BIOLOGY Volume 2 - A Guide to General Biology - 2004

12. MICROBIOLOGY AND BIOTECHNOLOGY

12.6. Measuring bacterial and fungal growth in culture

In the previous section, we analyzed a typical bacterial growth curve. We can expect the same curve to characterize the growth of Yeast (unicellular Fungi) or any culture of microorganisms.

When analyzing the growth of Bacteria or yeast, we can either directly count the number of Cells or measure certain parameters dependent on Cell number, such as solution turbidity or gas evolution. Typically, a small inoculum of microorganisms is added to a sterile nutrient medium, and the culture is grown in an incubator at the optimal growth Temperature. All other conditions should be kept as close to optimal as possible (sec. 12.1). Growth should be measured from the moment of inoculation.

As a rule of thumb in scientific research, it is good practice to run experiments in multiple replicates and include controls wherever possible and necessary. Some growth measurement techniques require a certain level of skill and, even in the hands of specialists, are not extremely precise. Therefore, it is advisable to set up duplicate samples (one replicate) for each experiment, if possible. A control sample, to which no microorganisms have been added, will verify whether proper sterile technique is being maintained. With sufficient experience, one can master all the described Methods to perfection, so we recommend practicing them before using them in your project work.

Cell numbers can be determined in two ways: by counting either the number of viable cells or the Total Cell Count. Viable cell count refers exclusively to the number of living cells. Total cell count is the combined total of both living and dead cells; this parameter is generally easier to determine.

12.6.1. Viable cell count

Sometimes It is important to know the number of viable cells. For example, the killing efficiency of certain bacteria during milk pasteurization can be assessed by measuring viable bacteria before and after pasteurization. In industrial production, only living cells drive the process, making it essential to know the number of live cells in a culture. Viable cell counts can be performed using pour-plate or spread-plate methods on Agar, while yeast viability can be determined using a hemocytometer method.

Spread-plate method

This method has already been described in sec. 12.4.1 (Fig. 12.5). A small, known volume of culture is applied to The surface of nutrient agar in a Petri dish. A drawback of this method is that a portion of the culture remains on the spreader and pipette, making it impossible to count cells with high precision. However, this is often negligible.

The method is based on the principle that each bacterium will, after a certain period—such as two days—form a single colony. Thus, the number of bacteria in the original added sample equals the number of colonies that develop after incubation. Only colonies consisting of 100,000 cells or more are visible to the naked eye. Typically, colonies contain several million bacteria.

Provided the sample contains neither too many nor too few bacteria, the colonies can be easily counted. As a precaution, it is best not to open the Petri dish lid during counting. Serial dilutions are usually required so that one of the dilution series yields an ideal number of colonies. Serial dilutions are a sequence of consecutive, uniform dilutions of a single sample (Fig. 12.9). Once a suitable dilution is found, the experiment is repeated at that dilution to increase accuracy and reproducibility. In Experiment 12.3 from sec. 12.9.2, the bacterial count in milk samples was determined using this method. Certain Limitations of the method are discussed at the end of this section.

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Fig. 12.9. Preparation of serial dilutions.

Pour-plate method

This method was previously described in sec. 12.4.1 (Fig. 12.6). Fundamentally, it does not differ from the spread-plate method, except that the sample is mixed with nutrient agar before the plates are poured, so that the colonies are distributed throughout the entire volume of the medium rather than growing only On the surface.

Yeast

Counting viable yeast cells can be performed using a hemocytometer and methylene blue. This method is described in more detail in the following section.

Problems associated with viable cell counting

Several problems are associated with determining viable cell counts.

1. Some bacteria form chains or clusters of cells, such as streptococci and staphylococci (Fig. 2,10). Each cell cluster gives rise to a single colony. Consequently, the results of viable cell counts are sometimes expressed not as the number of bacteria, but as the number of colony-forming units (CFUs).

2. If multiple types of bacteria are present, as in soil, milk, or Water samples, the growth conditions will not be equally favorable for every type. As a result, certain bacteria will grow much faster than others, and the number of visible colonies will not accurately reflect the actual number of bacteria in the sample.