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01/03/2013

MIKROBIOLOGI
BIOTEKNOLOGI A MULIGHED ’A’ KARAKTER 7-12

Mahdi Al-Dhahiri, Mathias Pius og Hadi Horani 2.e Cphwest HTX

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

FORMÅL
Øvelsen har 2 formål: 1) Identifikation af 6 forskellige bakterier. 2) Bestemmelse af resistensmønster (følsomhedsbestemmelse) for 2 af de identificerede bakterier.

RESULTATER
Se vedlagte bilag.    Skema 1: Identifikation af bakterier (bilag 1) Skema 2: Trinvis identifikation af en bakterie (bilag 2) Skema 3: Resistensbestemmelse 2 (bilag 3)

Side 1 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

DATABEHANDLING BESKRIV HVORLEDES DU HAR IDENTIFICERET DE FIRE BAKTERIER (SUPPLER MED BILLEDER). IKKE I DETALJER, MEN I OVERSIGT/FLOWDIAGRAM.

Flowdiagram over de 6 forskellige bakterier.

Bakterie 1

Gramfarvning: Negativ
• Der blev taget en koloni, og derefter blev den gramfarvet. da den fik en rød farve, kunne vi vurdere at den er gram-negativ

Form: Stave
• Ved at kigge på bakterien i et mikroskop, bestemte vi at bakterien lejrede sig i stave.

Vækst på blå plade observeret.
• Da der er vækst på den blå plade, fortsætter vi med at undersøge om bakterien er laktose positiv eller negativ.

Laktose: Positiv
• Der er gulpigmenterede områder under bakteriekolonierne og vi kan derfor se at det er en laktose positiv bakterie.

PGUA: Positiv
• Gule kolonier på PGUA-pladen betød at bakterien var PGUApositiv.

Konklusion: E. coli
• Eftersom bakterien var både PGUA-positiv og laktose-positiv, kan vi konkludere at bakterien var af arten E. coli

Side 2 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

Bakterie 2 Gramfarvning: Positiv
• Blåfarvning af bakterierne ved gramfarvning betød at bakterien var gram-positiv.

Form: Kokker
• Ud fra et mikroshop, kunne vi se at den havde en kokkeform.

Katalasetest: Negativ
• Der var ingen synlige bobler under katalasetesten, hvilket betyder at bakterien er katalase-negativ.

Lejring: Kæde
• Vi kunne se igennem et mikroshop, se at lejring var i kæde.

β-hæmolytisk: Negativ
• Ingen opklaring af blodagaren omkring kolonierne betød at bakterien ikke var β-hæmolytisk.

α-hæmolytisk: Negativ
• Bakterien blev også bestemt til at være α-hæmolytisk negativ, da der ikke sås nogen mørkfarvning af agaren.

Tellur: God vækst af sorte kolonier
• I tellur undersøgelsen, at der var god vækst af sorte kolonier, og derfor er den positiv og har navnet Enterococcus faecalis.

Konklusion: E. faecalis
• Bakterie 2 får så navnet E. faecalis.

Side 3 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

Bakterie 3 Gramfarvning: Negativ
• Vi fik oplyst, at bakterie 3 var gram negativ.

Form: Stave
• Ud fra mikroshopet, kunne vi se at den havde en stave-form.

Vækst på blå plade observeret
• Vi observerede at der var vækst på den blå plade.

Laktose: Positiv
• Ud fra laktose undersøgelsen, kunne vi aflæse at kolonierne blev gulpigmenterede.

PGUA: Negativ
• Vi aflæste at der var hvide kolonier uden gul agar, på PGUApladen og det betyder at den er negativ.

Forgæring: CBO A.1. / API
• Ud fra Enterobacteriaceae skemaet, og en del undersøgelser kom vi frem til at bakterien er en klebsiella pneunoniae.

Konklusion: Klebsiella Pneumoniae
• Bakterie 3 fik navnet Klebsiella pneumoniae.

