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) Positive for mannitol fermentationB) Negative for mannitol fermentationC) Positive for mannitol fermentationD) 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 distinguishingbetween 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

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

Gram positive rods

Spore formers

Aerobes & facultative

aerobesBacillus

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

aerobesBacillus

Strict anaerobes Clostridium

Non spore formers Aerobes Listeria

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

Diagnostics : Gram Negative Bacteria

Oxydase

Pseudomonaceae

NeisseriaceaeFermentation

of glucose

Neisseriaceae

Pseudomonaceae

EnterobacteriaceaeFermentation

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:– 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

Virus=Antigen

Epitopes

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

ELISAMethod

ENZ ENZENZ ENZ ENZ

ENZENZ

ENZ ENZ ENZ

Serum (source of Ag) is added to plastic wells

Antigen Present Antigen Absent

Blocking agent addedAb against Ag is addedWashDetecting Ab addedWashSubstrate added

Interpretation of Results• Serums of patients tested for the presence of HIV

– 1o Ab anti-HIV from mouse– 2o 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 1/128Control serums + —

1/8

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