MICRO 1_LEC 3_ Micrococcaceae_ Catalase (+) and Gram (+) Cocci.pdf
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ESSON 3:Micrococcaceae: Catalase (+) and Gram (+)Cocci L REACTIONS OF EACH GENERA IN DIFFERENT TESTS LECTURER:Pici Arcilie E. Malintad, RMT, MSMT (C) G...
ESSON 3:Micrococcaceae: Catalase (+) and Gram (+)Cocci L REACTIONS OF EACH GENERA IN DIFFERENT TESTS LECTURER:Pici Arcilie E. Malintad, RMT, MSMT (C) G enus Staphylococcus was included with the genus Micorcoccus thus there is a need to differentiate the two. LEARNING OBJECTIVES: STAPHYLOCOCCUS MICROCOCCUS Discuss the general characteristics of Staphylococcus P roducesacidfrom glucose spp. And Micrococcus spp. Including oxygenation, anaerobicallyincarbohydrate microscopic gram staining characteristics, and fermentation test. macroscopic appearance on blood agar. D oesnot produce Explaintheprincipleofthecoagulasetest,includingthe P roducesacidfrom glycerol in Acid. different principles associated with the slideversusthe the presence ofErythromycin It isnegative (-). tube test and the clinical significance. (0.4 mg/dl). Discuss the various types of diseases specifically S usceptible to lysis with associated with the Micrococcus spp., S. aureus. S. Lysostaphin. saprophyticus,andS. epidermidis. B. STAPHYLOCOCCUS I. CATALASE(+)AND GRAM(+)COCCI 3 Major Species: G ram (+) cocciare very heterogeneous group. ○ S. aureus Genera: ○ S. epidermidis ○ Staphylococcus ○ S. saprophyticus Closely related to theMicrococciFamily. These three (3) major species, which are the most ○ Micrococcus prominently associated with human infections, have the ○ Similar Organisms same microscopic morphology and cultural characteristics but can be differentiated by doing the A. GENERA AND SPECIES TO BE CONSIDERED following tests. 1 S taphylococcus aureus* S. aureus S. epidemidis S. saprophyticus Coagulase-negative Staphylococci(most commonly 2 Coagulase + - - encountered) Staphylococcus epidermidis* Hemolysis + +/- - Staphylococcus haemolyticus* Mannitol + - + Staphylococcus saprophyticus* ○ S. saprophyticus subsp.Bovis Trehalose + - + ○ S. saprophyticus subsp.Saprophyticus olden G emon L Staphylococcus lugdunensis Pigment White yellow Yellow Staphylococcus schleiferi ○ S. schleiferi subsp. Coagulans ECTURER’S NOTES: L ○ S. schleiferi subsp. Schleiferi Hemolysis 3 Coagulase-positive Staphylococci Most stains of Staphylococcus will produce Staphylococcus pseudintermedius beta-hemolysison the blood agar. Staphylococcus delphini ○ S. saprophyticus are usuallynon-hemolytic. Staphylococcus intermedius Staphylococcus hyicus Carbohydrate fermentation tests With the use of MannitolandTrehalose Ifpositive (+), they produce anacid. LECTURER’S NOTES: Only those belonging to the genus Staphylococcus are Pigment of clinical importance: One of the distinguishing characteristics in the genus Focus only onS. aureus*and thecoagulase-negative Staphylococcus. Staphylococci*as these arethe onescommonly Observed using enriched mediums like blood agar, encountered in clinical setups associated withhuman nutrient agar, or trypticase soy agar infections. The plate is incubated for18-24 hoursbefore examining. This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 1 MICROBIOLOGY | Lesson 3:Micrococcaceae 1. HABITATS B II. GENERA STAPHYLOCOCCUS For: Staphylococcus, Micrococcus, P lanococcus, and When we identify in the lab we have to base it on the Stomatococcus. following criteria: ○ Microscopic morphology P lanococcus Free-living saprophytes ○ Colonial appearance Micrococcus Describe the colonies after 18-24 hours of S taphylococcus incubation Isolated from clinically Micrococcus ○ Biochemical tests significant sources Stomatococcus Shape Spherical(grape-like masses) Surface of primates and Gram (+)cocci other mammals S taphylococcus