6/28/2016. Growth Media and Metabolism. Complex Media. Defined Media. Made from complex and rich ingredients

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Estella Jones
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1 Growth Media and Metabolism Complex Media Made from complex and rich ingredients Ex. Soya protein extracts Milk protein extracts Blood products Tomato juice, etc. Exact chemical composition unknown Can be selective and/or differential 2 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 3 1

2 Selective Media Contains compounds which inhibit or kill the unwanted organisms Ex. Medium containing penicillin only allows the growth of penicillin resistant microorganisms 4 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 5 Nutrition Macronutrients C,H,N,O,P,S 2

3 Carbon Required for the synthesis of all organic compounds : Carbohydrates Lipids Proteins Nucleic acids Carbon Sources Organic Monosaccharides Disaccharides Polysaccharides Proteins Lipids Nucleic acids Phenols, Etc. Inorganic CO 2 CO Phosphorous Required for the synthesis of : Nucleic acids Phospholipids ATP Used as a buffer; control of ph Sources: Organic and inorganic The inorganic form is the most used 3

4 Nitrogen Required for the synthesis of: Amino acids Nucleic acids Peptidoglycan Sources: Organic: Amino acids Inorganic: NH 3, NO 3, & N 2 Sulfur Required for the synthesis: Amino acids (Cysteine/Methionine) Vitamins (thiamine and biotin) Sources: Organic: Amino acids Cystein and methionine Inorganic: S, SO 4 Hydrogen and Oxygen Required for the synthesis of organics!! Carbohydrates Lipids Proteins Nucleic acids Sources: Organic: Organic carbon Inorganic: H 2 (Methanogens only) 4

5 Nutritional Classification Carbon sources Heterotrophs: Preformed organic compounds Autotrophs: Inorganic molecules CO 2 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 Autotrophs photolithotrophs Heterotrophs photoorganotrophes Autotrophs chemolithotrophs Heterotrophs chemoorganotrophs 5

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?

6 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 Sources of C,H,N,O,P,S? Type of media? Indicators? Selective agents? Allow growth of what bacteria? Possible reactions? Peptone - 17 g Proteose peptone - 3 g Lactose - 10 g Bile salts g Sodium chloride - 5 g Neutral red g Crystal Violet g Agar g Environmental Parameters Oxygen availability ph Temperature 18 6

7 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 7

Bacterial Metabolism (Cont d) Respiration Can occur aerobically or anaerobically Both use an inorganic final electron acceptor Aerobic respiration uses O 2 Anaerobic respiration uses an inorganic

8 Bacterial Metabolism (Cont d) Respiration Can occur aerobically or anaerobically Both use an inorganic final electron acceptor Aerobic respiration uses O 2 Anaerobic respiration uses an inorganic compound other than O 2 (Ex. NO3-) End product H 2 O 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 (CO 2 ) A few generate only acid or gas 8

Identification: Metabolic Tests Phenol red broth Allows determination of carbon source preferred and metabolism (Oxidation or fermentation) Contains simple carbon sources: Peptone (protein amino

9 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 + CO 2 Fermentation with accumulation of neutral products Glucose pyruvate acetoin 2 butanediol + CO 2 9

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.

10 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 + O 2 acetoin Usual results of MR/VP: MR+/VP-; MR-/VP+ MR-/VP- VP + = red VP - = Yellow Neutral - + Acid Acid produced No acetoin Neutral Acetoin 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 10

IMViC: Indole Test Principal Some microorganisms can metabolize tryptophan by the tryptophanase Tryptophane Tryptophanase Indole + acide Pyurvic + NH 3 Kovac s reagent Red color IMViC Test Methyl

11 IMViC: Indole Test Principal Some microorganisms can metabolize tryptophan by the tryptophanase Tryptophane Tryptophanase Indole + acide Pyurvic + NH 3 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 + CO 2 Voges Proskauer Test Fermentation with accumulation of butanediol Glucose pyruvate acetoine 2 butanediol + CO 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 11

