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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
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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
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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