Staining atalase (+) C resence asendogenous P Stomatococcus Reactions a test usually done to differentiate floraallows many spp. to Staphylococcus from Streptococcus. cause infections Motility Non-motile C. GENERAL CHARACTERISTICS Strongly pigmentedonenriched media O ne important distinguishing characteristic that Such as: agar, chocolate agar, BHI, or differentiates the different spp. of the coagulase-negative nutrient agar. Staphylococcus group is the Novobiocin susceptibility Pigment S. testing. S. aureus S. epidermidis saprophyticus taphylococcus epidermidis S olden g lemon Staphylococcus haemolyticus white yellow yellow Staphylococcus capitis Staphylococcus hominis subsp. AnaerobicorFacultatively anaerobic Susceptibleto Except: (obligate anaerobes) hominis Novobiocin ○ S. aureus subsp. anaerobius Staphylococcus lugdunensis Staphylococcus saccharolyticus Oxygen ○ S. saccharolyticus Staphylococcus warneri Requirements F acultative anaerobescan grow Other spp. aerobically or anaerobically. taphylococcus saprophyticus S Obligate anaerobescan grow in the Resistantto Staphylococcus cohnii absence of oxygen. Novobiocin Staphylococcus kloosii ircular, opaque,smooth colonies. c Staphylococcus xylosus Colonies After 18-24 hours of incubation. M icrococcus spp. Skin-Colonizing A. STAPHYLOCOCCUS AUREUS Kocuria spp. Organisms Kytococcus spp. Description ost virulent Speciesof Staphylococci M NOTE: encountered. Those in bold are the only ones considered, since these are the onescommonly associated with human Important sources forfood poisoning. infections. AlthoughS. lugdunensisandS. schleiferican alsobe ecovered R Invade in theBloodstream. encountered, they arerarely associatedwith human from a variety Metastasizeandappearin theurine. infections. ofhuman Form abscess in body organs thus infections producingseptic shock. OTHER COAGULASE(+)STAPHYLOCOCCUS 1 S taphylococcus intermedius ecovered R nterior nares A Important agent:Dog bite infection fromas many Perineum Isolated4x more frequentlythan S. aureus from the as10-15%of Nasopharynx oral cavity of dogs. healthy people. otherskin sites MisidentifiedasS. aureus. arrier states C T hecarrierstateiscommonamongthe ○ Because the organismproduce enzyme are the human population. coagulasejust likeS. aureus reservoir of Infectionsarefrequentlyacquiredwhen 2 Staphylococcus hyicus infection. the colonizing strains gain access to Never implicated as a human pathogen. the normal sterile sites. That is why This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 2 MICROBIOLOGY | Lesson 3:Micrococcaceae A3. CULTURAL CHARACTERISTICS t hey invade the bloodstream causing bacteremia. W hen you do ID of bacteria, you base it on both microscopicandcultural characteristics(both ofthem) A1. MICROSCOPIC MORPHOLOGY 1. Identifyits microscopic morphology 2. Inoculate the specimen to the respective culture Description ram (+) cocci — like bunches of G media. grapes ✅ On 5% sheep blood and rowin most G chocolate agars (blood and (3) CELL WALL COMPOSITION: laboratory-enriched chocolate agars) culture media ❌ OnMacConkey medium ightly crosslinked T orprotection from lysisunder F Collect blood from the patient and peptidoglycanand harsh osmotic condition bacteria to In patients with inoculate blood to the routinely teichoic acid mucosal cell receptor sites. bacteremia used blood culture medium. olysaccharide P rotects them from phagocytosis P row wellin G capsules by PMNs (polymorphonuclear broth-blood culture T hioglycolate broth (some strains only) neutrophils) systemsand Dextrose common nutrient Brain-heart infusion (BHI) broth indstheFc segment of IgG, thus B broths Protein A preventingantibody-mediated phagocytosisby PMNs. . Collect sample from patient and 1 inoculate A2. DAMAGES TO THE HOST 2. After 24H incubation,examineand