Antibiograma difuzimetrica standardizata
Principiu • O cantitate de substanta antimicrobiana este depusa pe suprafata unui mediu de cultura agarizat preinsamantata cu bacteria testata • Doua fenomene se produc concomitent: difuzarea antibioticului si cresterea bacteriei • In zonele unde antimicrobianul realizeaza concentratii mai mari decat CMI, bacteria nu creste • O data cu intrarea culturii in faza exponentiala, momentul critic, bacteria se divide mai repede decat difuzeaza medicamentul si se acumuleaza o panza vizibila de cultura , care nu mai este influentata de modificari ulterioare ale concentratiei de antibiotice • Diametrul zonei de inhibitie variaza invers proportional cu CMI • Bacteria testata este clasificata in categorii de sensibilitate, sensibila, intermediara, rezistenta prin raportarea la tabele interpretative( CLSI)
necesar • Placi cu agar Muller-Hinton, turnat in strat uniform de 4 mm grosime • Solutie salina sterila • Tulpina culturii de testat • Etalonul nr.0,5 Mc Farland cu sulfat de bariu • Tampoane de vata pe tija de lemn • Trusa cu discuri antimicrobiene comercializate de diferite firme • Pensa oftalmologica sau repartizor automat de discuri • Subler
procedura • Se prepara inoculul prin prelevarea a cca 5 colonii izolate din bacteria testata care se omogenizeaza in solutie salina sterila pana la obtinerea turbiditatii de 0.5 McFarland • Se usuca suprafata placilor agarului Muller Hinton prin mentinerea lor cu capacul intredeschis timp de 20-30 min • Se imerseaza tamponul de vata in suspensia bacteriana etalonata( inocul). Se scurge excesul de lichid prin rotirea ferma a tamponului pe perete tubului • Se descarca tamponul in struiri paralele peste toata suprafata mediului succesiv in 3 directii prin intoarcerea placii cu cate 60 grade. In final parcurge cu varful tamponului toata circumferinta mediului la limita cu sticla. In acest mod inoculul este dispersat uniform • Se lasa placa insamantata timp de 3-5 minute( niciodata mai mult de 15 min) pentru adsorbtia inoculului
• Se depun discurile cu substante antimicrobiene la distante de minim 15 mm de marginea placii si 30 mm intre centrele a doua discuri vecine, folosind o pensa sau mai bine un aparat automat de repartizare • Pe o placa cu diametrul de 9 cm pot fi depuse 8 discuri . • Se preseaza ferm cu pensa fiecare disc pe suprafata mediului • Se incubeaza placile la 37° C, timp de 18-20 ore
Citirea si interpretarea • Se masoara cu sublerul sau rigla gradata in mm diametrul zonelor de inhibitie completa, in lumina reflectata, pe fond negru mat • Se noteaza diametrele in mm ale zonelor de inhibitie si se compara cu ghidurile CLSI pentru a determina daca bacteria este sensibila, intermediara sau rezistenta la antibioticul respectiv
• FIG. 17. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the test plate. Step 2. Rotate the swab against the side of the tube while applying pressure to remove excess liquid from the swab prior to inoculating the plate. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 17. Kirby-Bauer disk diffusion susceptibility test protocol, inoculation of the test plate. Step 2. Rotate the swab against the side of the tube while applying pressure to remove excess liquid from the swab prior to inoculating the plate. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 19. Inoculation of the Mueller-Hinton agar plate, diagram illustrating the pattern the swab should follow as it is drawn across the plate. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 19. Inoculation of the Mueller-Hinton agar plate, diagram illustrating the pattern the swab should follow as it is drawn across the plate. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 19. Inoculation of the Mueller-Hinton agar plate, diagram illustrating the pattern the swab should follow as it is drawn across the plate. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 22. Place the palm of your hand on the top of the handle. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 23. Press down firmly and completely to dispense the disks. The springloaded handle will return to the original position when pressure is removed. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 24. Kirby-Bauer disk diffusion susceptibility test protocol, placement of antimicrobial disks using forceps to manually place the disks. Step 1. Antimicrobial disks can be manually placed on the Mueller-Hinton agar plate if desired. Place the MuellerHinton agar plate over the disk template. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 25. Remove one disk from the cartridge using forceps that have been sterilized. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 25. Remove one disk from the cartridge using forceps that have been sterilized. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 27. Press the disk with the forceps to ensure complete contact with the agar surface. Replace the lid of the plate between disks to minimize exposure to air-borne contaminants. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS
• FIG. 29. Measuring zones of inhibition. Gray shading represents a confluent lawn of bacterial growth. The white circle represents no growth of the test organism. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• FIG. 30. Kirby-Bauer disk diffusion susceptibility test protocol, measuring zone sizes. Using a ruler or caliper measure each zone with the unaided eye while viewing the back of the petri dish. Hold the plate a few inches above a black, nonreflecting surface illuminated with reflected light. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
• IG. 31. The size of the zone for this organismantimicrobial combination is 26 mm. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
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FIG. 32. Kirby-Bauer disk diffusion susceptibility test protocol, an alternate method for measuring zones. If the zones of adjacent antimicrobial disks overlap, the zone diameter can be determined by measuring the radius of the zone. Measure from the center of the antimicrobial disk to a point on the circumference of the zone where a distinct edge is present. Multiply this measurement by 2 to determine the diameter of the zone of inhibition. In this example, the radius of the zone is 16 mm. Multiply this measurement by 2 to determine a zone size of 32 mm for this organismantimicrobial combination. (Jan Hudzicki, University of Kansas Medical Center, Kansas City, KS)