12 TSI Three Sugars and Iron Three sugars Glucose (limiting) Sucrose Lactose Proteins Cysteine Indicator Phenol red SIM H 2 S, Indole and Motility Semi-solid medium Allows to visualize motility Cystein metabolism Cysteine H 2 S; H 2 S+ FeSO 4 Black precipitate Tryptophan metabolism (A) Tryptophan Indole + NH 4 + Pyruvate (B) Indole + Kovac reagent Red Non inoculated Non-motile Indole + - H 2 S and motile 12

Negative Urea Utilization Enzyme tested Urease Positive ph Indicator Phenol red

Urea carbonate Amino acids (alkaline) Complex Carbon Utilization Too large to be

smaller units Polysaccharides Starch (amylase) Lipids (lipase) Tributyrin Proteins

13 Negative Urea Utilization Enzyme tested Urease Positive ph Indicator Phenol red (turns pink) H 2 N C O + 2 H 2 O CO 2 + H 2 O + 2 NH 3 (NH 4 ) 2 CO 3 H 2 N ammonium Urea carbonate Amino acids (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 13

Q 2 e- 2 e- Fe-S 2 e- Fp interior NADH + H + FADH 2 Cyt b

14 Caseinase Milk Agar Lipase Spirit Blue Aerobic Respiration Electron Transport Chain 2 H + exterior 2 H + Q 2 e- 2 e- Fe-S 2 e- Fp interior NADH + H + FADH 2 Cyt b 2 H + 2 e- 3 H OH - 3 H 2 O H + Cyt o 3 H + + 1/2 O 2 H 2 O 14

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

2H 2 O 2 catalase 2H 2 O + O 2 Product of respiration Damaging for DNA Add 3% H 2 O 2 to bacterial growth bubbles (O 2 ) Aerobic metabolism requires catalase Anaerobic Respiration 2 H +

15 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 We add this. Does bacteria make this? Detect bubbles. 2H 2 O 2 catalase 2H 2 O + O 2 Product of respiration Damaging for DNA Add 3% H 2 O 2 to bacterial growth bubbles (O 2 ) Aerobic metabolism requires catalase Anaerobic Respiration 2 H + Exterior 2 H + Q 2 e- 2 e- Fe-S 2 e- Fp NADH + H + FADH 2 Interior 2 e- Cyt b Nitrate reductase 2 H + 3 H OH - 3 H 2 O NO H + (N = +5) nitrate Final e- acceptor NO H 2 O (N = +3) nitrite 15

Nitrate Reductase NO - 3 + 2 H + + 2 e- H 2 O + NO - 2 NO, N 2 O, nitrate nitrite NH 2 OH, NH 3, N 2 Step 1: Test for nitrite NO 2 - + sulfanilic acid and alpha naphthylamine HNO 2 Nitrate is reduced

16 Nitrate Reductase NO H e- H 2 O + NO - 2 NO, N 2 O, nitrate nitrite NH 2 OH, NH 3, N 2 Step 1: Test for nitrite NO sulfanilic acid and alpha naphthylamine HNO 2 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) NO H e- H 2 O + NO - 2 NO, N 2 O, nitrate nitrite NH 2 OH, NH 3, N 2 Step 2: Test for the presence of nitrate NO Zn (s) NO 2 - Nitrate is present Reduction to Nitrite Red Nitrate is absent Nitrite was reduced Yellow Multi Test: Enteropluri-test Tube of multiple metabolic tests Uses a constant inoculum Quick Reading can be automated Preparations and inconsistencies are normalized 16

Gram-negative rods. Enterobacteriaceae. Biochemical Reactions. Manal AL khulaifi

Gram-negative rods. Enterobacteriaceae. Biochemical Reactions. Manal AL khulaifi Gram-negative rods Enterobacteriaceae Biochemical Reactions Bacteria Gram positive Gram negative Cocci Bacilli Cocci Rods Characters of Enterobacteriaceae All Enterobacteriaciae Gram-negative rods Reduce