Microbiology

Reproduction patterns. During their growth cycles, microorganisms undergo reproduction many times, causing the numbers in the population to increase dramatically.

In fungi, unicellular algae, and protozoa, reproduction involves a duplication of the nucleus through the asexual process of mitosis and a splitting of the cell in cytokinesis. Reproduction can also occur by a sexual process in which haploid nuclei unite to form a diploid cell having two sets of chromosomes. Various changes then follow to yield a sexually produced offspring. Sexual reproduction has the advantage of mixing chromosomes to obtain genetic variations not possible with asexual reproduction. However, fewer individuals normally result from sexual reproduction than from asexual reproduction. More details on these methods are provided in the chapters on fungi and protozoa.

Bacteria reproduce by the asexual process of binary fission. In this process, the chromosomal DNA duplicates, after which the bacterial membrane and cell wall grow inward to meet one another and divide the cell in two. The two cells separate and the process is complete.

One of the remarkable attributes of bacteria is the relatively short generation time, the time required for a microbial population to double in numbers. The generation time varies among bacteria and often ranges between 30 minutes and three hours. Certain bacteria have very brief generation times. Escherichia coli, for example, has a generation time of about 20 minutes when it is dividing under optimal conditions.

The growth curve. The growth of a bacterial population can be expressed in various phases of a growth curve. The logarithms of the actual numbers in the population are plotted in the growth curve along the side axis, and the time is plotted at the base. Four phases of growth are recognized in the growth curve.

In the first phase, called the lag phase, the population remains at the same number as the bacteria become accustomed to their new environment. Metabolic activity is taking place, and new cells are being produced to offset those that are dying.

In the logarithmic phase, or log phase, bacterial growth occurs at its optimal level and the population doubles rapidly. This phase is represented by a straight line, and the population is at its metabolic peak. Research experiments are often performed at this time.

During the next phase, the stationary phase, the reproduction of bacterial cells is offset by their death, and the population reaches a plateau. The reasons for bacterial death include the accumulation of waste, the lack of nutrients, and the unfavorable environmental conditions that may have developed. If the conditions are not altered, the population will enter its decline, or death phase (Figure 1 ). The bacteria die off rapidly, the curve turns downward, and the last cell in the population soon dies. 

Figure 1

A growth curve of a bacterial population showing the four major phases of the curve.

Microbial measurements. In order to measure the number of bacteria in a population, various methods are available. In one method, known as the plate count method, a sample of bacteria is diluted in saline solution, distilled water, or other holding fluid. Samples of the dilutions are then placed in Petri dishes with a growth medium and set aside to incubate. Following incubation, the count of colonies is taken and multiplied by the dilution factor represented by that plate. Generally, plates with between 30 and 300 colonies are selected for determining the final count, which is expressed as the number of bacteria per original ml of sample.

Another measuring method is to determine the most probable number. This technique is often used to determine the number of bacteria in a sample of contaminated water. Water samples are added to numerous tubes of single-strength and double-strength lactose broth. If coliform bacteria (such as E. coli) are present, they will ferment the lactose and produce gas. Judging by the number of tubes that contain gas at the end of the test, one may approximate the original number of bacteria in the water sample.

Another evaluative method is by a direct microscopic count. A specially designed counting chamber called a Petroff-Hausser counter is used. A measured sample of the bacterial suspension is placed on the counter, and the actual number of organisms is counted in one section of the chamber. Multiplying by an established reference figure gives a number of bacteria in the entire chamber and in the sample counted. The disadvantage of this method is that both live and dead bacteria are counted.

Turbidity methods can also be used to assess bacterial growth. As bacteria multiply in liquid media, they make the media cloudy. Placing the culture tube in a beam of light and noting the amount of light transmitted gives an idea of the turbidity of the culture and the relative number of bacteria it contains.

The dry weight of a culture can also be used to determine microbial numbers. The liquid culture is dried out, and the amount of microbial mass is weighed on a scale. It is also possible to measure the oxygen uptake of a culture of bacteria. If more oxygen is used by culture A than by culture B and all other things are equal, then it may be deduced that more microorganisms are present in culture A. A variation of this method called the biochemical oxygen demand (BOD) is used to measure the extent of contamination in a water sample.