Prosedur pembiakan bakteri dari spesimen klinikDEPARTEMEN MIKROBIOLOGI FAKULTAS KEDOKTERAN USU

TUJUANMELAKUKAN ISOLASI & IDENTIFIKASI BAKTERI PADA MEDIA PERBENIHAN DARI SPESIMEN KLINIK DARI DARAH, USAP TENGGOROK, DAHAK, URINE, KEROKAN KULIT DAN CAIRAN SPINAL

ALAT-ALAT YANG DIPERLUKANInkubatorObjek glassDeck glassLampu spiritus/lampu bunsenMikroskopSengkelit/OseSungkup lilin/Candle jarAnaerobic jarGas packIndicator anaerob

MEDIA & REAGENT YANG DIBUTUHKANBlood AgarThayer Martin AgarMac Conkey AgarSaboraud Dextrose AgarReaksi BiokimiaPewarnaan GramPewarnaan Ziehl NeelsenTSI Agar (Triple Sugar Iron)Media Semi solid

CARA MELAKUKAN KULTURSengkelit ujung bulat dipanaskan hingga pijar di atas nyala api Kemudian dinginkan sebelum mengambil spesimen dengan menggunakan sengkelit tersebutBuka tutup piring petri secukupnya untuk menghindari kontaminasi dari udaraJika menggunakan tabung reaksi, tutup kapas dari tabung yang berisi media harus dibuka di dekat nyala api, dan sebelum menutupnya kembali maka terlebih dahulu ujung tabung dipanaskanSengkelit ujung bulat yang telah mengandung bahan spesimen digoreskan pada lempeng agar pada medium padat atau dilarutkan pada medium cair

CARA MELAKUKAN KULTURSengkelit ujung lurus digunakan untuk mengkultur pada agar semisolidMedia yang telah ditanam bahan spesimen langsung dieramkan di dalam inkubator dengan suhu yang sesuai (suhu optimum 37 C) untuk bakteri AerobUntuk bakteri mikroaerofilik, media yang telah ditanam bahan spesimen dimasukkan dalam Candle jar dieramkan di dalam inkubator dengan suhu yang sesuai (suhu optimum 37 C)

TEKNIK ISOLASI BAKTERI ANAEROBDALAM ANAEROBIC JAR YANG MENGGUNAKAN GAS PACK SEBAGAI GENERATOR H2 DAN CO2 DAN PALLADIUM SEBAGAI KATALISATOR

CARA KERJA BEJANA ANAEROB MEMAKAI GAS PACKMasukkan perbenihan yang telah ditanam ke dalam anaerobic jarBuka pembungkus indikator dan letakkan dalam anaerobic jar sedemikian rupa hingga dapat terlihat.Indikator segera berubah menjadi berwarna biru setelah kontak dengan udara, tetapi akan hilang warnanya jika suasana anaerob telah tercapaiBuka generator H2 & CO2 (gas pack) dan diaktivasi dengan memasukkan 10 cc air, kemudian letakkan dalam posisi tegak dalam anaerobic jarAnaerobic jar segera ditutup rapat, lalu eramkan dalam inkubatorPengamatan koloni dilakukan 24 jam kemudian

PENANAMAN PADA PERMUKAAN MEDIA PADATSengkelit atau kapas lidi steril digunakan untuk mengambil bakteri dari bahan pemeriksaan Atau untuk pengambilan koloni pada medium cair yang telah mengandung kultur bakteriSengkelit atau kapas lidi steril tersebut disemaikan pada permukaan medium padat dengan cara goresan (menstreak) secara berulang-ulangPenggoresan yang berulang bertujuan untuk mendapatkan koloni yang terpisah pada proses penyentuhan terakhir yaitu :Pada proses penggoresan 1x pertumbuhan koloni bakteri masih rapat satu sama lainPada proses penggoresan ke-2x koloni bakteri semakin renggangPada proses penggoresan ke-3x koloni bakteri semakin terpisah-pisahPada penggoresan terakhir, diharapkan bakteri dapat tumbuh dengan membentuk isolated colony (koloni yang terpisah)

Conventional diagnosis methods

PENANAMAN PADA PERMUKAAN MEDIA MIRINGMasukkan sengkelit steril berujung bulat untuk menggores pada permukaan agar miringMasukkan sengkelit steril berujung lurus ditusukkan tegak lurus ke dalam kira-kira 1 1,5 cm dari dasar tabung/ butt

Medium TSI yang belum ditanami kuman (medium steril)B,C,D : Medium TSI yang telah ditanami kuman :B. alkali/alkali; gas (-);H2S (-)C. acid/acid : gas (+); H2S (-)D. alkali/acid ; H2S (+)

PENANAMAN PADA PERMUKAAN MEDIA SETENGAH PADAT (SEMI SOLID)Sengkelit ujung lurus yang steril untuk mengambil isolated colony dari bakteriTusukkan sengkelit tersebut tegak lurus ke dalam media semi solid kira-kira 1 1,5 cm dari dasar

