1. Bacteria identification : Gram positive cocci

2. 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

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 Positive for mannitol fermentation
Negative for mannitol fermentation
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

14. Diagnostics : Gram Positive Cocci
Streptococcaceae
Catalase -
Aerobes & facultative anaerobes
Streptococcus
Micrococcaceae
Catalase +
Aerobes
Micrococcus
Staphylococcus

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
Spore formers
Aerobes & facultative aerobes
Bacillus
Strict anaerobes
Clostridium
Non spore formers
Aerobes
Listeria

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
Bacillus
Strict anaerobes
Clostridium
Non spore formers
Aerobes
Listeria

20. 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

21. Diagnostics : Gram Negative Bacteria
Oxydase
Pseudomonaceae
Neisseriaceae
Fermentation of glucose
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. 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

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
Epitopes
Virus=Antigen

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 –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

30. 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

31. Interpretation of Results
Assay for Ag of virus X
ELISA plate Absorbance readings
Standard
Patient 1
Patient 2
Patient 3
Neg. control
1/ / / / / /64 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

32. Interpretation of Results
Assay for Ab against virus X
Dilutions
1/ / / / / /64 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

33. 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