microbiology OSPE (BLOOD)

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staphylococci

arrangement ?

gram ?

catalase ?

coagulase ?

dnase ?

aerobes , anaerobes or facultative anaerobes ?

s. aureus selective media properties ?

Staphylococci Gram positive cocci

arranged in clusters

Catalase: positive

Coagulase: S. aureus (positive) & S. epidermidis, S. saprophyticus (negative)

DNase: S. aureus (positive)

Facultative anaerobes

S. aureus grows in presence of 7.5% sodium chloride and ferment mannitol

samples for staphylococci laboratory diagnosis ?

1- Sample:

Pneumonia [sputum]

Abscess [pus / swab]

Food poisoning [Stool/ vomitus] -

Endocarditis - Bacteremia [Blood]

UTI [Urine]

2-direct film from the sample

identify the sample

Direct film: from the sample

Gram stain of Staphylococcus aureus in pus

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ordinary media for s.aureus —> golden yellow endopigment

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enriched media (blood agar)

s. aureus —-> Beta hemolysis

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selective media mannitol salt agar

s. aureus —> ferment mannitol —-> yellow

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gram positive cocci arranged in clusters

identify the test ?

use ?

principle ?

method ?

Catalase test

Use: Differentiate bacteria that produce catalase enzyme, Staphylococci, from non catalase producing bacteria such as Streptococci.

Principle: • some organisms produce catalase enzyme which breakdown the hydrogen peroxide to oxygen and water.

Method: Few drops of 3% hydrogen peroxide solution, are placed on a clean glass slide; coloney of the organism is removed and immersed in the hydrogen peroxide solution. Interpretation: Immediate bubbling indicates positive results.

identify the test ?

use ?

principle ?

method ?

Coagulase test:

Use: differentiate Staphylococcus aureus that produce coagulase enzyme from non coagulase producing Staphylococcus (S. epidermidis and S. saprophyticus).

Principle: Coagulase causes plasma to clot by converting fibrinogen to fibrin.

Method: Slide coagulase test Tube coagulase test • 0.5 ml of the diluted plasma is put into tube. 5 drops of the suspension of test organism is added to the plasma in the tube. The tube is mixed gently, incubated at 35-37˚C, 1 hour Interpretation of test: • positive result :presence of fibrin clot.

identify the test ?

use ?

principle ?

method ?

DNase agar

Use: differentiate S. aureus which produce the DNAase enzyme from other staphylococci.

Principle: • Deoxyribonuclease enzyme hydrolyses DNA

Method: • The test organism is cultured on medium containing DNA. • Incubation at 37˚C for 18-24 hours, then the plate is flooded with a few millimeters of hydrochloric acid to precipitate unhydrolysed DNA. • The plate is examined against a dark background. • Interpretation: positive result: Clear zone around culture is detected.

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uses ?

one example ?

Typing:

Antimicrobial susceptibility:

- Selection of effective antibiotic.

- Differentiation between species.

Ex. Novobiocin sensitivity sensitive: S. epidermidis resistance: S. saprophyticus

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viral particles direct detection by electron microscope

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virus direct detection through inclusion bodies by inverted microscope

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mechanism ?

how is HIV detected by this method

direct detection of viral antigen by ELISA

color change is detected by spectrophotometer

Direct detection of P24 antigen

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direct detection of the viral antigen through immunofluorescence

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direct detection of the viral nucleic acid through PCR and RT-PCR, probes

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virus isolation through tissue culture (viruses are obligate inrtra-cellular)

identify and compare between the 3 cell lines of virus cell culture

what virus causes CPE

- Cell death & detachment from surface

- Rounding & grape-like cluster formation

- Syncytium ‘giant cell formation’

(polio v.)

(adeno v.)

(measles, mumps)

what are the 6 other ways to detect virus that doesnt produce CPE and describe each (مستحيل يطلب 6 بس تخيل )

1- Haemadsorption: attachment of erythrocytes to the surface of virus infected cells e.g. in mumps, parainfluenza and influenza viruses

2-Haemagglutination: some viruses (influenza) can agglutinate sheep RBCs.

3- Hemagglutination inhibition: It is a test used for detection of specific antibodies that could prevent haemagglutination by the virus.

4- Detection of the virus antigens or its genome in infected cells.

5- Inclusion bodies in some infected cells.

6- Viral growth can be identified serologically by neutralization test.

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mention the mechanism

virus isolation in the embryonated egg

• Some viruses will replicate in the living tissues and membranes of embryonated hen’s eggs, such as influenza virus.

• Egg-adapted strains of influenza virus replicate well in eggs and very high virus titers can be obtained.

identify ?

egg adapted strain of influenza virus

how does this mechanism of virus isolation works

by inoculating a susceptible host (laboratory animal) with infectious material derived from a patient and then observing that animal for signs of disease - suckling mice are used

- limited by virus ‘species specificity’, human viruses may need primates for replication.

identify ?

indirect serologic detection of antiviral antibodies = hist response

by IF, ELISA, CF, WB

(detection of IgM / or at least 4 fold increase of IgG)

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how are the antibodies detected in HIV

• Serology: Screening of blood for antibodies to HIV is first done by ELISA which is very sensitive test.

• Positive results should be confirmed using more specific tests like Western Blot (WB) or immunofluorsence (IF)