Comparison of microplate, agar drop and well diffusion plate methods for evaluating hemolytic activity of Listeria monocytogenes

Abstract

Hemolytic activity is an important characteristic for the differentiation of Listeria monocytogenes from apathogenic Listeria species within of conventional laboratory practices. We compared the efficacy of the agar well diffusion method with respect to two previously described methods such as the agar drop and microplate methods in quantifying hemolysis of L. monocytogenescultures. The hemolytic activities of 13 strains of L. monocytogenes were tested. Two culture media (Mueller Hinton blood agar and Mueller Hinton blood agar supplemented with 0.2% activated charcoal and 1 mmol/L CaCl2) were evaluated, using the agar drop and well diffusion methods as plating procedure. The agar well diffusion method was the best plating procedure for detecting the hemolysis of all strains studied after 24 and 48 h of incubation (p < 0.01). In addition, this plating procedure showed a greater sensitivity compared to microplate method at a read time of 6 h, giving positive reactions with all strains at an inoculum of 108 cfu/ml. The supplementation of charcoal on blood agar had a positive effect only when the plates were incubated after 48 h (p < 0.01). The results indicate that the agar well diffusion method can detect and quantify L. monocytogenes hemolytic activity faster and with higher sensitivity than the other two methods here studied. Key words: Listeria monocytogenes, hemolytic activity, microplate, agar drop and well diffusion methods