Qualitative and Quantitative Analysis in Microbiology

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Qualitative and quantitative analysis in microbiology

Various techniques have been devised to permit the analysis of the structure and function of microorganisms . Some techniques are qualitative in their intent. That is, they provide a "yes or no" answer. Other techniques are quantitative in their intent. These techniques provide numerical information about a sample.

Assessing the growth of a bacterial sample provides examples of both types of analysis techniques. An example of a qualitative technique would be the growth of a bacterial sample on a solid growth medium, in order to solely assess whether the bacteria in the sample are living or dead. An example of a quantitative technique is the use of that solid growth media to calculate the actual number of living bacteria in a sample.

Microscopic observation of microorganisms can reveal a wealth of qualitative information. The observation of a suspension of bacteria on a microscope slide (the wet mount) reveals whether the bacteria are capable of self-propelled motion. Microorganisms, particularly bacteria, can be applied to a slide as a so-called smear, which is then allowed to dry on the slide. The dried bacteria can be stained to reveal, for example, whether they retain the primary stain in the Gram stain protocol (Gram positive) or whether that stain is washed out of the bacteria and a secondary stain retained (Gram negative). Examination of such smears will also reveal the shape, size, and arrangement (singly, in pairs, in chains, in clusters) of the bacteria. These qualitative attributes are important in categorizing bacteria.

Microscopy can be extended to provide qualitative information. The incorporation of antibodies to specific components of the sample can be used to calculate the proportion of the samples in a population that possess the target of interest. Fluorescent-labeled antibodies, or antibodies combined with a dark appearing molecule such as ferritin, are useful in such studies. The scanning confocal microscope is proving to be tremendously useful in this regard. The optics of the microscope allows visual data to be obtained at various depths through a sample (typically the sample is an adherent population of microorganisms). These optical thin sections can be reconstructed via computer imaging to produce a three-dimensional image of the specimen. The use of fluorescent-tagged antibodies allows the location of protein within the living biofilm to be assessed.

The self-propelled movement of living microorganisms, a behavior that is termed motility, can also provide quantitative information. For example, recording a moving picture image of the moving cells is used to determine their speed of movement, and whether the presence of a compound acts as an attractant or a repellant to the microbes.

Bacterial growth is another area that can yield qualitative or quantitative information. Water analysis for the bacterium Escherichia coli provides an example. A specialized growth medium allows the growth of only Escherichia coli. Another constituent of the growth medium is utilized by the growing bacteria to produce a by-product that fluoresces when exposed to ultraviolet light. If the medium is dispensed in bottles, the presence of growing Escherichia coli can be detected by the development of fluorescence. However, if the medium is dispensed in smaller volumes in a grid-like pattern, then the number of areas of the grid that are positive for growth can be related to a mathematical formula to produce a most probable number of living Escherichia coli in the water sample. Viable bacterial counts can be determined for many other bacteria by several other means.

The ability of bacteria to grow or not to grow on a media containing controlled amounts and types of compounds yields quantitative information about the nutritional requirements of the microbes.

The advent of molecular techniques has expanded the repertoire of quantitative information that can be obtained. For example, a technique involving reporter genes can show whether a particular gene is active and can indicate the number of copies of the gene product that is manufactured. Gene probes have also been tagged to fluorescent or radioactive labels to provide information as to where in a population a certain metabolic activity is occurring and the course of the activity over time.

Many other qualitative and quantitative techniques exist in microbiological analysis. A few examples include immunoelectrophoresis , immunoelectron microscopy, biochemical dissection of metabolic pathways, the molecular construction of cell walls and other components of microorganisms, and mutational analysis. The scope of the techniques is everexpanding.

See also Laboratory techniques in immunology; Laboratory techniques in microbiology