emolysisand H describe the colonies In the bloodstream, aggregates ofIgG bound to protein Aon the surfaces willfix complement mediated tissue color of colonies T ake note of hemolysis on blood damageto the host. agarplate andcolor of colonies. The colors are variable in ue to the presence of avirulencefactorintheircellwalls, D characteristics. they become: 1 Anticomplementary MEDIA USED:SELECTIVE MEDIA Inhibitthe complement present in the serum involved in the antigen-antibody reaction IsolateStaphylococcifrom clinical 2 Antiphagocytic material Kill leukocytes Eliminate contaminationbyGram (-) 3 Platelet Injury PURPOSE organisms, especially if specimen was collectedfrom feces. 4 Elicit Hypersensitivity ○ Feces are heavily contaminated with 5 Mitogenic Gram (-) organisms. 6 Potentiate natural killer activity of human lymphocytes P henylethyl Alcohol(PEA) agar Columbia Colistin-Nalidixic Acid(CNA) COMMONLY agar USED MEDIA Since the media is selective,onlyGram (+) cocci growson these media. Incubated at A fter primary inoculationof the 35-37°CinCO2or specimen ambient air. T he detectable growth of S. aureus fter 18-24 hoursof A will be seengrowing on the solid incubation mediumlikebloodorchocolate agar. rowmost rapidly G H owever, itforms pigmentsbest at at 37°C room temperature (20-25°C). Since thesmearwas obtained from asputum sample, ○ you will seebacteriaandpuscells/PMNs/PMLs(red circles) This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 3 MICROBIOLOGY | Lesson 3:Micrococcaceae mooth S A4. AGARS olonies on solid C Entire 1. BLOOD AGAR mediumaremedium Slightly raised to large(0.5-1.5 mm) Low convex Opaque white utyrous B Meaning,butter-likeconsistency consistency Easily emulsifiable P igmentedcreamy yellowto golden yellow Pigments produced ○ Forms gray or deep golden yellow colonies. Two different variants of S. aureus on a blood agar plate Complete lysis of cellsaround the Two different variants of S. aureus on a blood agar plate: colonies. eta-hemolyticor B A. Presence of a small gray colony variant. In sheep-blood agar, the colonies produceBeta Some strains of S.aureus will produce this. are surrounded by azone of Hemolysis B. Presence of normal golden-yellow variant. complete hemolysisand we call this beta-hemolysis. 4.1.1 BETA-HEMOLYSIS – Blood Agar such as: n primary isolation, O ○ Nutrient agar golden yellow ○ Trypticase soy agar colonies can be These pigments grow very well observed growing on when incubated atroom primary plates temperature. Thegolden yellow pigment produced by some strains ofS. Beta-hemolysis on blood agar Staphyloxanthin aureus It is acarotenoid pigment. Blood agar - colonies are white to creamy. Various degrees of hemolysis are ○ The white opaque colonies are surrounded by a clear In blood agar plate zone of hemolysis. produced Especially, Beta- hemolysis Typical of most strains to produce beta-haemolysis. S. aureuswill be(+) positive. 4.1.2 ISOLATED COLONY – Blood Agar To differentiate staphylococcus from different organisms Catalase tests As itproduces catalasewhich differentiate them from streptococci. Isolated Colony of S, Aureus Isolated colonies of S. aureus that produce the yellow pigment. ○ ItgrowsverywellonNutrientAgarandSheep’sBlood Agar. S. aureusPrimary Culture Media Plate. Shows typicalgolden-yellow pigment that is produced by staphyloxanthin. Typical Colonies of S.aureus after incubation on Nutrient Agar. This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 4 MICROBIOLOGY | Lesson 3:Micrococcaceae 2. MANNITOL SALT AGAR Microscopic appearance of S.aureus (on the left) White opaque non-hemolytic colonies of S.aureus on blood agar (on the right) EDITOR’S NOTES: [SOURCE] Colonial morphology of S.aureus in MSA Some of the information Miss discussed were reiterated from the previous table. A selectiveanddifferential medium used toisolate Staphylococciin clinical specimens. ○ Selective due tohigh salt concentration. 