Inoculum preparation Table 1. Recommended media, inoculum and To obtain reproducible MICs from a gradient based Use the inoculum guide in TABLE 1. Emulsify several incubation1). system, the stability of the gradient must be maintai- well-isolated colonies from an overnight agar plate in lêÖ~åáëã= ^Ö~ê= fåçÅìäìã fåÅìÄ~íáçå ned throughout the critical period when the position a suitable suspension medium to achieve the specifi ed Öêçìé ãÉÇá~ of the growth/inhibition edge for a particular bac- inoculum turbidity by comparing to a McFarland pìëéÉåëáçå qìêÄáÇáíó= qÉãéÉê~íìêÉ= ^íãçëéÜÉêÉ qáãÉ= Antimicrobial Susceptibility Testing terium/antibiotic combination is determined. Due turbidity standard. For fastidious organisms such as EjÅc~êä~åÇF Eœ=O=ø`F EÜçìêëF For In Vitro Diagnostic Use to the stability and precision of the Etest predefi ned pneumococci, streptococci, gonococci, anaerobes and gradient, MIC values have been shown to be repro- Haemophilus spp., use the suspension prepared in INTENDED USE ducible and equivalent to those of the CLSI reference broth within 15 minutes. ^ÉêçÄÉë jìÉääÉê= MKURB=k~`ä MKR================== PR=ø` ~ãÄáÉåí NSJOM Etest is a quantitative technique for determining dilution procedures. eáåíçå EN=áÑ=ãìÅçáÇF the antimicrobial susceptibility of Gram negative Inoculation and Gram positive aerobic bacteria such as En- REAGENTS Soak a sterile, non-toxic swab in the inoculum terobacteriaceae, Pseudomonas, Staphylococcus and Etest is supplied in a package of 100 or 30 (some suspension and remove excess fl uid by pressing it lop^L=lopb jìÉääÉê= MKURB=k~`ä MKR PR=ø` ~ãÄáÉåí OQ=lop^==== Enterococcus species and fastidious bacteria, such as reagents) test strips of one antimicrobial agent. against the inside wall of the test tube. Remove more anaerobes, N. gonorrhoeae, S. pneumoniae, Streptococ- eáåíçå=H=OB= QU=lopb fl uid when streaking a 90 mm plate and less for a k~`ä cus and Haemophilus species. Th e system comprises STORAGE 150 mm plate. Carefully streak the entire agar surface a predefi ned antibiotic gradient which is used to All packages must be stored either at controlled room three times, rotating the plate 60 degrees each time determine the Minimum Inhibitory Concentration temperature (18-22 °C), in a refrigerator to evenly distribute the inoculum. Alternatively, use ^å~ÉêçÄÉë _êìÅÉää~= _êìÅÉää~=çê= N PR=ø` UMJURB= OQJQUJTO= (MIC), in μg/mL, of diff erent antimicrobial agents (4-8 °C) or freezer (-18- -22 °C) as specifi ed on the Retro C80 (rota-plater) to effi ciently streak the ino- _äççÇ jìÉääÉê= kOLRJNMB= ÇÉéÉåÇáåÖ= against microorganisms as tested on agar media using product label, until the given expiry date. culum over the agar surface. Allow excess moisture eáåíçå=ÄêçíÜ çå=íÜÉ= overnight incubation. `lOLNMB=eO to be absorbed for approximately 15 to 20 minutes ëéÉÅáÉë Etest gradient strips left over from an opened package so that the surface is completely dry before applying SUMMARY AND EXPLANATION must be kept dry. Th e opened package should be the Etest gradient strips. e~ÉãçéÜáäìë= eqj jìÉääÉê= MKR================== PR=ø` RB=`lO OMJOQ Current Antimicrobial Susceptibility Testing (AST) either re-sealed with a sealing clamp or placed in áåÑäìÉåò~É eáåíçå=çê= EN=áÑ=ãìÅçáÇF methods are based either on quantitative dilution an airtight storage container with colour indicating Notes: eqj=ÄêçíÜ techniques or qualitative diff usion procedures. Dilu- desiccant, and stored at the temperature stated on 1. When the inoculum and inoculation are optimal, tion methods are based on two-fold serial dilutions the label or at -20 °C. Left-over strips in storage con- an even confl uent growth will be obtained. jìÉääÉê= jìÉääÉê= MKR================== PR=ø` RB=`l OMJOQ of antibiotics in broth or agar media. Th ese methods tainers can be used until the expiry date if correctly 2. McFarland turbidity standards do not guarantee píêÉéíçÅçÅÅìë= O generate the MIC value i.e. Minimum Inhibitory stored and handled. Ensure that the batch number correct number of viable cells in the suspension. éåÉìãçåá~É= eáåíçå=H=RB= eáåíçå=ÄêçíÜ EN=áÑ=ãìÅçáÇF Concentration of a given antibiotic in μg/mL that and expiry date are marked on the storage container. Perform colony counts regularly to verify that the ~åÇ= ëÜÉÉé=ÄäççÇ will inhibit the growth of a particular bacterium inoculum procedure gives the correct number of píêÉéíçÅçÅÅá=OF under defi ned experimental conditions. Prevent moisture from penetrating into or forming viable cells in CFU/mL. Please refer to the QUA- kÉáëëÉêá~= d`J~Ö~ê= jìÉääÉê= MKR PR=ø` RB=`l OMJOQ within the package or storage container. Etest strips LITY CONTROL section. O Th e MIC value is not an exact entity and the “true” must be kept dry. ÖçåçêêÜçÉ~É Ä~ëÉ=H= eáåíçå=ÄêçíÜ MIC is between the lowest concentration that Application ÇÉÑáåÉÇ= inhibits the organism’s growth and the next lower