Standardization of in vitro susceptibility tests by the clinical and laboratory standards institute clsi and the european committee for antimicrobial susceptibility testing eucast, and current availability of reference methods constituted the major remarkable steps in the field. Comparison between disk diffusion and microdilution. Disc diffusion method 6mm sterile filter paper discs were purchased and sterilized. Disc diffusion method principle paper discs impregnated with specific antibiotics or the test substances are placed on the surface of the muller hinton agar medium inoculated with. The disc diffusion method was used to screen the antibacterial activity of both azadirachta indica leaf extractand synthetic antibiotics. Antifungal susceptibility testing of dermatophytes by agar. When a 6mm filter paper disk impregnated with a known concentration. Comparison of tablet and disk diffusion methods for. Pdf comparative study of disk diffusion and microdilution methods. Pdf comparative study of disk diffusion and microdilution.
Kirbybauer disk diffusion susceptibility test protocol asm. The standard disk diffusion method was used to test antifungal drugs against yeasts, and nondermatophyte filamentous fungal isolates. Antimicrobial activity of essential oils and other plant. Antimicrobial susceptibility was tested using paper disc agar diffusion method bauer et al. The disk diffusion method of kirby and bauer has been standardized and is a viable alternative to broth dilution methods for laboratories without the resources to utilize the newer automated methods for broth microdilution testing. Kirby bauer method and the liquid media based microbroth dilution method. Antimicrobial activity of methanol extracts of mosses from serbia. Antibacterial activity of azadirachta indica neem leaf. Paper discs 5 mm were sterilized by autoclave and soaked in a. Clsi document m44, clinical and laboratory standards institute, wayne, pa 2009. It uses antibiotic discs to test the extent to which bacteria are affected by those antibiotics. Study of the antibacterial and antifungal activities of synthetic benzyl bromides, ketones, and corresponding chalcone derivatives muhamad ali k shakhatreh,1 mousa l alsmadi,2 omar f khabour,1,3 fatima a shuaibu,1 emad i hussein,4 karem h alzoubi51department of medical laboratory sciences, 2department of applied chemical sciences, jordan university of science and technology, irbid, jordan. We tested 55 strains of dermatophytes against 4 commonly used antifungal agents viz.
Volume 9, issue 2, 2020, 961 967 issn 22846808 letters. Secondly, the method used to assess antimicrobial activity, and the choice of test organisms, varies between publications janssen et al. A comparison of disc diffusion and microbroth dilution methods for the detection of. Current status of antifungal susceptibility testing methods. Invitro activities of caspofungin compared with those for fluconazole and itraconazole. We concluded that the wellvariant of the diffusion method was more sensitive. The best antifungal efficacy 100 % was shown by clove, cinnamon and oregano at the concentration of 30 %. Gary young2 1chemical ecology laboratory, department of integrative biology, brigham young university, provo, utah, usa. Media are supplemented with 12% sodium chloride nacl if intended for. Comparative study of disk diffusion and microdilution. Other methods are used especially for antifungal testing, such as poisoned food technique.
Antimicrobial activity of some ethnomedicinal plants used. We describe a newly developed agarbased method employing disk diffusion assay to test the. The mics were determined as the oils were screened for their antifungal activity against lowest concentration of oil inhibiting the visible growth of a. Full length research paper in vitro antibacterial and. Each purified extracts were dissolved in dimethyl sulfoxide, sterilized by filtration using sintered glass filter, and stored at 4c. An invitro study abstract plant derived products have been used for medicinal purposes for centuries.
The disk diffusion test, or agar diffusion test, or kirbybauer test disc diffusion antibiotic susceptibility test, disc diffusion antibiotic sensitivity test, kb test, is a test of the antibiotic sensitivity of bacteria. Advantages of a standardized disk diffusion based assay for evaluating the antifungal susceptibility of dermatophytes include the ease of use, reproducibility, accuracy, and low cost 1, 3, 10, 15, 16, 22. Antibacterial and antifungal activities of plant part extracts against four pathogenic bacteria two grampositive and negative and three pathogenic fungi were investigated by the agar disk diffusion method. Antifungal susceptibility testing is a very dynamic field of medical mycology.
Measure from the center of the antimicrobial disk to a point on the circumference of the zone where a distinct edge is. The activity was quantitatively assessed on the basis of inhibition zone and their activity index was also calculated along with minimum inhibitory concentration mic. Well diffusion for antifungal susceptibility testing. In addition, the use of nonsupplemented muellerhinton agar in. Disc diffusion method in order to test antifungal activity of extracts of mosses against candida albicans, the disc diffusion method was used. In vitro antimicrobial activity of some medicinal plants.
A stock solution of extract was prepared by dissolving 0. In traditional indian medicine or ayurveda, zingiber officinale and many other herbs have been used as medicine. Optimization and isolation of dermatophytes from clinical. M44 method for antifungal disk diffusion susceptibility. Uses of antibiotic sensitivity testing antibiotic sensitivity test. Chloramphenicol disc was used as a positive control. A comparison of disc diffusion and microbroth dilution. Evaluation of antimicrobial activity of different solvent. If the zones of adjacent antimicrobial disks overlap, the zone diameter can be determined by measuring the radius of the zone. The disk diffusion assay method for antifungal susceptibility testing of dermatophytes species in this in vitro study may give insights into the application in therapeutic strategy against dermatophytic infections. Antibacterial and antifungal activities from leaf extracts.