3. CHROMagar O riginally invented byAlain Rambach. COMPONENTS A selective and differential media for the isolation and 1 H IGH SALT (10%) identification of Methicillin Resistant Staphylococcus Makes the mediumselective. Aureus (MRSA). Allows the growth of some genusStaphylococci ○ A type of bacteriaresistant to several antibiotics. membersbecause they cantolerate high saline Widely usedfordirectdetection ofnasal colonization. levels. Selective—ContainsanantibioticcefoxitinwhichMRSAis ○ Other organisms grow very weak in this medium. resistant to. 2 SUGAR MANNITOL Has a chromogenic substrate added to hydrolyze Serves as thedifferential ingredientin the agar. organisms to produce amauve-colored colony. The organisms capable of using mannitol as a food ○ Thesemauve-coloredcoloniesseenafterincubationis source will produceacidicbyproductsof fermentation also onebasisused for MRSA identification. that willlower the pHof the media. Other organisms can also hydrolyze the chromogenic ○ Because of this acidity, if the organisms ferment the substances within the media, resulting to colored colonies mannitol, the pH indicator turns toyellowfromred. fromwhite tobluetogreen. This explains theS. aureuscolonies surrounded bya yellow haloon the media after incubation. ○ It is a result ofacid production altering the pH. Other Staphylococci that ferments mannitol: ○ Staphylococcus saprophyticus ResemblesStaphylococcus aureus 3 PH INDICATOR PHENOL RED If the organismsferment mannitol, thered color becomesyellow. If the organismsdo not ferment mannitol,pink-colored Formation of Mauve-colored Colonies in CHROMagar (MRSA) coloniesare produced. A5. BACTERIAL METABOLITES CULTURAL CHARACTERISTICS –Mannitol Salt Agar S.aureusisthemostvirulentspeciesofallStaphylococci A notherbiochemicaltestfortheidentificationofStaphisto encountered and has a wide spectrum of factors which dosugarfermentationinwhichS.aureuscanfermentmany contribute to its ability to establish infections and cause carbohydrates (CHO)without gas. several diseases. Staphylococcusaureusfermentsmannitolandappearsas It can produce: yellow colonies. ○ Enzymes A useful selective medium for recoveringS.aureusfrom ○ Toxins fecal specimenswhen investigatingfood poisoning. 1. ENZYMES Contributes to spread of infection: ○ Phosphatase ○ Thermostable DNA ○ Lipase, Gelatinase, Protease, and Fibrinolysin Typical growth of S.aureus in MSA. ○ Hyaluronidase This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 5 MICROBIOLOGY | Lesson 3:Micrococcaceae 2. TOXINS A6. PATHOGENESIS AND SPECTRUM OF DISEASE S .aureus producestoxinsthatcontributesubstantiallytoits ability tocause disease. 1. POLYSACCHARIDE CAPSULE –S. aureus&S. epidermidis B oth produce a polysaccharide capsule that inhibits 6 types(A, B, C, D, E, F) cause phagocytosis. ENTEROTOXIN Produced in:various amounts by individual clinicalisolates staphylococcalfood poisoning. Appear as:aslime layer or biofilm C ausesSSS (Scalded Skin Syndrome Allows the organisms:toadhere to inorganic surfaces. EXFOLIATIVE in neonates). Impairs or inhibitsthepenetration of antibiotics. A ssociated withTSST-1 (Toxic Shock Thepeptidoglycanresembles theendotoxin effectof Syndrome Toxin), a multiorgan gram (-)by: involvement. ○ activating complement,interleukin-1 (IL-1,) PYROGENIC ○ Has several systemic effects, ○ acting as a chemotactic factor for the recruitmentof XOTOXIN C E includingfever,desquamationand polymorphonuclear cells (PMNs). hypotensionleading to shockand Thiscascadeofeventscausesswellingandmayleadtothe death. exacerbation of tissue damage. 