Medium steril Test motility positifTest motility negatif

PENANAMAN PADA MEDIA CAIRKoloni diambil dari sediaan menggunakan sengkelit atau kapas lidi steril lalu masukkan dalam media cairAgar koloni bakteri terlepas dari sengkelit atau kapas lidi tersebut, maka sengkelit di goyang2kan dalam medium cair ituJika terdapat pembentukan gas atau tidak, dapa dinilai dengan tabung terbalik (tabung Durham)

Classification & DiagnosisType of coloniesAppearanceColor, shape, size and smoothnessOn differential mediaBlood, MacConkey, EMBOn selective mediaMacConkey, Thayer-Martin

Classification & DiagnosisMetabolismUtilization of specific substratesLactose (Sal/Shi/Yer/)-Citrate (E. coli-/Klebsiella+)Production of certain end productsFermentation end products Acid (acetate, propionic acid, butyric acid etc.)AcetoinAlcoholAmineH2S

Classification & DiagnosisSpecialized testsImmunologicalO-, H- & K-Ag (serotype)Precipitation, agglutinationSpecialized enzymesCatalase--- Staph+. vs. Strep-.Coagulase---S. aureus+ vs. S. epidermidis-Oxidase---Neisseria gonorrhoea+Urease---Proteus+, Helicobacter+Antibiogram patternPhage typingFatty acid profile

Molecular diagnosisRibotypingRestriction fragment length polymorphism (RFLP)DNA hybridizationPCR, RT-PCR and RAPDNucleic acid sequence analysisPhage-GFP (TB)

RFLPGGATCCCCTAGG

DNA hybridization

In situHybridization

RAPD of P. aeruginosa

Rate of increase2nPCRRT-PCR

Molecular diagnosisReduce reliance on cultureFasterMore sensitiveMore definitiveMore discriminatingTechniques adaptable to all pathogensTechnically demandingRelatively expensiveCan be too sensitiveProvides no information if results are negative

Differentiating Staphylococci from StreptococciGram stain and morphologyBoth Gram +Staphylococci: bunched cocciStreptococci: chained cocci (S. pneumoniae form diplococcus)Enzyme testsStaphylococci: catalase +Streptococci: catalase -GrowthStaph.: large colonies (non-fastidious), some hemolyticStrep.: small colonies (fastidious), many hemolytic (a or b)

StaphylococciS. aureus: coagulase +S. epidermidis: coagulase -

Streptococci

Streptococci

On blood agar

Growth inhibition disc

S. pyogenes (group A)

-hemolytic

Sensitive to bacitracin

S. agalactiae (group B)

-hemolytic

Resistant to bacitracin

S. pneumoniae (pneumococcus)

-hemolytic

Sensitive to optochin

Viridans

-hemolytic

Resistant to optochin

Differentiating the Gram- bacteriaCocciNeisseria Rods Type of disease they causeEnteric Gram- rodsAPI testCurvedVibrio, Campylobacter, HelicobacterSpiral Gram- organismsSpirochetes

Gram negativeStraight rodsCurved rodsLactose+ Lactose-Citrate+ Citrate-H2S+ H2S-Klebsiella E. coli Salmonella ShigellaCampy blood agar42oC+ 25oC-

CampylobacterTCBS agarYellowOxidase+

Vibrio

BacteriaGram+Gram-Acid FastIntraCellularWallLessCocciRodCocciRodSpiralStaph.Strep.Non-sporeSporeStraightCurve+O2-O2+/-O2OtherS. a.S. e.S. s.ABPnVirFilRodA.i.C.d.L. m.M.t.M.l.N.c.+O2-O2B.a.B.c.C.b.C.t.C.p.C.d.TreponemaBorreliaLeptospiraNeisseriaMoraxella

P.a.EntericBact.Resp.ZooGUBordetella.H. influenzaeLegionellaYersiniaPasteurellaBrucellaFrancisellaStreptobacillusH. ducreyiGardnerellaCalymmatobacteriumRickettsiaCoxiellaErlichiaChlamydiaMycoplasmaVibrioCampylobacterHelicobacter

Processing specimens from CSFPoints to consider for bacterial/fungal/TBPriority of selection - based on clinical presentationCrystal-clear fluid, no cellular/chemical abnormalities do not consider fungal/TB cultureNo features suggestive of past or current TB do not do TB culture

Processing of specimenDescribe appearanceClearBloodyTurbidClots

Bloodytraumatic LP - less blood in subsequent tubesHaemorrhage in central nervous system equally blood stained tubes

Yellow-red CSF subarachnoid haemorrhage, jaundice

ClotsDue to increase in fibrinogenSpinal constriction, pyogenic meningitisTuberculous meningitis spider web clot on surface of fluid

