Micropipette Method, A New Technique for Detecting Ice-Nucleation Activity of Bacteria and It's Application

Abstract

A new micropipette method was developed to determine the ice nucleation activity of bacterial cells. The micropipettes used for the experiments were the scaled capillary tubes (Drumond Scientific Co., USA). Deionized water could be overcooled lower than -20C. Ordinary physical shock did not significantly influence on the freezing temperatures with slight deviations. Temperatures of the samples in micropipettes could be controlled easily and accurately. Difference was rarely detected between the temperatures of ethanol in cooling bath and water samples in micropipettes dipped in the former. Ice nucleation temperature, however, varied with the capacities of the micropipettes, showing higher value with the larger capacities. Positive correlation was observed between ice nucleation temperatures and logarithms of capacity with 0.93 or higher values of correlation coefficient. This method was compared with the procedure described by Vali. Ice nucleation temperatures by the present method showed small deviation (1 to 3% in CV). The present method was considered to be superior to other methods including Vali's method and its modifications. The ice nucleation activity of plant pathogenic bacteria was measured by the method. Isolates of Pseudomonas syringae pv. pisi were very active and showed ice nucleation at -2.9C in contrast to P. marginalis pv. marginalis, P. s. pv. maculicola and P. s. pv. tabaci. The other species of Pseudomonas showed no activity. No activity was also observed with follows: Corynebacterium michiganense pv. michiganense, C. flaccumfaciens pv. oortii, Erwinia carotovora subsp. carotovora, E. herbicola, E. milletiae, and X. campestris pv. campestris. Cultures of epiphytic bacteria isolated from Actinidia cinensis, Cyclobalanopsis spp., Eriobotrya japonica, Ficus carica and prunus avium showed ice nucleation activity at -2.7 to -3.0C. Deionized water in which plant buds were dipped for 3 days increased significantly ice nucleation activity due to bacteria. This method may be effective in detecting the active gemmisphere bacteria as ice nuclei.