4 types of cytolytic toxins:alpha,beta, 2. PROTEIN A –S. aureus delta, andgammatoxins which are S . aureus produces a surface protein known as, protein A hemolysins That is bound to the cytoplasmic membrane of the ○ It is because they cause lysis of organism,andhasahighaffinityfortheFcreceptoronIgG cells. molecules as well as complement. ○ These hemolysins are also called Theseserioussofttissue“community-associated”infections leukocidin(Panton Valentine are frequently mediated bymethicillin-resistantS.aureus Leukocidin)which act onWBC(kill (c ommunity-acquired MRSA or CA-MRSA). the leukocytes). Produces:toxin-mediated diseases THE 4 CYTOLYTIC TOXINS: ○ scalded skin syndrome 1. ALPHA TOXIN ○ toxic shock syndrome Most strainsof S. aureus produce ○ food poisoning. alpha toxin, whichdisrupts smooth Most infections associated with S.aureusgenerallyinvolve musclein blood vessels and is intense suppuration and destruction/necrosis of tissue toxicto erythrocytes, leukocytes, infections hepatocytes, and platelets. OST INFECTIONS ASSOCIATED WITH S. AUREUS: M 2. BETA TOXIN Are grouped as localized skin Believedtoworkinconjunctionwith Involve: infections such as: thealpha toxin. CYTOLYTIC uruncles (boils) F A heat-labile sphingomyelinase TOXINS Folliculitis ones B which catalyzes the hydrolysis of Impetigo Joints membrane phospholipids resulting Carbuncles Deep organs in cell lysis. Various wound infections Respiratory 3. DELTA TOXIN Deep infections that spread from system Three (3) spp. of Staphylococcus skin to cause bacteremia (w/ or Tissue identified as capable of producing w/out endocarditis) delta toxin: ○ S. aureus A7. STAPHYLOCOCCAL SCALDED SKIN SYNDROME ○ S. epidermidis (RITTER DISEASE) ○ S. haemolyticus A skin infection caused by Staphylococcus bacteria in Cytolytic to erythrocytesand which theskin becomes damaged and sheds. demonstratesnonspecific Usually afflictsneonates. membrane toxicityto other It is anexfoliative toxin, aserine protease mammalian cells. Splits the intercellular bridges oftheepidermidis,resulting 4. GAMMA TOXIN in extensive sloughing of epidermistoproduceaburn-like ProducedbyallstrainsofS.aureus effecton the patient. and may actually function in association with the Panton Valentine Leukocidin (PVL). This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 6 MICROBIOLOGY | Lesson 3:Micrococcaceae It is amultisystem illness, characterized by: ○ High Fever ○ Conjunctival reddening ○ Headache ○ Hypotension ○ Vomiting ○ Skin rashes ○ Diarrhea ○ Kidney failure Staphylococcal Scalded Skin Syndrome A8. LOCALIZED SKIN OR TISSUE INFECTIONS (CUTANEOUS INFECTIONS) INFECTION DESCRIPTION Inflammationof the hair follicles with two types: ○ Asmall red bump or pimple FOLLICULITIS Develops at theinfection siteof the hair follicle. ○ Sty Very common Folliculitis affecting one or Toxic Shock Syndrome more hair follicles on the edge of the upper or lower 10. EPIDEMIOLOGY A eyelid. The prevalence of S.aureus carriers ishighamong: ○ healthcare providers and immunocompromised or A deep-seated infection, originating immunosuppressed individuals. FURUNCLE / fromfolliculitis(if the infection Staphylococcicanbeefficientlytransmittedfrompersonto BOILS extends from the follicle to neighbor person. tissue). Upon transmission, the organism may becomeestablished Causesredness,swelling, and as a part of the recipient’snormal microbiota. severe pain. ○ And is later introduced to sterile sites by trauma and Commonly found on theneck, invasive medical surgery. armpit, andgroin regions. A11. DIFFERENTIATION AMONG COAGULASE (-) CARBUNCLE Aggregation of infected furuncles. STAPHYLOCOCCI, MICROCOCCI, AND STOMATOCOCCUS ○ May formlarge abscesses. It is a large area ofredness, swelling Can be differentiated based on the following: andpain, punctuated by several Either be micrococci or sites of drainage pus. Catalase (+);coagulase (-) staphylococci A very superficial skin infection