Synchronous Cultures of Bacteria

After reading this article you will learn about the synchronous cultures of bacteria.

Synchronous cultures are composed of populations of cells that are at the same stage of their life cycle. All the cells in the culture will divide at the same time will grow for a generation time, and all will divide again at the same time. Thus the entire population is kept uniform with respect to growth and division.

It is not possible to analyse a single bacterial cell to obtain the information about growth behaviour, i.e. organisation, differentiation, and macro-molecular synthesis. Synchronous culture provides the entire cell crop in the same stage of growth. Measurement made on such cultures is equivalent to the measurement made on individual cells.

Synchronous cultures of bacteria can be obtained by a number of techniques. Two fundamentally different experimental approaches have been employed. In the first approach, a synchronous population of cells can be sorted out according to age or size by physical separation of cells.

In methods of second type, a culture is induced by manipulating the physical environment or the chemical composition of the medium to obtain a synchronously dividing population. The techniques based on selection are preferable to those based on induction, since induction is likely to introduce distortions in the physiologic state of the cells.

1. Selection by Size and Age:

A population of cells is fractionated on the basis of size. The cells are filtered so that smallest cells pass through the filter. These small cells are the youngest, and must go through their whole life-cycle before dividing. Alternatively, the largest cells, which are ready to divide, may be retained or retarded by a filter.

These are then collected separately and used to obtain a synchronous culture. The most widely used method for obtaining synchronous cultures is the Helmstetter-Cummings technique. A population of cells is passed through a membrane filter of pore size small enough to trap bacteria in the filter.

The filter is then inverted, and fresh nutrient medium is allowed to flow through it (Fig. 18.29). After loosely associated bacteria are washed from the filter, the only bacterial cells in the effluent stream of the medium are those which arise through division.

If a sample of this stream is collected over a short period of time, all the cells in this sample are newly formed, and are therefore of the same age and divide synchronously.

The method has one disadvantage, that the population size is very small and changes in cellular constituents, therefore, cannot be measured. Instead of filtration, density gradient centrifugation is also used to separate the cells. A population of unsynchronized cells is separated into fractions, each composed of the cells of the same density and at the same stage in their life cycle.

2. Selection by Induction Technique:

A synchronous culture is also obtained by the use of shock treatments. These include variation in temperature, starvation, exposure to light (for photosynthetic organisms), drugs, and sub-lethal doses of radiation. A commonly used technique involves submitting a culture of microorganisms to single or multiple changes in temperature.

An exponentially growing culture at 37°C is held for about 30 minutes at 20°C. The lower temperature retards cell division. During the interval of 30 minutes all the cells mature to the point of fission. However, at 20°C none divide. On sudden return of the culture to 37°C, all the cells divide synchronously. By repeating the alterations of temperature, synchrony can be maintained in the culture for several generations.

Methods of inducing synchronous division based on changes in medium composition have also been used. Auxotrophic micro-organisms can be subjected to a period of starvation in a medium lacking an essential growth factor before being placed in a nutritionally complete medium.

A phasing of cell division is observed in cultures of a thymine-requiring mutant, following withdrawal and re-addition of thymine to the culture medium.

The time-course pattern of synthesis of various macro-molecules in the cell cycle is studied by removing portions of a synchronously dividing culture. The cells are then analysed for the content of macro-molecules or enzyme activity, Fig. 18.30 illustrates the behaviour of a synchronous culture.

The logarithmic plots show stepwise increase in cell members. However, the optical density of the culture increases exponentially, since optical density measures cells mass and mass is increasing. Similarly, the total synthesis of DNA, RNA, and protein increases exponentially.