Side 4 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

Bakterie 4

Gramfarvning: Negativ
• Vi fik oplyst at bakterie 4 var gram negativ.

Form: Stave
• Den havde en stave-form.

Oxidase: Positiv
• Vi kunne se at reaktionen indenfor 10 sekunder, blev farvet dybt blå. Det ville sige at den er positiv.

Bevægelighed: Polær
• Igennem et mikroskop, kunne vi ikke se om den havde en flageller, da de er rigtig små, men vi fik oplyst af læreren at den havde polær bevægelighed.

Halvfl. agar: Strikt Aerob
• Som ses på billedet, kunne vi aflæse at det var en strikt aerob, da aerobe bakterier vokser øverst i glasset.

Metalskær + Karakteristisk lugt: Positiv
• Vi kunne observere at den havde metalskær og lugtede karakteristisk. Positiv

Konklusion: Pseudomonas Aeruginosa
• Bakterie 4 fik navnet Pseudomonas aeruginosa.

Side 5 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

Bakterie 5

Gramfarvning: Positiv
• Vi har fået oplyst at bakterien er gram-positiv

Form: Kokker
• Vi får oplyst at bakterien har form som kokker.

Katalasetest: Positiv
• Vi lavede en katalase undersøgelse, og kom frem til at den boblede, det betyder så at den er positiv.

Lejring: I Hobe
• Bakterien lejrede sig i hober.

Clumping/koagulase vha. Slidex staphkit/hestecitratplasma
• Ud fra kortet, kunne vi observere at reaktionen var positiv, da den aggulererede.

Konklusion: Staphylococcus Aureus
• Bakterie 5 fik navnet Staphylococcus aureus.

Side 6 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

Bakterie 6

Gramfarvning: Positiv
• Det er blevet oplyst at den er gram-positiv kokker.

Form: Kokker

Katalasetest: Negativ
• Negativ katalase, da den ikke boblede.

Lejring i: Kæde
• Igennem et mikroskop, kunne vi observere det til at være i kæde.

β-hæmolytisk: Positiv
• Ud fra blodagarpladen, kunne vi konkludere at bakterien kan producere hæmolysin. Den er så positiv.

Hæmolytiske streptokokker gruppe: A/B/C/D/F/G eller E. faecalis (tellur:+)
• Efter vi har udført, på alle seks felter på kortet, kan vi se at den aggulerer i Gruppe A.

Konklusion: Streptococcus Pyogenes
• Bakterie 6 fik navnet Streptococcus pyogenes.

Side 7 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

BESKRIV HVORLEDES DU TESTEDE DE TO BAKTERIER FOR ANTIBIOTIKARESISTENS (SUPPLER MED BILLEDER).
Antibiotika resistens også kaldet agardiffusions metoden, der startede vi med at bruge, først vores to gramfarvning plader(plade 1 og 5). Hvor gramfarvning-1 er negativ og gramfarvning-2 er positiv, så indsætte vi antibiotika, i form af tre forskellige tabletter(Penicillin ‘Pen l’, Chloramphenicol ’CLR30’ og Tetracyclin ’TET 30’). Derefter lagde vi dem lidt spredt fra hinanden, på blodagarpladen og efter inkubationstiden kan man se hvor meget bakterievækst, der er rundt om tabletten eller om der slet ikke er dannet nogen vækst. Vi målte så diameteren i millimeter rundt omkring de tre forskellige tabletter og så kunne vi finde ud af hvor følsom, antibiotika er for bakterien.    (S)ensitive: følsomme bakterier har ingen påviste resistensmekanismer og kan ikke vokse ind til tabletten, og danner en zone omkring den. (I)ntermediær: Bakterien har nedsat følsomhed for pågældende antibiotikum, og vokser ind til tabeletten. (R)esistente: Bakterien har en eller flere signifikante resistensmekanismer, og kan derfor vokse helt tæt omkring tabletten.