In vitro antibacterial activity was assessed by disc diffusion and microdilution methods. Currently, this method is validated only for select. Method for antifungal disk diffusion susceptibility testing of yeasts. Bsac disc diffusion method for antimicrobial susceptibility. Thus, a varied range of natural products of plant, fungi and lichen origin were tested against two bacterial species, staphylococcus aureus atcc 25923 and escherichia coli atcc 25922, by two variants of the agar diffusion. Antibacterial and antifungal activities of spices mdpi. Disk diffusion method is a simple and costeffective for susceptibility testing of dermatophytes.
Methods for in vitro evaluating antimicrobial activity. Screening of essential oils for antibacterial activity was done by the disk diffusion method, which is normally used as a preliminary check and to select between efficient essential oils. Bsac disc diffusion method for antimicrobial susceptibility testing version 2. Antibacterial activity of leaf extracts of baeckea frutescens. Disc diffusion method was used for antibacterial activity. A variety of laboratory methods can be used to evaluate or screen the in vitro antimicrobial activity of an extract or a pure compound. Measurement of antimicrobial activity using agar well diffusion method. Correlation between microdilution, etest, and disk diffusion methods for antifungal susceptibility testing of posaconazole against candida spp. The agar diffusion method is a practical, agarbased method which enables the determination of the activity of various antifungal drugs.
Agar well diffusion method is widely used to evaluate the antimicrobial activity of plants or microbial extracts. The disc diffusion method for antibiotic susceptibility testing is the kirbybauer method. Diekema, md, facp ana espinelingroff, phd cynthia l. The determination of antibacterial and antifungal activities. Then, the antifungal activity was evaluated using the well diffusion method 41, and each well contained 150 l of the supernatant or methanol extract. Antifungal assay agar was formulated by berger and lazecka for convenience in assaying antifungal activity of pharmaceutical products and other materials by both base and seed layers for assays by cylinder plate or disc methods. Hospital proposed a single disk method for antimicrobial susceptibility testing 6. Agar diskdiffusion testing developed in 1940 8, is the official method used in many clinical microbiology laboratories for routine antimicrobial susceptibility testing. A laboratory test which determines how effective antibiotic therapy is against a bacterial infections. In vitro antibacterial and antifungal activities of crude propolis extract 1determination of activity by disk method. It is calibrated to eucast clinical breakpoints using broth microdilution for mic determination. The eucast disk diffusion test is based on mh media and disks of a good quality. The hexane and methanol extracts were obtained by cold percolation method and the antimicrobial activity was found using paper disc diffusion method.
Pdf correlation between microdilution, etest, and disk. Disc diffusion assay the disc diffusion assay was performed in radiationsterilized petri plates of 10 cm diameter tarsons as per the method described in indian phamiacopoeia. In order to test the effect of ph on the antifungal stability, ph values of the supernatant was adjusted to ph 3. The disk diffusion method is performed using muellerhinton agar mha, which. The determination of the efficacy was based on a modified disc diffusion method clsi m44a2. Comparative study of disk diffusion and microdilution methods. Other methods are used especially for antifungal testing. The agar diffusion method is a practical, agarbased method which enables the determination of the activity of various antifungal drugs against various fungal genera and species. Broth macro and microdilution assays can be used to determine antifungal susceptibility of dermatophytes, but these methods are expensive and require specific media. Agarbased disk diffusion assay for susceptibility testing. The standard disk diffusion method to test antifungal drugs for nondermatophyte filamentous fungi isolates m51a and supplement m51s1 provides qualitative results in 824 h when caspofungin, triazoles, and amphotericin b are used, faster than the clsi reference microdilution method17. Approved guidelinesecond edition volume 29 number 17 john h.
Agarbased disk diffusion assay for susceptibility testing of. Ethanolic and methanolic extracts in different concentrations 25%, 50%, 75% and 100% was prepared and tested against test organisms using disc diffusion method. We adopted agar based disk diffusion abdd method to test the sensitivity of common dermatophytes against two azoles fluconazole, itraconazole, griseofulvin and terbinafine. Preliminary screening of antibacterial activity using crude. In vitro antifungal activity was determined by using agar disc diffusion technique and the minimum inhibitory concentration mic was. Agarbased antifungal susceptibility testing, such as the disk diffusion dd method, can be an alternative that has a lower cost and the need for a reduced amount of labor compared with those required for the reference bd assay, although the results obtained by the dd assay can be plagued by the same variables that alter the mics obtained by. This medium is prepared according to manufacturers instructions, dispensed in tubes at 45 ml and sterilized. Evaluation of antifungal effect and toxicity of xanthyletin.
