indole + acide Pyurvic + NH3
Kovac’s reagent
Red color
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
Negative
Positive
• pH Indicator
– Phenol red (turns pink)
H2 N
H2 N
C O + 2 H2O  CO2 + H2O + 2 NH3  (NH4)2CO3
Urea
Amino acids
ammonium
carbonate
(alkaline)
Urea Utilization – Phenol Red
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
Phenylalanine Slants
A
B
A: Positive for phenylpyruvic acid
B: Negative for phenylpyruvic acid
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
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
CysteineH2S; H2S+ FeSO4 Black precipitate
• Tryptophan metabolism
(A) Tryptophan Indole + NH4 + Pyruvate
(B) Indole + Kovac reagent Red
Non inoculated
Non-motile
+
H2S and motile
Indole
-
Anaerobic Respiration :
Nitrate Reductase
2 H+
2 e-
Exterior
Fp
Fe-S
2 H+
Interior
2 e-
Q
NADH + H+
FADH2
2 e-
Cyt
b
2 H+
3 H+ + 3 OH -
2 e-
Nitrate
reductase
3 H2O
NO3- + 2 H+ (N = +5) nitrate
Final e- acceptor
NO2- + H2O (N = +3) nitrite
Anaerobic Respiration :
Nitrate Reductase (con’t)
NO3- + 2 H+ + 2 e-  H2O +
nitrate
NO2-
nitrite

NO, N2O,
NH2OH,
NH3, N2
Step 1: Test for nitrite
NO2- + sulfanilic acid and alpha naphthylamine  HNO2
Nitrate is reduced
Production of Nitrite
Red
Nitrate is reduced to nitrite
Nitrite is reduced
No Nitrite
Yellow
Nitrate is not reduced
No Nitrite
Yellow
Anaerobic Respiration :
Nitrate Reductase (con’t)
NO3- + 2 H+ + 2 e-  H2O +
nitrate
NO2-
nitrite

NO, N2O,
NH2OH,
NH3, N2
Step 2: Test for the presence of nitrate
NO3- + Zn (s)  NO2-
Nitrate is present
Reduction to Nitrite
Red
Nitrate is absent
Nitrite was reduced
Yellow
Oxidase Test : Aerobic Respiration
Electron Transport Chain
2 H+
2 e-
Fe-S
2 H+
exterior
Fp
interior
2 e-
Q
NADH + H+
FADH2
2 e-
Cyt b
3 H+ + 3 OH-
2 e-
H+
2 H+
Cyt o
3 H+ + 1/2 O2
H2O
3 H2O
Oxidase Test : Aerobic Respiration
phenylenediamine
• 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 (reduced
state) will turn blue (oxidized state)
Differential Tests for the Identification
of Gram Positive Cocci
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)
Catalase
Does bacteria make this?
2H2O2
We add this.
catalase
2H2O + O2 
Detect bubbles.
Product of respiration
Damaging for DNA
Add 3% H2O2 to
bacterial growth
bubbles
(O2)
Aerobic metabolism requires catalase
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
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Uses a constant inoculum
Quick
Reading can be automated
Preparations and inconsistencies are normalized