A Scanning Electron Microscopic Method for the Study of Bacterial Growth Inhibition by the Paper Disc Method

Abstract

SummaryWe previously found an inverse correlation between the number of bacteria and the number of fungi collected throughout the year from tatami mats in judo halls. Antibacterial substances produced by the fungi are presumably responsible for the decline in bacterial populations. Culture extracts from fungi isolated from the tatami mats were prepared and their antibacterial activity against Micrococcus luteus, Staphylococcus warneri, and Bacillus subtilis isolated from the mats was determined by the paper disc method. For all 3 bacteria, clear inhibitory zones were obtained indicative of the presence of a growth inhibitory compound in the fungal culture extract. A sampling method was developed for the scanning electron microscopic observation of bacteria around the inhibitory zones produced by the fungal culture extract. Using this method, changes in the surface microstructures of bacteria existing inside the inhibitory zone and growth region were successfully compared by scanning electron microscopy.Key words Bacteria, Fungi, Inhibitory zones in antibacterial assays, Scanning electron microscope.The microbial flora, the number of bacteria and the number of fungi on the tatami mats of the judo hall in Nippon Sport Science University change during the course of a year. The number of bacteria on the tatami mats decreases when the number of fungi increases, and the number of bacteria increases when the number of fungi decreases (Nakamura 2006). This inverse correlation between the number of bacteria and fungi is presumably caused not only by mutual competition for nutrients but also by antibacterial compounds produced during fungal growth. The paper disc method, a simple inexpensive method for determining the antibacterial activity of culture extracts, was used to show that bacteria frequently isolated from the tatami mats of the judo hall are sensitive to compounds in extracts prepared from fungi isolated from the tatami mats. A newly developed sampling method for the scanning electron microscopic analysis of bacteria within the fungal extract inhibitory zone has allowed a comparison of the surface microstructure of bacteria inside the inhibitory zone and those within the growth region. Materials and methods Freeze-drying of fungal cultureThe fungi isolated from the vinyl chloride tatami mats in the judo hall of Nippon Sport Science University were pure-cultured, followed by shake culture in Sabouraud's liquid medium at 27°C for 12 weeks (Nara et al. 2007). Fungi were separated from the culture fluid by centrifugation at 3,000 rpm for 15 min. The culture fluid was filtered through a 0.22-mm membrane (Millipore,