The aim of this study was to establish a reproducible protocol using the methodology
of hyaline zones around the colonies on specific agar plates for phospholipase and proteinase
production. This was an in vitro double-blind experiment, in which the dependent
variables were the enzymatic activity measurements (Pz) for the production of phospholipase
(Pz-ph) and the production of secreted aspartyl proteinases (Pz-sap). Three
independent variables give rise to different measurement protocols. All measurements
were carried out at two different moments by four examiners (E1, E2, E3, and E4). The
minimum sample size was 30 Candida albicans clinical isolates. Specific agar plates for
phospholipase and SAPs production were prepared according the literature. The intraand
inter-examiner reproducibility for each protocol was estimated using the Intraclass
Correlation Coefficient (ICC) and its confidence interval (95% CI). Based on the results
obtained for both phospholipase and SAPs, there appears to be no consensus on the
protocol chosen for each particular examiner. Measuring the colonies in triplicate may
be the main factor associated with the increase in measurement accuracy and should
therefore take precedence over measuring only one colony. When only one examiner is
responsible for taking measurements, a standard protocol should be put in place and the
statistical calibration of this researcher should be done prior to data collection. However,
if two or more researchers are involved in the assessment of agar plates, our results
suggest that the protocols using software to undertake plate reading is preferred.