atalase (-) organisms C common inchildren. s tomatococcus IMPETIGO resemblingstaphylococciby ○ Usually producesblistersor mucilaginosus gram stain and colonial soreson theface,neck,hands, morphology anddiaper area. It is characterized bywatery Micrococcus bristles, which becomepustules R esistant to200 mg/ml lysostaphin and thenhoney coloured crust. (+) positiveto modified oxidase test and benzidine Sensitive to0.04 mg bacitracinso has(+) positivegrowth A9. TOXIC SHOCK SYNDROME C ausedwhenToxicshocksyndrometoxin(TSST)liberated by S.aureusenters the bloodstream. This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 7 MICROBIOLOGY | Lesson 3:Micrococcaceae OMMENTS REGARDING SPECIFIC ORGANISMS C READING OF RESULTS —Catalase Test Micrococcus spp. & Staphylococci related genera Not lysedwith lysostaphin Lysed with lysostaphin R esistant to antibiotic R esistant to 0.04 U of furazolidone bacitracin S usceptible to 0.04 U of S usceptible to to bacitracin furazolidone Results of the Catalase Test on S.aureus Microdase (+) Microdase (-) T he immediate formation ofoxygen u sually will only grow Facultativelyanaerobic POSITIVE reaction bubblesis evidence that the aerobically organism produces catalase. A12. IDENTIFICATION NEGATIVE reaction Few or no bubbles after20 seconds. W hen we identify the organism, we have to base our identification on the microscopic morphology, cultural TWO WAYS TO PERFORM CATALASE TEST —Catalase Test characteristicsandbiochemical tests. Slide method Onceanorganismhasbeencharacterizedasgram-positive, ○ You havetopick2-3coloniesfromtheplate,putiton catalase-positive,coccoid bacterium, the glass slide and add3% hydrogen peroxide. ○ Complete identification may involve a series of tests, Plate method including: ○ You can add 3% hydrogen peroxide directly on the purecolonies ofStaphylococcus aureus. 1 A tmospheric requirements. 2 Resistanceto 0.04 U ofbacitracinandfurazolidone. SOURCES OF ERROR —Catalase Test Possession ofcytochrome C, as determined by the 3 microdase (modified oxidase) tests. 1 D o not use colonies taken from the blood agar. Sinceblood cellscontain the enzymecatalaseso it A13. BIOCHEMICAL TESTS could give afalse positiveresult. Blood agarcan interfere with catalase test sinceblood 1. CATALASE TEST cells arecatalase positive. U sed for theidentification ofS.aureus. It is recommended to use another agar which does not The test that detects thepresenceof thecatalaseenzyme. have blood. Catalase — isanenzymethatremoveshydrogenperoxide ○ Nutrient AgarorTrypticase Soy Agarmay be (H2O2) by catalyzing the breakdown of the molecules into used instead. water and oxygen. Do not use a metal loop or needle with H₂O₂ as it will Primarily usedtodifferentiatebetweenGram (+)cocci. 2 give a false positive result and degrade the metal. The enzyme is produced by all species of Staphylococcus. ○ So they can bedifferentiatedfromStreptococci,which 2. COAGULASE TEST can not produce the enzyme. C oagulase(+)isthemostdistinguishingcharacteristicof c Catalase (+) genusStaphylococcus S.aureus which differentiates itself fromthecoagulase(-) Staphylococci. g eneraStreptococcusand Performedinthelaboratoryusingtheslidetesttodetectthe Catalase (-) Enterococcus bound coagulase or the clumping factor and the true coagulase which detects the free coagulase. PROCEDURE –Catalase Test PRINCIPLE —Coagulase Test ake a colony of the organism fromovernight growthon T 1 T he enzyme coagulase produced by S.aureus binds theblood agar plate. plasma fibrinogen and activates a cascade of reaction Use a disposable loop to carefully remove1-2 coloniesof 2 causing plasma to clot. Staphylococci. ○ An organism can produce two types of coagulase, Place it on the glass slide. referred to asboundandfree. Make sureany of the blood agar is not transferredto 3 the slideas