3Gram Stain 2. Saturate the smear with crystal violet for 1 minute. spread as a thin layerOne drop of water+Loop of Bacteria1-allowed to air drysmear2-fixedclean glass slide2. 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
4Gram staining Results Gram 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.
5Cells Description under the microscope 1- Cell response : GV + or GV-
6Cells Description under the microscope 2- Cell shape and arrangement :Cocci:single- Diplo – Grape like-Strepto
7Cells Description under the microscope Road: Short or longSingle or Chain
11Catalase testThe catalase test is distinguished streptococci from staphylococciflood culture with drops of 3% H2O2Catalase-positive cultures bubble at onceThe test should not be done on blood agar because blood itself will produce bubblesH2O2CatalaseH2O + O2 (gas, ↑)Staphylococci
12Species of Satphylococci Three species of staphyloccoci have medical importance:S. aureus: Pathogenic & commensally found in nose (nares)S. epidermidis: non pathogenic & common commensals in nares & skinS. saprophyticus: Cause UTI in female & occasionally commensally found skin
13Coagulase Test Principle: This test used to differentiate between S. aureus (CPS) & other Staphylococcus species (CNS)Fibrinogen(Plasma)CoagulaseFibrin(Clot)
14Staphylococci General characters: Gram Positive Cocci Grape-like Non MotileNon Spore FormingNon CapsulatedNon FastidiousFacultative AnaerobesFermentativeCatalase positive
16Laboratory diagnosis of Staphylococcus Gram Stain:Gram Positive Cocci, arranged in clusterCulture: 1- Blood agar , 2- MSA mediaBlood agar (Non-Selective Media)Coagulase Positive Staphylococci are Pigmented & hemolyticCoagulase Negative Staphylococci are non-pigmented & non-hemolytic
17MSA is selective differential medium for staphylococci It contains: NaCl (7.5%), Mannitol, & Phenol RedThe cause of selectivity due to presence of high salt concentrationThe cause of differential because contains mannitol (sugar) and phenol red (pH indicators turns yellow in acidic pH and turns red in alkaline pH).
18Coagulase Test The tube coagulase test (Free): Procedure: Advantage Mix 0.1 ml of culture ml of plasmaIncubate at 37C for 4 hObserving the tube for clot formationAny degree of clotting constitutes a positive testAdvantageMore accurateDisadvantageTime consumedS. aureusS. epidermidis
19Coagulase Test Two Methods: The slide Method Tube Method The slide coagulase testUsed to detect bound coagulase or clumping factorAdd one drop heavy bacterial suspension and one drop of plasma on clean slideMixing well and observing for clumping within 10 secondsAdvantageRapid diagnosisDisadvantageLess accurate
20Deoxyribonuclease (DNAase) test Principle:DNA is insoluble in acidDNA is hydrolyzed into oligo nucleotides by the action of DNaseNucleotides soluble in acid
21DNase Test Procedure & result: Inoculate DNA agar with tested organism in circular motionIncubate at 37C for 24-48hObserve DNase activity by adding 1N HCl to the agar surface, a zone of clearing indicates a positive testThe zone represents the absence of DNAThe medium around colonies not producing DNase remains opaque, which is a reflection of the precipitation of DNA by the added acid.
22Novobiocin Sensitivity A simple disk diffusion test for estimating novobiocin susceptibility used to distinguish S. saprophyticus from other clinically speciesInoculated overnight culture on Mueller-Hinton agarAdd novobiocin disk on inoculated plateIncubate at 370C overnightNovobiocin resistance is intrinsic to S. saprophyticus but uncommon in other clinically important species.
23Practical Work Gram stain Catalase test Mannitol fermentation on MSA Coagulase Test by Tube and Slide MethodDNAase Test