Abstractwe have compared a commercially available tablet diffusion method for the in vitro antifungal susceptibility testing of fluconazole fcz and voriconazole vcz with the disk diffusion method m44 clsi with 282 clinical yeast isolates. A method frequently used to screen plant extracts for antimicrobial activity is the agar disc diffusion technique morris et al. The disc diffusion method showed excellent correlation with the etest and the nccls methods. The antifungal activity of the methanol extract was tested using a microdilution method. Comparison between disk diffusion and microdilution methods. Methods are available for testing antifungal activity against dermatophytes but no simple reference method is available. The inoculum for the disk diffusion method is prepared using a suitable broth. Similarly to the procedure used in disk diffusion method, the agar plate surface is inoculated by spreading a volume of the microbial inoculum over the entire agar surface. Plants were collected from palni hills of southern western ghats and the ethnobotanical data were gathered from traditional healers who inhabit the study area. This method provides qualitative results after 24 hours incubation.
Comparison of broth micro dilution and disk diffusion. Azadirachta indica neem leaf extract was taken to test its antifungal activity against three fungal species aspergillus flavus, alternaria solani and cladosporium. Effective treatment of a disease entails the development of new pharmaceuticals or some potential source of novel drugs. Disk diffusion could be easily used to screen the antibacterial activity of plant extracts, however results are scarcely comparable with microdilution assay, because of problems related to. Azadirachta indicaleaf extract showed strong antimicrobial activity against all bacterial species studied at all the concentrations tested. The most known and basic methods are the diskdiffusion and broth or agar dilution methods. Antibacterial activity of leaf extracts of baeckea. The superior stability of antifungal agents in tablets can explain the differences for each category of susceptibility by both methods. In vitro activities of 8 antifungal agents against 37 strains of dermatophytesa. Here 20 ml quantities of sabouraud dextrose were plated in petri dish. In our laboratory, the antifungal activity of a range of plant extracts and chromatographic fractions was evaluated by the disk diffusion agar method and the microdilution method in accordance with m27a2 clinical and laboratory standards institute clsi, 2002 with modifications european committee on antibiotic. In this study, two methods for the determination of antifungal activity of natural products, agar diffusion and broth microdilution, the clsi reference methods. Antifungal activity of essential oils from indian medicinal.
The methanol extract showed strong antifungal activity. Antibacterial and antifungal activities from leaf extracts of. The use of standard methodology for determination of. Method for antifungal disk diffusion susceptibility testing of yeasts, 2nd ed. We describe a newly developed agarbased method employing disk diffusion assay to test the susceptibility of 47 isolates of dermatophytes against 8 antifungals. In vitro antibacterial activity of some plant essential. Study of the antibacterial and antifungal activities of. Kirbybauer disk diffusion susceptibility test protocol, an alternate method for measuring zones. The antifungal activity of the extract was tested by disc diffusion method 10,12 against the five pathogenic fungi at the concentrations of 150 gdisc and 300 gdisc for each. Method for antifungal disk diffusion susceptibility testing of nondermatophyte filamentous fungi, 1st. In phase i, the antibacterial and antifungal activity of the leaves, bark, flowers and fruit pulp of couroupita guianensis were assayed. The antibacterial and antifungal activity was evaluated as in traditional antibiotic susceptibility testing using the disc diffusion method 2.
Antifungal activity of natural products has been tested by adapting methods designed for synthetic drugs. The agar used is meullerhinton agar that is rigorously tested for composition and ph. Bsac disc diffusion method for antimicrobial susceptibility testing. Comparative study of disk diffusion and microdilution methods for evaluation of antifungal activity of natural compounds against medical yeasts candida spp and cryptococcus sp. The antifungal activity of the agent was tested in the following dilution range 1g, 2g, 4g. Assessment of antimicrobial activity of fourteen essential. Each purified extracts were dissolved in dimethyl sulfoxide, sterilized by filtration using sintered glass filter, and. On the basis of our results, it appears that the disc diffusion test is a useful method for testing the activity of voriconazole against aspergillus spp. The aromatogram method 4 is an analytical method similar with the disc diffusion assay. Commonly used medicinal plants of our community could be an excellent source of drugs to fight off this. Method for antifungal disk diffusion susceptibility. The emergence and spread of antibiotic resistance, as well as the evolution of new strains of disease causing agents, are of great concern to the global health community. Clearly, the disc diffusion method has the potential to provide a simple means of performing in vitro tests, but not all antifungal agents are available in discs. Despite the many guide lines that nccls have published for susceptibility tests of moulds m27a, m28a, there is no clear method and routine test for the evaluation of dermatophyte antifungal activity.
Our results show that the method is reproducible, is simple, and could be used to determine the antifungal susceptibility of. In vitro activities of 8 antifungal agents against 37 strains. We describe a newly developed agarbased method employing disk diffusion assay to test the susceptibility of 47. Antifungal effect of selected essential oils on malassezia. Pdf antifungal activity of natural products has been tested by adapting methods designed for synthetic drugs. Currently, no agarbased susceptibility testing method has been standardized for testing dermatophytes. Kirbybauer disk diffusion susceptibility test protocol. Further the depth of the agar in the plate is a factor to be considered in the disc diffusion method.