Laboratory techniques in microbiology

A number of techniques are routine in microbiology laboratories that enable microorganisms to be cultured, examined and identified.

An indispensable tool in any microbiology laboratory is the inoculating loop. The loop is a piece of wire that is looped at one end. By heating up the loop in an open flame, the loop can be sterilized before and after working with bacteria . Thus, contamination of the bacterial sample is minimized. The inoculating loop is part of what is known as aseptic (or sterile) technique.

Another staple piece of equipment is called a petri plate. A petri plate is a sterile plastic dish with a lid that is used as a receptacle for solid growth media.

In order to diagnose an infection or to conduct research using a microorganism, it is necessary to obtain the organism in a pure culture . The streak plate technique is useful in this regard. A sample of the bacterial population is added to one small region of the growth medium in a petri plate and spread in a back and forth motion across a sector of the plate using a sterile inoculating loop. The loop is sterilized again and used to drag a small portion of the culture across another sector of the plate. This acts to dilute the culture. Several more repeats yield individual colonies. A colony can be sampled and streaked onto another plate to ensure that a pure culture is obtained.

Dilutions of bacteria can be added to a petri plate and warm growth medium added to the aliquot of culture. When the medium hardens, the bacteria grow inside of the agar . This is known as the pour plate technique, and is often used to determine the number of bacteria in a sample. Dilution of the original culture of bacteria is often necessary to reach a countable level.

Bacterial numbers can also be determined by the number of tubes of media that support growth in a series of dilutions of the culture. The pattern of growth is used to determine what is termed the most probable number of bacteria in the original sample.

As a bacterial population increases, the medium becomes cloudier and less light is able to pass through the culture. The optical density of the culture increases. A relationship between the optical density and the number of living bacteria determined by the viable count can be established.

The growth sources for microorganisms such as bacteria can be in a liquid form or the solid agar form. The composition of a particular medium depends on the task at hand. Bacteria are often capable of growth on a wide variety of media, except for those bacteria whose nutrient or environmental requirements are extremely restricted. So-called nonselective media are useful to obtain a culture. For example, in water quality monitoring, a non-selective medium is used to obtain a total enumeration of the sample (called a heterotrophic plate count). When it is desirable to obtain a specific bacterial species, a selective medium can be used. Selective media support the growth of one or a few bacterial types while excluding the growth of other bacteria. For example, the growth of the bacterial genera Salmonella and Shigella are selectively encouraged by the use of Salmonella-Shigella agar. Many selective media exist.

Liquid cultures of bacteria can be nonspecific or can use defined media. A batch culture is essentially a stopped flask that is about one third full of the culture. The culture is shaken to encourage the diffusion of oxygen from the overlying air into the liquid. Growth occurs until the nutrients are exhausted. Liquid cultures can be kept growing indefinitely by adding fresh medium and removed spent culture at controlled rates (a chemostat) or at rates that keep the optical density of the culture constant (a turbidostat). In a chemostat, the rate at which the bacteria grow depends on the rate at which the critical nutrient is added.

Living bacteria can also be detected by direct observation using a light microscope , especially if the bacteria are capable of the directed movement that is termed motility. Also, living microorganisms are capable of being stained in certain distinctive ways by what are termed vital stains. Stains can also be used to highlight certain structures of bacteria, and even to distinguish certain bacteria from others. One example is the Gram's stain, which classifies bacteria into two camps, Gram positive and Gram negative. Another example is the Ziehl-Neelsen stain, which preferentially stains the cell wall of a type of bacteria called Mycobacteria.

Techniques also help detect the presence of bacteria that have become altered in their structure or genetic composition. The technique of replica plating relies on the adhesion of microbes to the support and the transfer of the microbes to a series of growth media. The technique is analogous to the making of photocopies of an original document. The various media can be tailored to detect a bacteria that can grow in the presence of a factor, such as an antibiotic, that the bacteria from the original growth culture cannot tolerate.

Various biochemical tests are utilized in a microbiology laboratory. The ability of a microbe to utilize a particular compound and the nature of the compound that is produced are important in the classification of microorganisms, and the diagnosis of infections. For example, coliform bacteria were traditionally identified by a series of biochemical reactions that formed a presumptive-confirmed-completed triad of tests. Now, media have been devised that specifically support the growth of coliform bacteria, and Escherichia coli in particular.

Various laboratory tests are conducted in animals to obtain an idea of the behavior of microorganisms in vivo. One such test is the lethal dose 50 (LD50), which measures the amount of an organism or its toxic components that will kill 50 percent of the test population. The lower the material necessary to achieve the LD50, the more potent is the disease component of organism.

See also Antibiotic resistance, tests for; Blood agar, hemolysis, and hemolytic reactions; Microscopy; Qualitative and quantitative analysis in microbiology