STANDARDIZATION STUDY FOR AGAR WELL DIFFUSION METHOD

Abstract

Antibiotic sensitivity tests are important to determine antibiotics that can be used against disease-causing microorganisms. Although different standardization studies for disk diffusion and MIC tests were established by several organizations, no standardization study was done for agar well diffusion method. Standardization for disk diffusion method, antibiogram, was done first by World Health Organization (WHO). Today many organizations including, The European Committee on Antimicrobial Susceptibility Testing (EUCAST) in Europe, Clinical and Laboratory Standards Institute (CLSI) in USA, and Standardization of Antibiotic Sensitivity Tests (ADTS) group of Turkish Microbiology Society (TMC) in Turkey developed their own standardization methods. TMC advises the use of EUCAST protocols for studies in Turkey.

The aim of the present study was standardization of agar well diffusion method. Disk diffusion method from EUCAST was used as a reference method to compare results of agar well diffusion test. For both disk diffusion method as well as agar well diffusion test 0.5 McFarland bacterial suspension and Mueller Hinton Agar (MHA) media were used. For agar well diffusion test 3 different thicknesses, 4 mm, 6 mm and 8 mm, of MH agar was used. Also three different well diameter, 4 mm, 6 mm and 8 mm were tested. All tests were performed as three repetitions. Two antibiotics were used for disk diffusion tests and agar well diffusion tests. The concentration of erythromycin and chloramphenicol were 15 µg and 10 µg, respectively. A Gram positive and a Gram negative strain were used for susceptibility tests, S. aureus ATCC 25923 and E. coli DH10B strains, respectively. All of plates were incubated at 37°C for 16-18 hours. At the end of incubation, the inhibition zones around wells were measured.

The inhibition zones of S. aureus ATCC 25923 with erythromycin disk were 29, 30 and 31 mm on 4, 6 and 8 mm agar, respectively. Agar well diffusion with 4, 6 and 8 mm MHA media, erythromycin inhibition zones were 21, 25 and 29 mm, 22, 26 and 30 mm, and 23, 27 and 31 mm, respectively. The inhibition zones of E. coli DH10B with chloramphenicol disk were 25, 26 and 27 mm on 4, 6 and 8 mm agar, respectively. Agar well diffusion with 4, 6 and 8 mm MHA media, chloramphenicol inhibition zones were 5, 6 and 7 mm, 15, 16 and 17 mm, and 25, 26 and 27 mm, respectively. It was observed that the disk diffusion inhibition zones were in accordance with the results of 8 mm agar well zone, in every MH agar thicknesses. As the standard for disk diffusion test the MHA thickness is 4 mm, the agar well diameter for this thickness is 8 mm. So for standardization of agar well diffusion tests, the MHA thickness should be 4 mm and the diameter should be 8 mm.  

A standardization method for agar well diffusion test is necessary to compare results obtained elsewhere. Our study is the first study for standardization of agar well diffusion test. Standardization assays should be done by other laboratories to determine a worldwide usable and comparable agar well diffusion method. 

Keywords

• Agarwell,Standardization,Media thickness,Well diameter

AGAR WELL DİFÜZYON YÖNTEMİNDE STANDARDİZASYON ÇALIŞMASI

Abstract

Antibiyotik duyarlılık testleri hastalık etkeni mikroorganizmalara karşı kullanılabilecek antibiyotiklerin belirlenmesinde önemlidir. Antibiyogram testlerinde farklı kuruluşlar tarafından standardizasyon çalışması yapılmasına karşın agar well testleri için standardizasyon çalışmasının yapılmadığı görülmüş ve bu çalışmada disk difüzyon testiyle karşılaştırmalı standardizasyon çalışması yapılması amaçlanmıştır. Testler 0,5 McFarland bulanıklıkta bakteri süspansiyonu ile 3 farklı kalınlıkta (4, 6, 8 mm) Mueller Hinton agar besiyeri ve 3 farklı çapta kuyucuk (4, 6, 8 mm) kullanılarak üç tekrarlı olacak şekilde yürütülmüştür. S. aureus ATCC 25923 ve E. coli DH10B bakterileri için sırasıyla eritromisin (15µg) ve kloramfenikol (10µg) antibiyotikleri kullanılmıştır. Tüm petriler 16-18 saat, 37°C’de inkübe edilmiştir. İnkübasyon sonunda disklerin ve kuyucukların etraflarında oluşan inhibisyon zonları ölçülmüştür. Antibiyotik duyarlılık testinde kullanılan agar well yöntemi için yaptığımız standardizasyon çalışmasında en uygun Mueller Hinton Agar besiyeri kalınlığı ve kuyucuk çapı saptanmıştır. Agar kalınlığı ne olursa olsun, disk difüzyon zon çapıyla agar well kuyucuk zon çapının 8 mm kuyucukla eşleştiği görülmüştür. Agar kalınlığı arttıkça inhibisyon zonu artmaktadır. Disk difüzyon testinde standart 4 mm kalınlığındaki besiyeri olduğu için bu kalınlıktaki besiyerinde disk difüzyon sonucuna uyan inhibisyon zonu oluşturan kuyucuk çapı 8 mm olarak saptanmıştır. Standart ölçüm için 4 mm agar kalınlığı ve 8 mm kuyucuk çapı olmalıdır.

Çalışmamız agar well difüzyon konusundaki ilk standardizasyon çalışmasıdır. Diğer laboratuvarların benzer standardizasyon çalışmaları yapmaları halinde agar well difüzyon yöntemini tüm dünyada geçerli olabilecek bir standarda kavuşturabiliriz.

Keywords

• Agarwell,Standardizasyon,Besiyeri kalınlığı,Kuyucuk çapı

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