Transcript STAPHYLOCOCCI By - OUR SITE

THERE ARE 3 SPECIES:
 Staph.
aureus: pathogenic and is
responsible for most of the
Staphylococcal infections.
 Staph. epidermidis: members of the
normal flora of skin and mucous
membranes and can cause disease.
 Staph. saprophyticus: non-pathogenic
S. AUREUS CAUSES
Boils, styes , frunculosis , pneumonia,
mastitis, phlebitis, meningitis, urinary
tract infections , bacteremia ,
osteomyelitis and endocarditis .
 Toxigenic diseases in humans as
Scalded skin syndrome in neonates
(exfoliative toxin) and food poisoning
(enterotoxins) .
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Samples: differ according to the clinical
site of infection, may be swabs ,pus,
sputum, C.S.F, blood , urine ,…….) ,
SAMPLES WILL BE EXAMINED BY :
1.Direct film
 Stained with Gram stain for characteristic
morphology, but we cannot differentiate
between S.aureus and other staphylococci
on smears .
 S.aureus is Gram positive cocci arranged in
clusters (The clusters arise because
staphylococci divide in two planes), non
spore forming and non motile.
Staphylococci in pus
Staphylococci in culture
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Gram stain of Staphylococcus aureus in pus
2. CULTURE
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Staphylococci are facultative anaerobes, optimum
temperature: 37C.
It can grow in normal atmospheric Co2 concentration.
Staphylococci can be grown on ordinary media
On blood agar S. aureus cause  hemolysis .
S. aureus can grow on mannitol salt agar producing
(yellow colonies).which is selective for its isolation , as
S.aureus but not other staphylococci ferment mannitol
and salt inhibits other normal flora but not S.aureus
S. aureus produce golden yellow endopigments.
GOLDEN YELLOW ENDOPIGMENTS.
STAPHYLOCOCCUS AUREUS ON
BLOOD AGAR
3. IDENTIFICATION OF COLONIES BY :
a. Film stained by Gram for characteristic
morphology .
 b. Biochemical reactions :
All staphylococci are catalase positive.
(This test differentiate staphylococci from
streptococci which are catalase negative) .
 S. aureus can be identified by being
coagulase and DNAase positive ,and by
mannitol fermentation.
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CATALASE TEST:
Use:
The test is used to differentiate those
bacteria that produce the enzyme catalase,
such as Staphylococci, from non catalase
producing bacteria such as Streptococci.
Principle:
 the test is based on that some organisms
produce catalase enzyme.
 Catalase acts as a catalyst in the breakdown
of hydrogen peroxide to oxygen and water.
CATALASE TEST:
 Method:
 Few drops of 3% hydrogen peroxide solution,
are placed on a clean glass slide; a good
growth of the organism is removed and
immersed in the hydrogen peroxide solution.
The culture should not be more than 24 hours
old and from a blood-free culture medium
such as nutrient agar.
 Interpretation: Immediate bubbling indicates
positive results.
CATALASE TEST:
COAGULASE TEST:
Use:
The test is used to differentiate Staphylococcus
aureus that produce the enzyme coagulase, from
non coagulase producing bacteria such as S.
epidermidis and S. saprophyticus.
Principle:
Coagulase causes plasma to clot by converting
fibrinogen to fibrin.
COAGULASE TEST:
Two types of Coagulase are produced by most strains
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of Staph aureus :
Free coagulase which converts fibrinogen to fibrin by
activating a coagulase reacting factor present in plasma.
Free coagulase is detected by the appearance of a fibrin
clot in the tube test.
Bound coagulase (clumping factor) which converts
fibrinogen directly to fibrin without requiring a coagulase
reacting factor. It can be detected by the clumping of
bacterial cells in the rapid slide test.
COAGULASE TEST:
Slide coagulase test
(to detect bound coagulase):
 a colony of the test organism is emulsified in a
drop of physiological saline on a slide.
 A drop of plasma is added and mixed gently.
 Interpretation: Clumping of the organism within
10 seconds denotes positive results.
COAGULASE TEST:
Tube test (to detect free coagulase):
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0.5 ml of the diluted plasma is pipetted into
tube. 5 drops of the suspention of test
organism is added to the plasma in the tube.
The tube is mixed gently, incubated at 3537˚C.
The tube is examined after 1 hour.
If no clotting occurred, the tube is examined
at 30 minute intervals for up to 6 hours.
During examining the tube for clotting tilt the
tube gently.
COAGULASE TEST:
 Interpretation
of tube test:
 positive result is indicated by the
presence of fibrin clot.
 S. aureus strains produce fibrin clot
within 1 hour of incubation.
