طريقة تخطيط الاطباق Streak Plate Method التخطيط المتعامد.
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Transcript of طريقة تخطيط الاطباق Streak Plate Method التخطيط المتعامد.
Gram StainOne drop of water
+Loop of Bacteria
clean glass slide
spread as a thin layer 1-allowed to air dry
2-fixed
smear
2 .Saturate the smear with crystal violet for 1 minute.3 .Rinse the slide gently with water.
4 .Saturate the smear with iodine for 30 Second.5 .Rinse the slide gently with water.
6 .Decolorize (alcohol)7 .Rinse the slide gently with water.
8 .Saturate the smear with safranin for 1 minute.9 .Rinse the slide gently with water.
10 .Carefully blot the slide dry with bibulous paper.11 .Observe the slide under the microscope
Gram staining ResultsGram staining ResultsGram positive bacteria will stain purple.
Gram negative bacteria will stain red/pink.
Gram positive bacteria have a thick cell wall made of peptidoglycan, whereas gram negative bacteria have thin layer of peptidoglycan.
2 -Cell shape and arrangement:
Cocci: single- Diplo – Grape like-Strepto
Cells Description under the microscope
Gram-Positive BacteriaGram-Positive Bacteria
I- Gram Positive bacteria
A- Gram positive cocci B- Gram positive rods
Spore-formingNon spore-formingCorynebacterium
AerobicBacillus anthracis
AnaerobicClostridium
The catalase test is distinguished streptococci from The catalase test is distinguished streptococci from staphylococcistaphylococci
flood culture with drops of 3% H2O2 flood culture with drops of 3% H2O2
Catalase-positive cultures bubble at onceCatalase-positive cultures bubble at once
The test should not be done on blood agar because The test should not be done on blood agar because blood itself will produce bubbles blood itself will produce bubbles
H2O2 H2O + O2 (gas, ↑)Staphylococci
Catalase
Catalase test
Species of Species of SatphylococciSatphylococci
Three species of staphyloccoci have medical Three species of staphyloccoci have medical importance:importance:– S. aureusS. aureus:: P Pathogenicathogenic & commensally found in & commensally found in
nose (nares)nose (nares) S. epidermidisS. epidermidis:: non pathogenicnon pathogenic & common & common
commensals in nares & skincommensals in nares & skin S. saprophyticus:S. saprophyticus: Cause UTI in female & Cause UTI in female &
occasionally occasionally commensallycommensally found skin found skin
Coagulase TestCoagulase TestPrinciple:Principle:
This test used to differentiate between S. aureus (CPS) & other Staphylococcus species (CNS)
Coagulase test
Coagulase PositiveStaphylococus aureus
Coagulase-NegativeS. epidermidis & S. saprophyticus
Fibrinogen (Plasma)
Coagulase Fibrin )Clot(
StaphylococciStaphylococci
General characters:General characters:– Gram Positive CocciGram Positive Cocci– Grape-likeGrape-like– Non MotileNon Motile– Non Spore FormingNon Spore Forming– Non CapsulatedNon Capsulated– Non FastidiousNon Fastidious– Facultative AnaerobesFacultative Anaerobes– FermentativeFermentative– Catalase positiveCatalase positive
Laboratory diagnosis of Laboratory diagnosis of StaphylococcusStaphylococcus
Gram Stain:Gram Stain:– Gram Positive Cocci, arranged in clusterGram Positive Cocci, arranged in cluster
Culture: Culture: 1-1- Blood agarBlood agar , 2- , 2- MSA mediaMSA media
– Blood agarBlood agar (Non-Selective Media) (Non-Selective Media)Coagulase Positive Staphylococci are Pigmented & hemolyticCoagulase Positive Staphylococci are Pigmented & hemolytic
Coagulase Negative Staphylococci are non-pigmented & non-Coagulase Negative Staphylococci are non-pigmented & non-hemolytichemolytic
MSAMSA is selective differential medium for staphylococci is selective differential medium for staphylococci– It contains: NaCl (7.5%), Mannitol, & Phenol RedIt contains: NaCl (7.5%), Mannitol, & Phenol Red– The cause of The cause of selectivityselectivity due to presence of high salt due to presence of high salt
concentrationconcentration– The cause of The cause of differentialdifferential because contains mannitol because contains mannitol
(sugar) and phenol red (pH indicators turns yellow in (sugar) and phenol red (pH indicators turns yellow in acidic pH and turns red in alkaline pH).acidic pH and turns red in alkaline pH).
