Bacteria identification : Gram positive cocci

Bile-Esculin Enterococcus identification Selective media 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 Positive for mannitol fermentation Negative 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)  L-pyrrolidone carboxylic acid + β-napthylamine Detection of reaction: β-napthylamine + p-dimethyl-aminocinnamaldehyde  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/microlab/IDFlowcharts.pdf

Diagnostics : Gram Positive Cocci Streptococcaceae Catalase - Aerobes & facultative anaerobes Streptococcus Micrococcaceae Catalase + Aerobes Micrococcus 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 Spore formers Aerobes & facultative aerobes Bacillus Strict anaerobes Clostridium Non spore formers Aerobes Listeria

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 Bacillus Strict anaerobes Clostridium Non spore formers Aerobes Listeria

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

Diagnostics : Gram Negative Bacteria Oxydase Pseudomonaceae Neisseriaceae Fermentation of glucose Enterobacteriaceae Fermentation of lactose McConkey Enterobacter Escherichia Klebsiella 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: Immunity: 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 Y Y ENZ Y ENZ Y

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 Y Y ENZ Y ENZ Y

Interpretation of Results Assay for Ag of virus X ELISA plate Absorbance readings Standard Patient 1 Patient 2 Patient 3 Neg. control 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 Dilutions 3.8 1.9 0.8 0.4 0.2 0.1 0.04 0.03 0.05 3.6 3.0 1.6 0.12 0.06 Conclusions: Patient 1 is not infected Patients 2 & 3 are infected Patient 3 has an 8X higher Ag load

Interpretation of Results Assay for Ab against virus X Dilutions 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 Standard Patient 1 Patient 2 Patient 3 Neg. control 3.8 1.9 0.8 0.4 0.2 0.1 0.04 0.03 2.5 1.2 0.6 0.3 0.15 0.07 0.05 3.6 3.0 1.6 0.12 0.06 ELISA plate Absorbance readings Conclusions: Patient 1 & 3 are seropositive Patients 2 is seronegative

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