The Urea Method for Bacterial Viability Counts with the Electron Microscope and its Relation to other Viability Counting Methods

Abstract

SUMMARY: Live rod-shaped bacteria incubated on a medium containing 3 yo urea grow but do not divide. They thus become much longer than dead bacteria lying among them, and the numbers of each kind can easily be counted in an electron microscope. This urea method gives accurate results and has several advantages over colony-counting methods. The significance of various interesting differences in the counts given by the two methods is discussed. Slope cultures at 18 hr. gave live counts of over 95 yo by the urea method.A common need in quantitative bacteriology is to discover what proportion of bacteria in n given sample is alive. The standard procedure is to make counts of the colonies which form when measured volumes of the sample, suitably diluted, are incubated in nutrient media. On the assumption that each colony arises from one live bacterium, the number of the latter in the sample can then be determined. Such counts made before and after some treatment will enable the resulting fall in viability to be measured. In order to find the actual percentage of live cells in a sample a total count of the bacteria in the measured volume must also be made. In studying the survival of bacteria during drying from tiny droplets or thin films, an accurate measurement of the volume of the samples becomes difficult or impossible. To overcome this problem a new method has been developed for determining the proportion of live bacteria in such tiny samples. This paper gives details of the method and typical results of a test of its accuracy.The method is based on the property possessed by urea in concentrations of about 3 yo ( 0 . 5~) of preventing the division of certain bacteria without inhibiting their growth (Wilson, 1906). Hence after incubation of suitable bacteria for a few hours on nutrient agar containing urea, the live cells have grown into giant forms which can readily be distinguished from the shorter dead organisms (Pl. 1, fig. 5 ) . The numbers of live and dead can then easily be counted in an electron microscope. EXPERIMENTALThe action of urea We have no information about the mechanism by which urea exerts its effect. We have, however, investigated its action on the growth of a paracolon bacillus over a range of concentrations of urea from 1 to 10 yo in nutrient agar medium.The bacteria were taken from 18 hr. slope cultures, suspended in broth, and grown on filmed electron microscope specimen grids as described below. 23G. Microb. XI

The Measurement of Bacterial Viabilities by Slide Culture

“…In this usage an organism may be called dead if it does not multiply in those conditions, and certain philosophical difficulties in the concept of death as applied to micro-organisms (see, for example, Valentine & Bradfield, 1954;Powell, 1956) may be avoided. Many procedures are available for determining the viability in this sense, the most popular being that of obtaining simultaneous total and viable counts on the population.…”

The Action of Chemical Disinfectants on Bacteria in Droplets Compared with that in Large Volumes

“…Viability counts, Counts were made by the urea method in the manner previously described (Valentine & Bradfield, 1954). The results given are the mean counts made on three similar drops in each case.…”

“…Little has been published about the rate of killing of bacteria suspended in droplets owing, for the most part, to the difficulty of adapting the standard viability counting methods for use with such small samples. However, the urea method of measuring bacterial viability (Valentine & Bradfield, 1954) does allow the yo live organisms in small drops to be determined. In outline this method consists of incubating a drop of the suspension under investigation on the film of an electron microscope specimen support placed on a nutrient agar medium containing 3% (w/v) urea.…”

A Rapid Method for Determining the Proportion of Viable Bacteria in a Culture

“…differential staining (Strugger, 1948 (Valentine & Bradfield, 1953. Such methods work well with artificial mixtures of unequivocally living and dead organisms, but in natural mixtures there are often many intermediate forms which cannot be categorized with certainty in this way.…”

“…(Compare also the discussion of Valentine & Bradfield, 1954.) (i) I n samples taken from continuous cultures of the Monod type ( Table 1).…”