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?

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)

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 Yellow (acid) + gas = Fermentation of sugar Yellow (acid) no gas = Fermentaion of sugar Orange (neutral) no gas = Oxidation of sugar Red (alkaline) no gas = Oxidation of proteins Uninoculated

TSI — Three Sugars and Iron 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: Usual results of MR/VP: butanediol + -naphthol + KOH + O2  acetoin VP + = red VP - = Yellow - + Usual results of MR/VP: MR+/VP-; MR-/VP+ MR-/VP- Neutral Acid Acid produced No acetoin Neutral Acetoin

IMViC: Indole Test Principal Some microorganisms can metabolize tryptophane by the tryptophanase Tryptophanase Tryptophane 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 pH Indicator Urease Phenol red (turns pink) Positive Negative C O + 2 H2O  CO2 + H2O + 2 NH3  (NH4)2CO3 H2N Urea ammonium carbonate (alkaline) Amino acids

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-motile Non inoculated Indole + - H2S and motile

Anaerobic Respiration : Nitrate Reductase 2 H+ 3 H+ + 3 OH - 3 H2O NO2- + H2O (N = +3) nitrite NO3- + 2 H+ (N = +5) nitrate 2 e- Fp Fe-S Q Cyt b NADH + H+ FADH2 Nitrate reductase Interior Exterior Final e- acceptor

Anaerobic Respiration : Nitrate Reductase (con’t) NO3- + 2 H+ + 2 e-  H2O + NO2-  NO, N2O, NH2OH, NH3, N2 nitrate nitrite Step 1: Test for nitrite NO2- + sulfanilic acid and alpha naphthylamine  HNO2 Nitrate is not reduced No Nitrite Yellow Nitrate is reduced Production of Nitrite Red Nitrate is reduced to nitrite Nitrite is reduced No Nitrite

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 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 3 H2O H+ 2 H+ 3 H+ + 3 OH- H2O 3 H+ + 1/2 O2 2 e- Fp Fe-S Q Cyt b Cyt o NADH + H+ FADH2 interior exterior

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 2H2O2 2H2O + O2  catalase Product of respiration Does bacteria make this? Detect bubbles. 2H2O2 2H2O + O2  catalase Product of respiration Damaging for DNA We add this. 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