INORGANIC PYROPHOSPHATASE-BASED DETECTION SYSTEMS .1. DETECTION AND ENUMERATION OF CELLS

A novel technique, useful for detection and enumeration of both pro- a nd eukaryotic cells, has been developed. The method relies on the dete ction by a very sensitive assay of an enzyme, the inorganic pyrophosph atase, which is constitutively expressed in all cells. This technique has been used to demonstrate detection and enumeration of both Escheri chia coli and Saccharomyces cerevisiae cells. Cells were incubated, fo r a chosen time period and at a specific temperature, in a specific ly sis buffer which contained both lysing activity and inorganic pyrophos phate. The activity of the inorganic pyrophosphatase released from the cells after lysis was determined by an enzymatic luminometric inorgan ic pyrophosphate detection assay (ELIDA; P. Nyren and A. Lundin (1985) Anal. Biochem. 151, 504-509). The amount of PPi hydrolyzed was propor tional to the logarithm of the cell number. The sensitivity of the ass ay was dependent on several factors, such as cell type, incubation tim e, and incubation temperature. E. coli cells at concentrations of 1 X 10(4) cells/ml could be detected in an assay performed at 58 degrees C for 10 min and S. cerevisiae cells at concentrations of 2 X 10(4) cel ls/ml at 49 degrees C within 10 min. The assay could be designed both as a present/absent test, e.g., for determination of bacteriuria, or a s a quantitative assay. Determination and enumeration of all types of cells is in principle possible by the appropriate design of the experi mental parameters. Possible applications for the approach in a wide va riety of areas, such as fundamental and medical sciences, clinical lab oratories, food and dairy industry, pharmaceutical industry, water san itary plants, and biomass determination in general, are discussed. (C) 1994 Academic Press, Inc.