Centrifuge if >1 ml; 1,500-3,000g for 20 min. Do not brake.Vortex if < 1 ml Aspirate supernatant with sterile pipette, leave 0.5 to 1.0 mlReserve supernatant (additional studies)

Vortex sediment 30s to resuspendDont use pipette to mix sediment; bacteria may adhere to sides of tubes

Microscopic examinationTake from separate tubeSpread evenly on slideAllow to air-dry, fix with methanolGram-stainCan detect organisms if 104/ml

Gram-stain smearLook for pus cells, bacteria under 40x and 100x objectivesGram negative intracellular diplococci N. meningitidis

Gram positive diplococci or short streptococci S. pneumoniaeGram negative rods - filamentous or other polymorphic forms H. influenzaeGram negative rods E. coli, Chryseobacterium meningosepticum; esp. from newbornGram positive cocci in groups & singly S. aureus

Gram positive streptococci S. agalactiae (Group B streptococci)Gram positive rods L. monocytogenes esp. newborn infantGram positive yeast cells Cryptococcus neoformans. Capsules surrounding cell are unstained, best seen in Indian ink preparation

Ziehl-Neelsen smearIf suspect tuberculous meningitisTransfer several drops of CSF sediment on slideAllow one drop to dry before adding anotherAcid fast bacilli difficult to detect in CSF-need to centrifuge 20 30 minutes

Air dry slide, fix with methanol, then stain by Ziehl-Neelsen method.Examine smear first with 40x objective to determine distributionThen with 100x objective to detect red bacilliProlonged search may be required

Fluorochrome smearAuramine stained smear to detect M. tuberculosis, more sensitiveAuramine a fluorochrome dye which fluoresce when illuminated by UV lightUse fluorescent microscope fluorescing rods detected using 40x objective

India ink preparationIf cryptococcal meningitis is suspectedA small drop of CSF sediment to a slide & add a small drop of India ink (Pelikan brand)Mix, cover with cover-slipDo not make too thick preparation will not see capsules/cells.Can use nigrosin dye

Oval or rounded stained cells surrounded by unstained capsule

Culture of specimenCulture as soon as possibleNever refrigerate except for viral studiesKeep CSF at 35-37C.Inoculate 1 or 2 drops of vortexed sediment onto media plate

Culture MediaRoutineBlood agarIncubate 35-37C up to 48 hrsChocolate agarIncubate 35-37C up to 48 hrs in CO2Check growth after overnight incubation

AdditionalNewborn infantMacConkey agarIncubate plate 35-37C overnight

If suspect tuberculous meningitisLowenstein Jensen mediumInoculate slope at 35-37C, at 45 angle to ensure specimen is in contact with full length of slope.After 1 week, place media upright.Incubate cultures for further 5 to 6 weeks.Examine twice a week for growth.

Cryptococcal meningitisCapsulated yeast cells seen in microscopic examinationInoculate Sabouraud agarIncubate 35-37C up to 72 hrsCheck growth after overnight incubation

Bacteriological Examinationof Faecal SpecimensEnteric Pathogenic Bacteria :SalmonellaShigellaVibrio cholerae, Vibrio parahaemolyticusCampylobacterYersinia enterocoliticaEscherichia coli

Isolation MediaSelective-Differentiating Plating MediaMacConkey agar (MAC)Xylose Lysine Desoxycholate agar (XLD)Desoxycholate citrate agar (DCA)Bismuth sulfite agar (BSA)Salmonella Shigella agar (SSA)Brilliant green agar (BGA)Hektoen enteric agar (HEA)Thiosulfate citrate bile salts sucrose agar (TCBS)Taurocholate tellurite gelatin agar (TTGA)Meat extract agar (MEA)

Preparation and Inoculationsuspend faecal swab, rectal swab; fresh or in Cary-Blair in 1 ml of sterile saline (0.85% NaCl)wash swab, swirl against the side of the tube

only for formed stoolliquid: direct inoculationinoculum size depends on media, high or low selectivity and total plating media usedMacConkey: low selectivity

Salmonella, Shigella, E coli, Yersinia enterocoliticaVibrioCampylobacterMACXLDBSASel FXLDBSATCBSAPWTCBSSelective agar for CampylobacterInoculation Schema

Identification of IsolateScreening and differential testsurease and TSI/KIA

Lysine Indole Motility (LIM)Motility Indole UreaSulphide Indole Motility

Serological testsBiochemical tests

MAC, DCA, XLD, SSpick 2 4 NLF coloniesTSI, ureaseTSI reactionsK/A/g/H2SK/A/gUrease negativeK/A/H2SK/ALF coloniesA/A or A/A/gIdentification

Serological and Biochemical TestsSalmonella: PSO, PSH, O9, Vi, dHShigella : Dysenteriae, Boydii, Flexneri, SonneiVibrio cholerae: Polyvalent O, Ogawa, Inaba; (O139)E. coli: P2, P3, P4 and O128c

Biochemical confirmatory tests