Comparison of colony lift with direct spotting methods of blot preparation on the effect of colony hybridization in the detection of environmental organisms

Tai-Lee Hu, Shen-Chung Wu
Department of Environmental Engineering and Science, Feng Chia University, Taichung, Taiwan, ROC

Nucleic acid probes are used on site to detect or to identify individual microbial cells without cultivation. This molecular technique can avoid some limitations of traditional identification methods including time consuming and imprecise. This study examined the factors affecting colony hybridization and compared the effectiveness of membrane prepared by colony lifting with direct spotting procedures using the universal probe Eub 338. The results of hybridization varied depending on the type of colony morphology. For dry and rough colonies, colony hybridization was not suitable for detecting Acinetobacter sp. (CK2A, CK2B), Alcaligenes sp. (TH11 B), Xanthomonas sp. (TH7B), Arthrobacter globiformis (CCRC 10598) and Microbacterium sp. (CCRC 11036). Colonies of Acinetobacter sp. (CCRC 15425) and Alcaligenes spp. (CCRC 10828, H) on agar and membrane were thick and raised, and their detection signals of hybridization were diffused or blank. Colonies of Alcaligenes sp. (CM7A, ANV2) and Acinetobacter sp. (ANV8) isolated from the sludge of biological processes treating ABS wastewater were flat and smooth, and their hybridization signals were clear. For those strains suitable for colony hybridization, the colony blots prepared by colony lift and direct spotting procedures gave the same sensitivity for colony hybridization.

J Microbiol Immunol Infect 2000;33:123-126.