Blood can be transferred to the agar plate either by a pipette (after first having transferred blood to an empty tube) or by applying drops on the plate from the blood culture bottle ventilator or from a syringe. For this to give accurate volumes, it is important to calibrate the procedure to yield 125 ± 25 μL.
Yes, the method can be performed on plates of other sizes and shapes but the amount of blood in the inoculum has to be changed. Contact EUCAST for more information on the amount of blood for different plates.
The inhibition zones are different for each antibiotic and species combination, some are sharp and some fuzzy. To make it as easy as possible to read the zones, be careful when streaking the plates. Streak the plate to avoid patterns from the streaking (do not press too hard) and make sure that the inoculum is evenly distributed over the agar surface. A rotating plate inoculator is useful.
Isolated colonies in the inhibition zones for piperacillin-tazobactam, imipenem and meropenem zones with P. aeruginosa after 16-20 hours incubation is a result of the heavy inoculum in combination with the prolonged incubation. The same phenomenon appears with P. aeruginosa ATCC 27853 when processed according to RAST. Ignoring isolated colonies when reading zones for these specific combinations has been validated against reference broth microdilution for clinical isolates and does not result in erroneous categorization. For all other species-agent combinations, colonies within zones should be taken into account when reading.
Following a positive signal, the EUCAST RAST is validated for storage up to 3 hours in room temperature and up to 18 hours in the blood culture instrument. Any change in these procedures needs to be validated by the respective laboratory.
No, RAST should not be performed on bottles with mixed species.
The RAST breakpoints for 16-20 hours incubation were developed to allow interpreting and reporting RAST results when blood cultures bottles signal positive at a time in the day which, because of limited opening hours, will not allow reading of results after 4, 6 or 8 hours (e.g. in the afternoon). The 16-20h incubation shall only be used when plates cannot be read after 4, 6 or 8 hours incubation.
Breakpoints for A. baumannii are not validated for other species of Acinetobacter. All EUCAST RAST breakpoints are valid only for the species listed for each table.
At the moment we are not planning on setting breakpoints for more species, but we are intending to deliver breakpoints for more antibiotics for E. coli, K. pneumoniae and P. aeruginosa.
EUCAST does not have any formal recommendation for how many times the QC procedure should be repeated before the system is considered validated. A minimum of five tests on separate days is appropriate. The results should then be compared to the RAST QC targets and ranges. The perform adjustments and repeat QC as needed.
Either blood culture bottle (eg. aerobic and anerobic) can be used.
The method was developed for horse and sheep blood. The use of other types of blood or growth supplements might result in different zone sizes and has to be validated at the local laboratory.
Make a 0.5 McFarland solution and dilute the suspension 1:1 000 000 by transferring 10µl solution to a 990 µL tube of saline three times. Take the whole volume of 1 mL in the last tube and transfer to a blood culture bottle. See graph below.
a) Make a 0,5 McFarland dilution of a QC strain
b) Dilute according to dilution series above and add horse or sheep to the blood culture bottle
c) Incubate bottle in the blood culture instrument
d) Process the bottles according to the described RAST methodology when the instrument signals positive
e) Use the RAST QC criteria available in the RAST QC document to assess the results
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