HANDLING Check that the inoculated agar surface is completely ëìééäÉãÉåíë concentration. Even under the best of controlled Before using the Etest gradient strips from an unope- dry before applying Etest gradient strips. conditions, a dilution test may not give the same ned package, visually inspect to ensure the package Open the package and handle the Etest strips as de- Notes: endpoint each time it is performed. Th e reproduci- is intact. Do not use the Etest gradient strips if the scribed under HANDLING. A template can be used 1. Please consult Etest Technical Manual for further highest value on the MIC scale. When the inhi- bility of the conventional dilution test is within ± 1 package has been damaged. to optimally position Etest strips in an equidistant information on specifi c applications. bition ellipse is below the strip (does not intersect two-fold dilution of the endpoint. Th e MIC value pattern on an agar plate. Four to six (maximum) 2. Includes ß-haemolytic Streptococci groups A, B, the strip), report the MIC < the lowest value on obtained from a standardised CLSI procedure can be Allow the original package or storage container to Etest strips can be placed on a 150 mm agar plate C and G and viridans group S. mutans, S. mitis, S. the MIC scale. regarded as the reference criterium for defi ning the reach room temperature before opening (+4 °C/ (Figure 2a). For single MICs, one or two strips can sanguis and S .bovis. • Organisms such as staphylococci, Acinetobacter susceptibility of a microorganism. approx. 30 minutes, -20 °C/ approx. 60 minutes). be used on a 90 mm agar plate (Figure 2b). Strip 3. Use well defi ned and high quality medium that spp., anaerobes and gonococci may be susceptible Ensure that moisture condensing on the outer placement is automatically optimised when using supports good growth. Th e brand chosen should to sulbactam, tazobactam or clavulanic acid per PRINCIPLES OF USE surface has evaporated completely before opening the Simplex C76 (Figure 6). For organisms expected to have good batch-to-batch reproducibility to se. For Etest PTc and TLc, this may result in an Th e Etest gradient technology is based on a com- package. Packages stored at room temperature can be be highly susceptible, use fewer strips per 150 mm ensure that accurate and reliable MIC values are inhibition ellipse with an extended parallel band bination of the concepts of dilution and diff usion used immediately. plate and only one on a 90 mm plate. obtained. of inhibition alongside the strip. Extrapolate the principles for susceptibility testing. As with other 4. For trimethoprim and trimethoprim/sulfamet- upper elliptical curvature towards the strip to ob- dilution methods, Etest directly quantifi es antimicro- Open the package by carefully cutting off the top hoxazole, ensure that the brand and batch of agar tain the MIC (consult ETEST READING GUIDE, bial susceptibility in terms of discrete MIC values. of one blister compartment, or across the top of the has a low thymine/thymidine content to minimise Figure 9). However, in using a predefi ned, stable and conti- foil pouch. When handling Etest strips manually, antagonism of the activity of trimethoprim and • If inhibition ellipses for clindamycin, erythromy- nuous antibiotic concentration gradient, Etest MIC grip only the handle of the strip i.e. the area labelled sulphonamides. cin or chloramphenicol "dip" at the endpoint, values can be more precise and reproducible than E. Do not touch the surface of the strip with the 5. Th e inherent calcium content in Mueller Hinton extrapolate the MIC at the initial indentation, i.e. results obtained from conventional procedures based antibiotic gradient, i.e. the side opposite the MIC agar may vary between brands and batch to batch. 0.5-1 dilution above the intersection. on discontinuous two-fold serial dilutions. scale (Figure 1, B). Strips can be placed in an Etest Perform quality control of agar plates on a batch • For fosfomycin showing numerous (>5 ) macro- applicator tray until ready to use (Figure 3). Th e to batch basis to qualify it for use, particularly for colonies in the inhibition ellipse, read the MIC at Although processed like the disc diff usion test, i.e. vacuum pen Nema C88™ (AB BIODISK) can be Figure 2a. Template for 6 Figure 2b. Template for 2 testing of daptomcyin. complete inhibition. A few (< 5) colonies can be similar inoculum preparation, choice of agar media used to effi ciently apply Etest strips either from the strips per 150 mm plate. strips per 90 mm plate. 6. Ensure that an effi cient anaerobic system is used ignored. When zone edges are hazy, read the MIC and incubation conditions, Etest is not a diff usion applicator tray or the original foam cartridge (Figure to achieve rapid anaerobiosis to avoid false resis- at 80% inhibition. method and diff ers totally in concept from conven- 4).