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Diagnostics Gram Positive Rods Classification Aerobes & facultative aerobes Bacillus Strict anaerobes Clostridium Aerobes Listeria Spore formers Gram positive rods Non spore formers Characteristics of Spore Forming Rods • All are large Gram positive rods • Make endospores • Mainly found in soil, water and dust • Highly diverse nutritional requirements – Use simple and complex carbon sources – Their spores allow them to resist extreme conditions Endospore Morphology • Location: – Terminal (a, d, e) – Subterminal (b) – Central (c, f). • Shape: – Circular (b, d) – Ellipsoid (a, c, e, f) • Spore diameter: – Non-deforming (a, b, c) – Deforming (d, e, f). Medically Important Bacilli • Bacillus – Mostly harmless – A few opportunistic species • Bacillus cereus & Bacillus subtilis – Food poisoning – One pathogenic species • Bacillus anthracis – Anthrax Aerobes & facultative aerobes Bacillus Strict anaerobes Clostridium Aerobes Listeria Spore formers Gram positive rods Non spore formers Medically Important Bacilli • Clostridium – Several pathogenic species • Clostridium perfringens – Gas gangrene • Clostridium tetani – Tetanus • Clostridium botulinum – Botulism • Clostridium difficile – Diarrhea prolonged contraction of skeletal muscle fibers, neurotoxin produced by the bacteria Canned food that has not been sterilized properly; paralytic illness Identification: Metabolic Tests • Phenol red broth – Simple Carbon source: • Peptone (protein amino acids) • Desired sugar added – pH indicator • Phenol red – Yellow acid pH – Orange neutral pH – Red alkaline pH Phenol Red Broths • Carbon utilization: – Sugar • Acid reaction (yellow) • or neutral (original) – Protein • Alkaline reaction (red) 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 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 Diagnostics Gram Positive Cocci Classification Streptococcaceae Catalase - Aerobes & facultative anaerobes Gram positive cocci Streptococcus Micrococcus Micrococcaceae Catalase + Aerobes Staphylococcus Characteristics of Gram Positive Cocci • All are non sporulating • Mainly found amongst the natural flora of humans and animals • Fastidious (‘picky’) nutritional requirements – Use simple carbon sources Cellular Aggregation of Gram Positive Cocci Micrococcus & Streptococcus Streptococcus Micrococcus Staphylococcus Gram Positive Cocci of Medical Importance • Micrococcaceae – Staphylococcus aureus • Causes several types of infections, food infections and toxic shock (skin and respiratory tract) – Staphylococcus epidermidis • Cause opportunistic infections (catheters with biofilms) – Staphylococcus saprophyticus • Major cause of cystitis in women (bladder infection) Gram Positive Cocci of Medical Importance • Streptococcaceae – Streptococcus pyogenes • Strep throat and flesh eating disease – Streptococcus agalactiae • Genital infections – Streptococcus mutans • Endocarditis – Streptococcus pneumonia • Otitis, meningitis, and pneumonia – Enterococcus spp. • Opportunistic infections Identification: Metabolic Tests • Microccocus Vs Staphylococcus • Oxidase test – Micrococci are + – Staphylococci are – • Bacitracin (antibiotic) – Micrococci are sensitive – Staphylococci are resistant Identification: Metabolic Tests • Differentitation of Staphylococcus species • Coagulase test – Coagulase positive staphylococci » S. aureus – Coagulase negative » All the other Staphylococci • Mannitol fermentation – S. aureus and some S. saprophyticus are positive – S. epidermidis is negative S. epidermidis S. aureus Identification: Metabolic Tests • Differentiation of Streptoccocus species • Serological grouping according to Lancefield – Based on the type of carbohydrate in their cell wall » 8 groups (A-H and K-U) » Group A: β-hemolytic - Streptococcus pyogenes » Group B: β-hemolytic - S. agalactiae » Group C: α or γ hemolytic- S. viridans » Group D: γ –hemolytic – Enterococcus » Does not belong to any Lancfield group » α hemolytic- S. pneumoniae and S. mutans Blood Hemolysis • Blood agar: – Discrimination according to hemolysis patterns • Alpha hemolysis – Incomplete hemolysis • Beta hemolysis- Complete hemolysis • Gamma hemolysis – No hemolysis Identification: Metabolic Tests • Identification of Streptoccocus pneumoniae – Bile solubilization • Strep. pneumoniae is positive • Other Strep. are negative • Identification of Enterococcus – Bile-esculin test • Enterococcus is positive • Other Strep. are negative