Figur 2 Gramfarvning 1 negative ’antibiotika’

Figur 1 Gramfarvning 5 positive ’antibiotika’

DISKUSSIONSSPØRGSMÅL
a) Beskriv kort gramfarvningens princip, og hvilke fejlkilder kan der opstå ved gramfarvning?

Gramfarvning bruges i mikrobiologiske sammenhæng til at identificere forskellige celletyper på baggrund af dennes membran. Ved gramfarvning behandles en celleprøve først med krystalviolet der binder til cellevæggen, hvorefter en jodopløsning danner komplekse forbindelser mellem den tilførte krystalviolet og cellevæggen i de grampositive celler hvor cellevæggen typisk er tykkere. Dernæst skylles prøven med ren ethanol, hvorved cellerne, såfremt de er gramnegative, affarves, da deres cellevæg ikke har kunnet danne de samme komplekse forbindelser som tykkere cellevægge kan. Til sidst behandles prøven med kontrastfarve således at det er tydeligere at se om cellerne er grampositive eller negative. Hvis gramfarvningen ikke udføres nøjagtigt er der rig mulighed for at fejlkilder fremkommer. Allerede ved fikseringen af organismen på objektglasset kan der ske fejl, da organismen enten kan overophedes hvorved cellevæggen sprænges og testen vil være ubrugelig, eller påførelsen af varme kan være for striks, med resultatet ikke fikseres ordentligt og vasker væk ved den første væskebehandling. Ved affarvningen med ethanol

Side 8 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

kan det, ved overforbrug af ethanol, risikeres at selv de ellers-grampositive organismer affarves, og resultatet vil være ubrugeligt. Ligeledes kan underforbrug af ethanol få gramnegative organismer til at fremstå grampositive da de ikke affarves korrekt. For store prøveportioner kan betyde at farvevæsken fanges i selve prøvemediet i stedet for i cellevæggene, og kan være svære at affarve, hvorved et falsk-positivt resultat kan fremkomme. Cellerne i prøven skal derudover også være under 24-timer gamle, da grampositive organismer efter denne grænse kan fremstå som gramnegative ved prøvens konklusion. Organismerne bør heller ikke være antibiotikabehandlede på dette tidspunkt da antibiotika kan påvirke cellens evne til at vedligeholde cellevægge, og tilmed nedbryde dem. I et sådant tilfælde kan grampositive organismer fremkomme som gramnegative da de ikke vil kunne binde krystalviolet i deres cellevæg.

b) Forklar antibiotikas forskellige virkemåder Antibiotika har i alt tre forskellige måder med hvilken den kan forhindre en bakterie i at sprede sig. I penicillin virker ved at efterligne det peptid cellen bruger til at forstærke cellevæggen. Glycopeptid transpeptidase er et enzym der indsætter tværbroer bestående af peptider i cellevæggen for at forstærke den. Når penicillin bindes af dette enzym blokerer dens aktive β-lactamring, som ikke findes i det oprindelige peptid, transpeptidasen og sætter enzymet ude af funktion. Uden mulighed for at skille sig af med penicillin molekylet er transpeptidasen ikke længere i stand til at konstruere de forstærkende tværbroer i cellevæggen, hvorved væggen kollapser. Et andet lignende antibiotikum, vancomycin, efterligner ikke peptidet, men binder sig i stedet til allerede eksisterende D-alanin peptider og blokerer derved for glycopeptid transpeptidases funktion. Andre antibiotika fungerer ved at angribe bakteriens proteinsyntese. Eksempler på denne type antibiotika er tetracyclin, gentamicin samt quinoloner. Tetracyclin binder sig til bakteriens ribosomer og forhindrer translation i at finde sted. Gentamicin kan infiltrere en celle og binder, ligesom tetracyclin, til bakteriens ribosomer, hvor den i stedet for at deaktivere dem, interfererer i proteinsyntesen, og gør ribosomet ude af stand til at danne de proteiner som mRNA’et koder for. Uden evnen til at danne proteiner efter behov, dør cellen. Quinoloner går ulig tetracyclin og gentamicin ikke efter ribosomerne, men efter en gruppe enzymer kaldet topoisomeraser der åbner bakteriens lukkede cirkulære DNA når der skal transskriberes mRNA til nye proteiner. Ligesom de andre eksempler dør cellen også her, da den ikke kan producere proteiner.

