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Growth Media and Metabolism
Complex Media
• Made from complex and rich ingredients
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Ex. Soya protein extracts
Milk protein extracts
Blood products
Tomato juice, etc.
• Exact chemical composition unknown
• Can be selective and/or differential
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Defined Media
• Known chemical composition
May contain up to 80 different ingredients
– May be quite simple
– Allows the growth of a restricted number of
microorganisms
– Highly variable composition as a function of the
microorganism
–
• May be selective and/or differential
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Selective Media
• Contains compounds which inhibit or kill the
unwanted organisms
– Ex. Medium containing penicillin only allows the
growth of penicillin resistant microorganisms
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Differential Media
• Allows the discrimination of different species
• Often contain pH indicators
– Allows the discrimination of different metabolisms
Production of alkaline
products turns the
medium red
Production of acid
products turns the
medium yellow
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Nutrition
• Macronutrients
– C,H,N,O,P,S
Carbon
• Required for the synthesis of all organic
compounds :
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Carbohydrates
Lipids
Proteins
Nucleic acids
Carbon Sources
• Organic
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Monosaccharides
Disaccharides
Polysaccharides
Proteins
Lipids
Nucleic acids
Phenols, Etc.
• Inorganic
– CO2
– CO
Phosphorous
• Required for the synthesis of :
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Nucleic acids
Phospholipids
ATP
Used as a buffer; control of pH
• Sources:
– Organic and inorganic
• The inorganic form is the most used
Nitrogen
• Required for the synthesis of:
– Amino acids
– Nucleic acids
– Peptidoglycan
• Sources:
– Organic: Amino acids
– Inorganic: NH3, NO3, & N2
Sulfur
• Required for the synthesis:
– Amino acids (Cysteine/Methionine)
– Vitamins (thiamine and biotin)
• Sources:
– Organic: Amino acids
• Cystein and methionine
– Inorganic:
• S, SO4
Hydrogen and Oxygen
• Required for the synthesis of organics!!
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Carbohydrates
Lipids
Proteins
Nucleic acids
• Sources:
– Organic:
• Organic carbon
– Inorganic:
• H2 (Methanogens only)
Nutritional Classification
• Carbon sources
– Heterotrophs:
• Preformed organic compounds
– Autotrophs:
• Inorganic molecules
– CO2 and CO
Nutritional Classification (Cont’d)
• Energy sources
– Phototrophs:
• Light
– Chemotrophs:
• Oxidation of organic and inorganic compounds
• Source of e– Organotrophs:
• Reduced organic molecules
– Lithotrophs:
• Reduced inorganic molecules
Nutritional Types
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Autotrophs photolithotrophs
Heterotrophs photoorganotrophes
Autotrophs chemolithotrophs
Heterotrophs chemoorganotrophs
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
Environmental Parameters
• Oxygen availability
• pH
• Temperature
18
Oxygen Requirements
• Aerobic:
– Absolute need of oxygen to survive
– Used as a final electron acceptor
– Used by bacteria that carry out an oxidative or
aerobic respiratory metabolism
• Microaerophilic:
– Absolute need for low concentrations of oxygen
– High concentrations are detrimental
Oxygen Requirements (Cont’d)
• Anaerobic/Aerotolerant:
– Oxygen is not required for growth or survival but is
tolerated
• Facultative anaerobes:
– Facultative oxygen requirement
– May use oxygen or not
– Possesses an oxygen dependant and oxygen
independent metabolism
• Strict or obligate anaerobic :
– Oxygen is neither used nor tolerated; cannot survive
in the presence of oxygen
Bacterial Metabolism
• Most microorganisms initially channel the
carbon source through a glycolytic pathway →
pyruvate
• Different pathways are used to metabolize
pyryvate
– Respiration/Oxidation
– Fermentation
Bacterial Metabolism (Cont’d)
• Respiration
– Can occur aerobically or anaerobically
– Both use an inorganic final electron acceptor
• Aerobic respiration uses O2
• Anaerobic respiration uses an inorganic compound
other than O2 (Ex. NO3-)
– End product H2O
Bacterial Metabolism (Cont’d)
• Fermentation
– Pyruvate is metabolized anaerobically
– Makes use of an organic electron acceptor
– Many diverse electron acceptors used by different
microorganisms
• Different end products generated as a function of final
electron acceptor used
– Very useful for microbial identification
Fermentations
• By-products:
– Most generate acid + gas (CO2)
– A few generate only acid or gas
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 - Fermentation
– Orange - neutral pH - Oxidation
– Red - alkaline pH - Oxidation
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
Discrimination of Glucose
Fermentation
• Fermentation with acid accumulation:
– Glucose  pyruvate  lactic and/or acetic acid + CO2
• Fermentation with accumulation of neutral
products
– Glucose  pyruvate  acetoin  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 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 reactions allow the discrimination of
bacteria of the Enterobacteriaceae family
IMViC: Indole Test
• Principal
– Some microorganisms can metabolize tryptophan
by the tryptophanase
Tryptophane
Tryptophanase
Indole + acide Pyurvic + NH3
Kovac’s reagent
Red color
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
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
TSI — Three Sugars and Iron
• Three sugars
– Glucose (limiting)
– Sucrose
– Lactose
• Proteins
– Cysteine
• Indicator
– Phenol red
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
-
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)
Complex Carbon Utilization
• Too large to be transported inside
• Requires exocellular enzymes for the external
degradation into smaller units
– Polysaccharides
• Starch (amylase)
– Lipids (lipase)
• Tributyrin
– Proteins (protease)
• Casein (caseinase)
Amylase – Starch Agar
Before iodine addition
After iodine addition
Caseinase – Milk Agar
Lipase – Spirit Blue
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
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)
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
Anaerobic Respiration
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
Nitrate Reductase
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
Nitrate Reductase (Cont’d)
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
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