erythrocytes in the blood will causea false SLIDE TEST —Coagulase Test positive reaction. B ound coagulase (or clumping factor) is detectedusinga After that, add a drop of3% hydrogen peroxideintothe rapid slide test. 4 colony. This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 8 MICROBIOLOGY | Lesson 3:Micrococcaceae In the slide test, the bound coagulase or the clumping SOURCES OF ERROR IN COAGULASE TEST factor isboundtothebacterialcellwallandreactsdirectly ome strains of Staphylococcus producefibrinolysin, S withfibrinogen. which dissolves the clot after4 hoursof incubationat ○ This results in the precipitation of fibrinogen on the 1 35°Cand may appear negative if allowed to incubate Staphylococcal cell causing the cellstoclumpwhena longer than 4 hours. bacterial suspension is mixed with plasma. ○ We need to incubate the tube for 1-4 hours only. Most, but not all, strains of S.aureus produce clumping Plasma with citrate isnot suitablefor use becauseit gives factorsand thus are readily detected by this test. false (+) results In addition, false positives may occur as a result of auto 2 ○ Thus,plasma containing agglutination when colonies are grown on media withhigh ethylenediaminetetraacetic acid (EDTA)should salt concentrations. be used. Indicated when the organisms Positive (+) coagulaseis sufficient in naming theisolated POSITIVE agglutinatewhen mixed with S. aureus. 3 plasma. ○ Although other Staphylococcus spp. also produce the clumping factor. A pproximately10% to 15%of Serumcannotbe used since it lacks clotting factors, EGATIVE LATEX N strains. 4 including fibrinogen.Plasmais themedium of choice COAGULASE TEST As a result of the masking by Slide test shouldnot be performedwith organismstaken capsular polysaccharides. fromhigh salt media(e.g., Mannitol Salt Agar) asthe salt 5 content can createfalse positiveresults. Do notpick 3 TUBE COAGULASE TEST colonies from mannitol salt agar. P erformed by emulsifying several colonies in0.5 mlplasma Some S. aureus can produce astaphylokinase enzyme obtained from EDTAin order to give amilky suspension. along withstaphylocoagulase enzyme. When you pick the colony, you pick thepure or theisolated ○ Staphylokinase enzyme can dissolve clots colonyfrom the plate and emulsify that in 0.5 mlplasma ○ Thus, incubating the tube for a maximum of 4 Then incubate at35°-37°Cfor 1-4 hours, checkingforclot hours is important formation. 6 If it exceeds 4 hours, the test result would be negativebecause the clot has already been PERFORMANCE OF TUBE COAGULASE TEST – Tube dissolved Coagulase Test ○ When doing the Tube Coagulase test, check the P erformedbyinoculationofatubecontainingplasmaand tubeevery 30 minutes. incubating at35°C. Productionoftheenzymeresultsinaclotformationwithin1 STEPS IN PERFORMING SLIDE TEST IN THE LAB to 4 hoursof inoculation. utadropofplasmaobtainedfromEDTAonaclean,dry P Done only to confirm aslide negative result. 1 glass slide. ○ Isolates (not producing the clumping factors) Put a drop of distilled water orNSSnexttothedropof The clumping factor detected in the slide test 2 plasma as acontrol. should be tested for their ability to produce Using a loop or straight wire, pick a pure culture of extracellular coagulase 3 Staphylococcus or isolated colony and emulsify it into ○ The free coagulase causes clotting of plasma when each drop. incubated for1-4 hoursat35°-37°C. Inoculating the saline first, try to create a smooth Theresultofthetubetestwouldbeclotformationwithin1 4 suspension. to 4 hoursof inoculation. 