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Bacteria identification : Gram positive cocci Bile-Esculin • Enterococcus identification – Esculin Esculitin + Glucose – esculitin + Fe Ferric citrate (dark brown) • Selective media – Bile added to inhibit gram + bacteria – Enerococci can survive Bile-Esculin - + Novobiocine Sensitivity • Kirby Bauer Test • Discrimanates S. saprophyticus from other staphylococci • S. saprophyticus is the only resistant staphylococci Mannitol + Salt Agar • • • • High salinity (7.5%) : enriches staphylococcus Indicator : Phenol Red Carbon Sources : Mannitol and Proteins Detects Mannitol Fermention – Mannitol acid formation Mannitol + Salt Agar A) B) C) D) Positive for mannitol fermentation Negative for mannitol fermentation Positive for mannitol fermentation No growth Tellurite/Baird Parker Agar • Selective Media: – Lithium chloride – 1% Pottasium Tellurite Solution • Differential Media: – Egg Yolk : lecithinase (clearing) – Pottasium Tellurite : coagulase-positive (blackening) Tellurite/Baird Parker Agar PYR Test • Detected Enzyme : Pyrrolidonyl peptidase – L-pyrrolidonyl-β-napthylamide (PYR) Lpyrrolidone carboxylic acid + β-napthylamine • Detection of reaction: – β-napthylamine + p-dimethylaminocinnamaldehyde pink precipitate PYR Test - + Cellular Aggregation of Gram Positive Cocci Micrococcus & Streptococcus Streptococcus Micrococcus Staphylococcus -Aggregation can be used for distinguishing between genera Diagnostic of Medically important Gram Negative Bacteria Using the Identification Flow Chart http://mysite.science.uottawa.ca/jbasso/microl ab/IDFlowcharts.pdf Diagnostics : Gram Positive Cocci 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 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 Diagnostics : Gram Positive Rods Aerobes & facultative aerobes Bacillus Strict anaerobes Clostridium Aerobes Listeria Spore formers Gram positive rods Non spore formers 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 Diagnostics : Gram Negative Bacteria Neisseriaceae Pseudomonaceae Neisseriaceae Fermentation of glucose Pseudomonaceae Oxydase Enterobacter Escherichia Klebsiella Enterobacteriaceae Fermentation of lactose McConkey Serratia Proteus Salmonella Shigella Morganella Representative Gram Negative Bacteria • Gram-negative Cocci – Neisseria gonorrhoea – Neisseria meningitis • Gram-negative rods – Enterobacteriaceae family • Escherichia, Enterobacter, Salmonella, Shigella, Klebsiella, Proteus, Morganella – Pseudomonaceae family • Pseudomonas Immunology Immunology • Purpose of the immune system: – Discriminate self from non-self • Non-self –Antigens • Immunity: – All mechanisms used by the host to protect itself and fight non-self Non-Self - Antigens • Anything that can react with the participants of the immune system – Ex. antibodies • Epitope: Characteristic of the antigen which allows its recognition as being non-self – Ex. Lipids, proteins, lipopolysaccharides The Antigen Epitopes Virus=Antigen Immunological Diagnostic Methods • Determine the presence of an antigen: • An organism • A protein • A toxin • An antibody – ELISA method to determine quantity – Immunochromatography (Rapid tests) ELISA • Used to detect the presence of antigens or antibodies – High sensitivity – Quantitative ELISA –Antigen Detection Serum (source of Ag) Added to wells Blocking agent added Ab against Ag added Wash Detecting Ab added Wash Substrate added Antigen Present Antigen Absent ENZ ENZ ENZ ENZ ENZ ENZ ENZ ENZ 29 ELISA –Antibody Detection Target Ag for Ab to be detected added to wells Blocking agent added Test serum added Wash Detecting Ab added Wash Substrate added Ab Present Ab Absent ENZ ENZ ENZ ENZ ENZ ENZ ENZ ENZ 30 Interpretation of Results Assay for Ag of virus X Dilutions Standard Patient 1 Patient 2 Patient 3 Neg. control ELISA plate 1/2 1/4 3.8 1.9 0.1 1/8 1/16 1/32 1/64 1/128 1/256 0.8 0.4 0.2 0.1 0.04 0.03 0.05 0.03 0.03 0.03 0.03 0.03 0.03 0.8 0.4 0.2 0.1 0.05 0.03 0.03 0.03 3.6 3.0 1.6 0.8 0.4 0.2 0.1 0.05 0.12 0.06 0.03 0.03 0.03 0.03 0.03 0.03 Absorbance readings Conclusions: Patient 1 is not infected Patients 2 & 3 are infected Patient 3 has an 8X higher Ag load 31 Interpretation of Results Assay for Ab against virus X Dilutions Standard Patient 1 Patient 2 Patient 3 Neg. control ELISA plate 1/2 1/4 3.8 1.9 2.5 1/8 1/16 1/32 1/64 1/128 1/256 0.8 0.4 0.2 0.1 0.04 0.03 1.2 0.6 0.3 0.15 0.07 0.03 0.03 0.1 0.05 0.03 0.03 0.03 0.03 0.03 0.03 3.6 3.0 1.6 0.8 0.4 0.2 0.1 0.05 0.12 0.06 0.03 0.03 0.03 0.03 0.03 0.03 Absorbance readings Conclusions: Patient 1 & 3 are seropositive Patients 2 is seronegative 32 Next Week • Final Quiz • Practical Exam: – Determine which partner will complete the exam fist – Make sure everything on the exam is clear, you can ask any question concerning the methods required for the practical exam