c)

Hvilke mekanismer kan gøre bakterier resistente overfor antibiotika

Eftersom de fleste antibiotika fungerer ved at hæfte sig på eksisterende proteiner i cellen er de meget følsomme overfor ændringer i disse proteiner. Ved mutation af et bakteries DNA kan kodningen for et protein ændres, hvilket kan skabe problemer for antibiotikamolekylet når det for eksempel forsøger at binde sig til ribosomer i cellen, eller peptider. Eftersom der typisk er meget kort tid mellem bakteriegenerationer, og mangfoldigheden af celler i et passende miljø kan bakterier mutere og tilpasse sig meget hyppigt, hvilket gør mutationer i deres genom til en kæmpe trussel for antibiotikas effektivitet. Naturlig selektion betyder også at når først en sådan mutation er til stede i en population har den en seriøs fordel overfor andre bakterier af denne art, hvorfor den hurtigt bliver udbredt og antibiotikakuren bliver uvirksom. Penicilliner virker som tidligere nævnt ved at binde til glycopeptid transpeptidasen, hvor en aktiv β-lactamring forhindrer enzymet i at binde og forstærke cellevæggen. Mutationer i nogle bakterier har imidlertid givet dem evnen til at producere enzymet β-lactamase der kan nedbryde β-lactamringen, og dermed

Side 9 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

penicillinmolekylet, og gøre det ineffektivt til behandling af den pågældende bakterie. Udover tilfældige mutationer kan bakterier også udveksle resistensgener indenfor egen art, og i visse tilfælde også fremmede arter. En del af det kodende DNA i bakterier findes i små ringformede plasmider som findes i cytoplasmaet. Bakterier kan lave kopier af disse plasmider og udskille dem, hvorefter andre bakterier kan optage dem og tilføje dem til deres repertoire. På denne måde kan resistensgener frit udveksles imellem bakterier.

d) Nævn nogle eksempler på, hvordan der kan ske kontaktsmitte i laboratoriet Kontaktsmitte kan finde sted enten direkte eller indirekte. Direkte kontaktsmitte kan opstå når der arbejdes frit med bakterier, eksempelvis ved fejlagtig behandling af kontaminerede værktøjer så som øjepodenåle, objektglas, eller agarplader/petriskåle. Direkte kontaktsmitte er nødvendig for mikroorganismer der ikke tåler udtørring eller nedkøling. Indirekte kontakt kan ske hvis man kommer i berøring med et objekt der tidligere har været i berøring med en bakterie. Hvis en kittel har været i kontakt med en bakterie og det kontaminerede område senere kommer i kontakt med en persons hud. Forskellen fra direkte kontaktsmitte er typisk at disse mikroorganismer tåler at udtørre og blive nedkølet, hvorfor de udgør en smittefare længe efter at være fjernet fra gromiljø.

e) Hvad betyder inkubationstid Inkubationstiden for en organisme er et udtryk for hvor lang tid der går fra første kontakt organismen til de første symptomer præsenterer sig. Eftersom symptomer på en sygdom typisk først viser sig når en infektion af sygdomsfremkaldende organismer når en vis størrelse, afhænger inkubationstiden meget af organismens fordoblingstid og initialstørrelse.

f)

Hvad er formålet med en tretrinsspredning

Ved en tretrinsspredning sås kolonier ved flere forskellige fortyndinger, så kolonier kan studeres på flere forskellige størrelsesordener. Ved anvendelse af denne metode kan kolonier således studeres både på makroskopisk og mikroskopisk plan, altså hvordan både større og mindre kolonier gebærder sig.