POSITIVE TUBE COAGULASE TEST
DNASE AGAR
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Use:
This test is used to differentiate S. aureus which
produce the enzyme DNAase from other staphylococci
which don’t produce the enzyme DNAase.
It is particularly useful if plasma is not available to
perform coagulase test or when the results of
coagulase test are difficult to interpret.
Principle: Deoxyribonuclease enzyme hydrolyses
deoxyribonucleic acid (DNA)
DNASE AGAR
Method:
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The test organism is cultured on medium containing
DNA(DNAase agar ) , after incubation at 37˚C for 1824 hours ,then the plate is flooded with a few
millimeters of 1 mol/liter (3.6%) hydrochloric acid to
precipitate unhydrolysed DNA.
After standing few minutes the plate is examined
against a dark background.
Interpretation: Clear zone around spot culture is
detected in case of positive result.
DNase positive
breakdown of the DNA in the agar. There is a
clear zone
(arrow) around the bacterial growth where
there is no longer any DNA left in the agar to
precipitate out of solution after the HCl was added.
ANTIMICROBIAL SUSCEPTIBILITY
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Staphylococcal
isolates should be
tested to
antimicrobial
susceptibility to
select the effective
drug .
NOVOBIOCIN SENSITIVITY TEST
Principle:
It is done to differentiate between the coagulase
negative Staphylococci
Method:
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The test is done by placing a filter paper disc
impregnated with novobiocin on the surface of the
culture plate after inoculating it with the organism
A zone of inhibition of growth occurs around the
disc if the organism is Staph. epidermidis
No zone of inhibition of growth occurs if the
organism is Staph. saprophyticus
DIAGNOSIS OF STAPHYLOCOCCAL FOOD
POISONING:
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The food remnants, vomitus and stools are cultured
on mannitol-salt agar and the isolated staphylococci
are identified by their biochemical activities,
catalase, coagulase and DNase production.
To trace the source of contamination of food, the
food handlers are tested for the possibility of being
nasal or skin carriers of strains belonging to the
same phage type as the strain causing food
poisoning.
PHAGE TYPING:
1-Plates are inoculated with the test strain, and
dried.
2-A drop of each phage is placed in a marked
square area.
3-After incubation, lysis will occur in the areas of
the corresponding phage type.
PHAGE TYPING
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• Aerobe
Eukaryotic cells
• 25 – 30ºC
• 37ºC
 Saprophyte
• Opportunistic
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1ry pathogen
Shape
Moulds ?
Filaments
Yeast-like
- Candida - Cryptococcus
NonSeptated septated
Hyaline
Yeast ?
Brown
Dimorphic
-Histoplasma
-Sporotrichosis
Fungal Diagnosis
Sample:
•It's advisable to collect as much material
as possible due to scaricity of many fungi in
many specimens.
•The type of the sample collected is
determined according to the suspected site
of infection such as:-skin scrapping – nail
clipping – hair epilation – sputum – blood,
c.s.f.
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Storage and transport: Skin, nail and hair:
They are collected in air-tight containers such as small glass
bottles.
The fungi in test samples can remain viable for weeks or
even months.
They shouldn't be refergirated as the viability of some
species of dermatophytes is affected.
Other specimens should be collected, stored and
transported in a manner similar to that employed for
bacteriological investigation.
If specific fungus is suspected, the lab should be notified as
special media and culture procedures may be needed and
that also will be helpful for the safety of lab personnel.
Direct Ex.:
Value:
• Guide for Culture
•Sometimes diagnositics
1- Unstained:
- KOH
- India ink
2- Stained:
• Gram stain
• Calcofluor white: fluor. M.
• Giemsa: intracellular
• PAS, GMS: in tissue
Culture:
Culture Chs.:
• O2:
• Temp.:
Media:
• SDA
• Cyclohexamide ‘Actidion’ <> Saprophytes
For isolation of ?
• Chloramphenicol / Gentamicin <> Bacteria
- Enriched: Dimorphic f.
Colony identification:
• Chs
- Size, shape,
recto-verso, texture
• Stained film
• BRs:
- Sugar ferm.
- Urease test
Antigen detection: Ag as beta-glucan candida
Serology: Antibodies / limited /
Candida, Cryptococcus / ELISA
Histopathology: Yeast cells, granules
Molecular: PCR, probe
Skin test: ? No diff. () active , past / Candidin
Wood’s light: UV  Hair, skin / not all fungi /
if –ve ?
Animal inoculation:
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Identification of Candida albicans:-
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The colony appears as a rounded white creamy in colour and creamy in
texture.
Film stained by lactophenol blue show budding yeast and filaments
(pseudo-mycelium).
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If stained by Gram stain appear Gram positive.
Germ tube test : culture of the yeast on
serum causes rapid formation of filaments
when incubated at 37°C for 4 hours.
Fermentation of sugars.