5 Using an applicator stick, mix it thoroughly. The result is read within 1 to 4 hours from inoculation of 6 Rock the slide gently for5-10 seconds. suspectedS. aureus. RESULTS OF SLIDE TEST ACTIVATION OF COAGULASE REACTING FACTORS – Tube Coagulase Test T he clotting mechanism involves activation of plasma Coagulase ReactingFactors(CRF),amodifiedorderived thrombin molecule to form a complex and reacts with fibrinogen to form thefibrin clot. ○ ThecomplexiscalledtheCoagulaseReactingFactor Complex. Right side: test-positive result (plasma+colony) Left side: control (saline + colony) This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 9 MICROBIOLOGY | Lesson 3:Micrococcaceae A ppearance of microscopic clumping B reak ends, push agar plug and bacterial growth, and 5 in10secondsorlessinthecoagulated place on DNA agar. plasma drop (test) Incubate test agar at37 C for 1-4 hoursand observe 6 ○ This indicates the presence of a halo of pinksurrounding the inoculum. POSITIVE (+) coagulation factor. thermostable DNA(pink halo) Clumping factor is present S. aureus N o clumping in saline drop (control) Thermostable S. delphini The control is a negative control. Dna Test (+) S. schleiferi NEGATIVE No clumpingin either drops. S. intermedius S. hyicus Slide Test (Right side: test-positive result, Left side: control) INTERPRETING RESULTS FROM TUBE TESTS – Tube Presence of a halo pink surrounding the colony (right) Coagulase Test Observe the presence ofclot. 5. LYSOSTAPHIN TEST S usceptibility to lysostaphin is a rapid test method in differentiatingStaphylococcifromMicrococci SPECIMEN COLLECTION –Lysostaphin test N ot intended for the primary isolation of patient specimens Only used with the culture of isolated organisms Used inconjunctionwithotherbiochemicalteststoidentify cultures of isolated organisms PROCEDURE A llowdiskstoequilibratetoroomtemperaturebefore 1 opening the vial. Using a pure 18-24 hour culture, prepare a heavy 2 suspension (equivalent in density to a McFarland 2.0 Positive result:presence of a clot of any size standard) in 2 ml of sterile saline. Negative result:no clot is formed. Transfer 1 ml of the bacterial suspension into a second 3 test tube. 4. THERMOSTABLE DNA TEST Add one lysostaphin disk to one of the bacterial T hermostabledeoxyribonuclease(DNase)testisusedas 4 suspensions and shake vigorously. a rapid screening method for the detection of ○ Second suspension is used as a negative control. staphylococcal enterotoxininmilk and milk products. Incubate both suspensions aerobically at 35-37 C for ThermostableDNaseissuperiortotubecoagulasefordirect 5 2.5 hours in an incubator, or for 2 hours in a 35-37 C detection of Staphylococcus aureus in positive blood water bath. Do not disturb suspension before 2 hours. cultures After incubation,compare the turbidityof the bacterial ○ Toluidine blue DNA agar → incubate for1hourbefore 6 suspension with the lysostaphin disk against the performing the test control suspension. PROCEDURE RESULTS 1 C olony in 5% BA C omplete clearingof the bacterial Place in the center of the microcapillary tubeandseal suspension 2 the ends. ositive (+) Markeddecrease in its turbidityas P 3 Heat in aboiling water bathfor 15 min. compared with the negative control 4 Allow it to cool. suspension This transcript is strictly confidential and is intended forAVR 5TH FLOORgroup members only; please don’tshare or distribute! 10 MICROBIOLOGY | Lesson 3:Micrococcaceae Indicatessusceptibilityto lysostaphin C ontain an interpeptide bridge consisting of glycine-rich (Staphylococcus spp.) peptides. ○ Sensitive to lysostaphin Lysostaphinisanendopeptidasethatcleavesthesepeptide linkages, rendering the cells susceptible to osmotic lysis S uspension remainsturbid ○ lysostaphin susceptible Does not clear The interpeptide bridge of Micrococcus does not contain egative (-) N Indicatesresistanceto lysostaphin glycine (Micrococcus spp.) ○ Sincethepresenceofglycineisessentialfortheaction of lysostaphin, the Micrococcus cells are not affected HOWINGPOSITIVE REACTION(LYSOSTAPHIN S lysostaphin resistant SUSCEPTIBLE) –Lysostaphin
