Diagnostics Gram Positive Rods

Classification Gram positive rods Spore formers Aerobes & facultative aerobes BacillusStrict anaerobesClostridium Non spore formers AerobesListeria

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 Gram positive rods Spore formers Aerobes & facultative aerobes BacillusStrict anaerobesClostridium Non spore formers AerobesListeria

Medically Important Bacilli Clostridium –Several pathogenic species Clostridium perfringens –Gas gangrene Clostridium tetani –Tetanus Clostridium botulinum –Botulism Clostridium difficile –Diarrhea Canned food that has not been sterilized properly; paralytic illness prolonged contraction of skeletal muscle fibers, neurotoxin produced by the bacteria

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

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) NeutralAcid

Voges-Proskauer Test VP + = red VP - = Yellow Usual results of MR/VP: MR+/VP-; MR-/VP+ MR-/VP- Reagents VP: butanediol +  -naphthol + KOH + O 2  acetoin NeutralAcid - + produced No acetoin NeutralAcetoin

Urea Utilization Enzyme tested –Urease pH Indicator –Phenol red (turns pink) C O + 2 H 2 O  CO 2 + H 2 O + 2 NH 3  (NH 4 ) 2 CO 3 H2NH2N H2NH2N Urea ammonium carbonate (alkaline) Amino acids Positive Negative

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 additionAfter iodine addition

Caseinase – Milk Agar

Lipase – Spirit Blue

Aerobic Respiration Electron Transport Chain 3 H 2 O H+H+ 2 H + 3 H OH - 2 H + H2OH2O 3 H + + 1/2 O 2 2 e- Fp Fe-S 2 e- Q Cyt b Cyt o NADH + H + FADH 2 interior exterior

Oxidase Test 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) phenylenediamine

Catalase 2H 2 O 2 2H 2 O + O 2  catalase Product of respiration Damaging for DNA Aerobic metabolism requires catalase bubbles (O 2 ) Add 3% H 2 O 2 to bacterial growth We add this. Does bacteria make this? Detect bubbles.

Anaerobic Respiration 2 H + 3 H OH - 3 H 2 O 2 H + NO H 2 O (N = +3) nitrite NO H + (N = +5) nitrate 2 e- Fp Fe-S 2 e- Q Cyt b NADH + H + FADH 2 Nitrate reductase Interior Exterior Final e- acceptor

Nitrate Reductase NO H e-  H 2 O + NO 2 -  NO, N 2 O, NH 2 OH, NH 3, N 2 nitrate nitrite Step 1: Test for nitrite NO sulfanilic acid and alpha naphthylamine  HNO 2 Nitrate is not reduced No Nitrite Yellow Nitrate is reduced Production of Nitrite Red Nitrate is reduced to nitrite Nitrite is reduced No Nitrite Yellow

Nitrate Reductase (Cont’d) NO H e-  H 2 O + NO 2 -  NO, N 2 O, NH 2 OH, NH 3, N 2 nitrate nitrite 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

Diagnostics Gram Positive Cocci

Classification Gram positive cocci Streptococcaceae Catalase - Aerobes & facultative anaerobes Streptococcus Micrococcaceae Catalase + AerobesMicrococcusStaphylococcus

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. aureus S. epidermidis

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