Mannitol fermentation on MSA
Mannitol fermentedYellow colonies:
S. aureus
Mannitol nonfermenterRed colonies:
S. epidermidis& S. saprophyticus
Coagulase TestCoagulase Test
The tube coagulase test The tube coagulase test (Free):(Free):
Procedure:Procedure: – Mix 0.1 ml of culture + 0.5 ml of plasmaMix 0.1 ml of culture + 0.5 ml of plasma– Incubate at 37C for 4 hIncubate at 37C for 4 h– Observing the tube for clot formationObserving the tube for clot formation– Any degree of clotting constitutes a Any degree of clotting constitutes a
positive testpositive test
AdvantageAdvantage– More accurateMore accurate
DisadvantageDisadvantage– Time consumedTime consumed
S. aureus S. epidermidis
Coagulase TestCoagulase TestTwo Methods:Two Methods:– The slide MethodThe slide Method– Tube MethodTube Method
The slide coagulase test The slide coagulase test – Used to detect bound coagulase or clumping factorUsed to detect bound coagulase or clumping factor– Add one drop heavy bacterial suspension and one drop of plasma on Add one drop heavy bacterial suspension and one drop of plasma on
clean slideclean slide– Mixing well and observing for clumping within 10 secondsMixing well and observing for clumping within 10 seconds
AdvantageAdvantage– Rapid diagnosisRapid diagnosis
DisadvantageDisadvantage– Less accurate Less accurate
Deoxyribonuclease (DNAase) testDeoxyribonuclease (DNAase) test
Principle:Principle:– DNA is insoluble in acidDNA is insoluble in acid– DNA is hydrolyzed into oligo nucleotides by the DNA is hydrolyzed into oligo nucleotides by the
action of DNaseaction of DNase– Nucleotides soluble in acidNucleotides soluble in acid
DNase test
PositiveStaphylococus aureus
NegativeS. epidermidis & S. saprophyticus
DNase TestDNase Test
Procedure & result:Procedure & result:– Inoculate DNA agar with tested organism in circular motionInoculate DNA agar with tested organism in circular motion– Incubate at 37C for 24-48hIncubate at 37C for 24-48h– Observe DNase activity by adding 1N HCl to the agar surface, a zone of clearing Observe DNase activity by adding 1N HCl to the agar surface, a zone of clearing
indicates a positive testindicates a positive test– The zone represents the absence of DNAThe zone represents the absence of DNA– The medium around colonies not producing DNase remains opaque, which is a The medium around colonies not producing DNase remains opaque, which is a
reflection of the precipitation of DNA by the added acid.reflection of the precipitation of DNA by the added acid.
Novobiocin SensitivityNovobiocin Sensitivity
A simple disk diffusion test for estimating novobiocin susceptibility A simple disk diffusion test for estimating novobiocin susceptibility used to distinguish used to distinguish S. saprophyticusS. saprophyticus from other clinically species from other clinically speciesInoculated overnight culture on Mueller-Hinton agar Inoculated overnight culture on Mueller-Hinton agar Add novobiocin disk on inoculated plateAdd novobiocin disk on inoculated plateIncubate at 370C overnightIncubate at 370C overnight Novobiocin resistance is intrinsic to Novobiocin resistance is intrinsic to S. saprophyticusS. saprophyticus but but uncommon in other clinically important species.uncommon in other clinically important species.
Novobiocin test
SensitiveS. aureus
S. epidermidis
ResistantS. saprophyticus