1. Bacteria identification : Gram positive cocci

2. Bile-Esculin Enterococcus identification –Esculin  Esculitin + Glucose –esculitin + Fe  Ferric citrate (dark brown) Selective media –Bile added to inhibit gram + bacteria –Enerococci can survive

3. Bile-Esculin - +

4. Novobiocine Sensitivity Kirby Bauer Test Discrimanates S. saprophyticus from other staphylococci S. saprophyticus is the only resistant staphylococci

5. Mannitol + Salt Agar High salinity (7.5%) : enriches staphylococcus Indicator : Phenol Red Carbon Sources : Mannitol and Proteins Detects Mannitol Fermention –Mannitol  acid formation

6. Mannitol + Salt Agar A)Positive for mannitol fermentation B)Negative for mannitol fermentation C)Positive for mannitol fermentation D)No growth

7. Tellurite/Baird Parker Agar Selective Media: –Lithium chloride –1% Pottasium Tellurite Solution Differential Media: –Egg Yolk : lecithinase (clearing) –Pottasium Tellurite : coagulase-positive (blackening)

8. Tellurite/Baird Parker Agar

9. 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

10. PYR Test -+

11. Cellular Aggregation of Gram Positive Cocci Micrococcus & Streptococcus Streptococcus Micrococcus Staphylococcus -Aggregation can be used for distinguishing between genera

12. Diagnostic of Medically important Gram Negative Bacteria

13. Using the Identification Flow Chart http://mysite.science.uottawa.ca/jbasso/microl ab/IDFlowcharts.pdf

14. Diagnostics : Gram Positive Cocci Gram positive cocci Streptococcaceae Catalase - Aerobes & facultative anaerobes Streptococcus Micrococcaceae Catalase + AerobesMicrococcusStaphylococcus

15. 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

16. 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)

17. 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

18. Diagnostics : Gram Positive Rods Gram positive rods Spore formers Aerobes & facultative aerobes BacillusStrict anaerobesClostridium Non spore formers AerobesListeria

19. 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

20. 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

21. Diagnostics : Gram Negative Bacteria Oxydase Pseudomonaceae Neisseriaceae Fermentation of glucose NeisseriaceaePseudomonaceae Enterobacteriaceae Fermentation of lactose McConkey Enterobacter Escherichia Klebsiella Serratia Proteus Salmonella Shigella Morganella

22. 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

23. Immunology

24. 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

25. 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

26. The Antigen Virus=Antigen Epitopes

27. 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)

28. ELISA Used to detect the presence of antigens or antibodies –High sensitivity –Quantitative

29. ELISA Method ENZENZENZ ENZENZ ENZ ENZ ENZ ENZ ENZ Serum (source of Ag) is added to plastic wells Antigen Present Antigen Absent Blocking agent added Ag Ab against Ag is added Wash Detecting Ab added Wash Substrate added

30. Interpretation of Results Serums of patients tested for the presence of HIV –1 o Ab anti-HIV from mouse –2 o Ab anti-Ab of mouse Conclusions –Patients 1 & 3 are positive for HIV –Patient 3 has a higher titer –Patient 2 is negative for HIV 1/2 1/4 1/8 1/16 1/32 1/64 1/128 Control serums + — 1/8

31. 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