g)

Hvilken test bruges til at skelne imellem stafylokokker og streptokokker

Da stafylokokker, ulig streptokokker, danner enzymet katalase kan disse differentieres mellem ved at udføre en katalasetest på mikroorganismen for at bestemme hvilken én man har med at gøre. Det kan dog ikke konkluderes at en mikroorganisme tilhører en af disse to grupper kun på baggrund af en katalasetest, og yderligere informationer er nødvendige til at identificere prøver. Er man dog allerede sikker på at man har med enten den ene eller anden at gøre, vil en katalasetest ved positivt resultat vise at man har med stafylokokker at gøre, og ved negativt at der er tale om streptokokker.

Side 10 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

h) Hvad er en hæmolyse Hæmolyse er en proces der fremkommer, når hæmolytiske bakterier (som streptokokker) lyserer erythrocytter (røde blodceller). α- og β-hæmolytiske bakterier producerer enzymet hæmolysin der opløser erythrocytters cellevæg, hvorved dennes indhold frigives. På en blodagarplade vil en β-hæmolytisk koloni præsentere sig ved at agarpladen opklares omkring kolonierne da erythrocyttens indhold af hæmoglobin frigives og derefter diffunderes væk. I αhæmolytiske celler vil denne opklaring modarbejdes af produktionen brintoverilte der forekommer som produkt ved aerob vækst hos bakteriecellen, som tilføjer en grøn-brun farve til blodagaren.

i)

Hvordan afgøres om en bakterie er katalase-positiv?

Der findes bakterier, der danner katalase, som er et enzym, der omdanner brintoverilte (H 2O2 hydrogenperoxid), til ilt (O2) og vand (H2O). Det der afgør om en bakterie er katalase-positiv er, hvis man følger disse trin, og der så er bobler: ”En dråbe 3% brintoverilte afsættes på et objektglas Med en øjepodenål opsamles lidt kolonimateriale, som afsættes på dækglas, der holdes i hånden Dækglasset placeres oven i dråben, således at kolonimaterialer vender nedad Der observeres for bobler under dækglasset” [1]

j)

Hvad gør, at bakterier kan bevæge sig, og hvordan kan det bedømmes om en bakterie er bevægelig?

Det der gør, at bakterier kan bevæge sig er, at de har flageller. Der er to typer flageller: Polære flageller: at de/den sidder i den ene ende, så de bevæger sig altså målrettet (polært bevægelige) Peritriche flageller: at de/den sidder rundt om hele flagellen, så de drejer rundt om sig selv (Peritricht bevægelighed) Det bedømmes, at de er bevægelige ved, at de/den bevæger sig i modsat retning end andre, altså den drejer rundt om sig selv, eller bevæger den/de sig målrettet.

k)

Hvad betyder det, at en bakterie er fakultativt anerob, og hvordan kan en bakteries iltkrav undersøges?:

Hvis en bakterie er fakultativt anerob, så kan den tåle alle iltkoncentrationer, og så vokser den igennem hele reagensglasset. Iltkravet hos en bakterie kan undersøges ved, at observere, hvor i glasset der er vækst. Hvis der er vækst i bunden, så det anaerobe bakterier (kan ikke tåle ilt) Hvis der er vækst i midten, så det mikroaerofile bakterier (kan tåle lidt ilt) Hvis der er vækst øverst, så er det aerobe bakterier (er iltkrævende) Hvis der er vækst igennem det hele, så er det som sagt en fakultativt anaerobe bakterier (kan tåle alle iltkoncentrationer)

Side 11 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

l)

Forklar den test, der får streptokokker til at agglutinere (antigen-antistof)?:

Denne test går ud på, at teste kokkerne for gruppeantigen A, B, C, D, F og G vha. et Stretex testkit. I testen fremstiller man først ekstraktionsenzym (et selv ”lavet” enzym), først derefter undersøger man, hvilken gruppeantigen der er, ved at kigge efter det felt, hvor der er klumper, og det vil ligne skilt mælk. Dette er agglutination.

m) Hvad er et selektivt-indikativt substrat, og giv eksempel på, hvor det er anvendt?: Et selektivt-indikativt substrat, er et medie, der både indikerer noget og hæmmer nogle bakterier. Et eksempel på et selektivt-indikativt substrat er PGUA-plade.

n) Hvad er årsagen til at forskellige kulhydratforgæringer kan anvendes til identifikation af bakterier?: Årsagen til det er, at man indikerer hvilken bakterie det er (ved farveskift), da bakterier (som os mennesker) har forskellige genetiske egenskaber. Det er en test, der påviser om bakterien kan forgære (fermentere) det kulhydrat som testbouillonen indeholder, og dermed danner syrer under forgæringen, som får pH til at falde, og dermed sker der et farveskift. [2]

o) Nævn forskellige metoder, der kan anvendes til at diagnosticere (=identificere) bakterier: Der findes forskellige måder, at identificere bakterier på, og her er nogle af dem: Enten-eller metoden: denne metode går ud på, at man undersøger et karakter ad gangen, og så finder man ud af, om det enten er positivt eller negativt, og sådan fortsætter man. Vi har f.eks. brugt et flowdiagram til denne metode. En fordel ved denne metode er, at man normalt kun undersøger få karaktertræk, men ulempen ved det er, at det tager for lang tid, da man skal have svar på hvert karaktertræk, så man kan fortsætte. Den trinvise identifikationsmetode: denne metode går ud på, at man starter med, at undersøge nogle få primære karaktertræk (gram-reaktion, bevægelse, iltkrav osv.), hvor man så, bruger enten-eller reaktion af gramfarvning af bakterien. Resultaterne fra testene bruges til, at se på identifikationsmodeller, hvorved det så skal vise hvor i bakteriesystemet, der skal søges efter bakterien (vi har anvendt denne metode, i skema 2). Identifikation ved brug af PCR (Polymerase Chain Reaction) – teknik: denne metode går ud på, at man isolerer og opformerer (danner et større antal) bakteriens DNA ved PCR. Det opformerede DNA kan så, påvises ved gelelektroforese (en adskillelsesteknik af DNA i dette tilfælde) [3]

Side 12 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

KONKLUSION
Vi har taget de 6 forskellige bakterier, trinvis (i kronologisk rækkefølge) i flowdiagrammet ’identifikation af bakterie’, ved hjælp af enten-eller metoden og trinvismetoden. Indtil vi nåede frem, til identifikation af bakterien. Vi kan konkludere at de 6 forskellige bakterier er blevet bestemt, og delforsøgene som blev udført, indtil vi kom til den endelige bakterie, gik meget godt, og formentlig uden fejlkilder. Vi kan også bestemt, resistensmønster for gramfarvning-positiv og gramfarvning-negativ. Hvilken følsomhed de tre forskellige antibiotika tabletter er, ’senstive, intermediær eller resistens’ og hvor stor eller lille væksten er, omkring tabletten, som vi måler.

BILAG
Bilag 1) Skema 1: Identifikation af bakterie.

Side 13 af 14

Identifikation af Bakterier Mahdi, Hadi, Mathias

Mikrobiologi Bioteknologi

01-03-2012 CPH WEST

Bilag 2) Skema 2: Trinvis identifikation af en bakterie(A1 på flowdiagram):

Bilag 3) Skema 3: Resistensbestemmelse (for en grampositiv og en gramnegativ bakterie)

LITTERATUR

[1] Kompendiet ”Identifikation og resistensbestemmelse af bakterier” s.44 [2] Hjælp af Charlotte Kragh Klausen [3] Viden til spørgsmålene er fra Kompendiet ”Identifikation og resistensbestemmelse af bakterier”

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