Techniques for Studying Microorganisms

Research into disease-causing microorganisms requires an adequate laboratory method. Koch and colleagues developed methodologies in the laboratory including procedures for coloring bacteria to be easily examined and observed, as well as techniques for growing microorganisms in the laboratory.

One technique developed is the use of media with a dense and transparent substrate at incubation temperatures (temperatures suitable for growth) to grow bacteria. Initially used gelatin for this purpose turned out to be inadequate because at body temperature it becomes liquid. Then, other media are used such as potato or carrot slices which results are not good, because of the lack of nutrients for microorganisms, especially those related to the human body.

In the end the problem can be overcome by using certain sea algae extracts. The algae extract, called "agar-agar" (often called agar), can be dissolved in a nutrient solution and when it becomes a gel it will remain solid over a wide temperature range.

Pure Culture Concept

The "agar" media are excellent substrates for the growth of microorganisms, with the "agar" mixture of microorganisms will grow separately so that it is easy to observe. The technique used to grow microorganisms on the media in order to cause microorganisms to grow somewhat apart from each other, or perhaps each cell grows together to form a colony, which is a group of cell masses that can be seen with ordinary eyes without tools (Figure).

Picture: Colonies of microorganisms on a Petri dish medium. Note that there are two kinds of colonies which show two different types of bacteria and one fungus colony.
Picture: Colonies of microorganisms on a Petri dish medium. Note that there are two kinds of colonies which show two different types of bacteria and one fungus colony.

All cells in the colony are the same meaning they are all considered to be one offspring (progeny) or one microorganism therefore representing what is referred to as pure culture. The use of agar on microbiological media proposed by Koch in the early 1880s is still used today. To get one species of culture must separate mixed cultures in the following way:

At first the samples are taken from the air, soil, or dirt and then spread on a sterile medium, then the colonies will grow which have different shapes and colors, depending on the specific characteristics of each bacterium. Furthermore, the different colonies are grown on sterile medium separately so that pure cultures will be obtained.

Pure Culture Technique

Nature around us has a very large population of microorganisms. Hundreds of species from various microorganisms inhabit parts of our body, including the mouth, digestive tract, and skin. These microorganisms are present in unusually large numbers. Research on microorganisms in various habitats requires techniques to be able to separate complex mixed populations or mixed cultures, into different species as pure cultures. Pure culture consists of a population of cells that all originate from one parent cell.

Breeding and isolation of pure culture

In the laboratory, microorganisms are cultured in nutrients called mediums. There are so many media available, their use depends on several factors, one of which is the type of microorganism that will be grown.

The procedure used in preserving bacteria is quite a lot. The method chosen depends very much on the nature of the microorganism to be cultured. In addition, it also depends on the purpose of preserving culture. Will the culture be stored for a short time (months) or is it stored for an infinite time (years). Short-term maintenance process, culture can be stored at refrigerator temperature (0-10°C), whereas for long-term maintenance, it can be stored in liquid nitrogen at -196°C or can also be dehydrated in a tube by being frozen and closed tightly in the room empty This process is called lyophilization (Figure).
Image: lyophilization process
Image: lyophilization process

Several small bottles of cotton-clogged bottles containing frozen organisms are placed in glass flasks, which are connected to condensers. The condenser is connected to a high vacuum pump, and this system dehydrates the culture. After dehydration, the bottle is removed, then placed one by one in a larger tube, covered with asbestos packaging, and tightly closed in a vacuum.

Technique for characterizing microorganisms

If the pure culture of a microorganism has been obtained, then it is then examined in a laboratory, so that information is obtained about the nature of microorganisms. The validity of laboratory tests is determined by the purpose of the test. Is the culture a new type of microorganism or a known microorganism. Cultures need to be characterized extensively. Various tests / methods for characterizing microorganisms can be seen in Table.

Table: Methods for Characterizing Microorganisms

 No. The main characteristic Method
 1Morphology Observation of specimens with the help of light or electron microscopy, whether or not colored. Electron microscopy technique allows the observation of ultra-thin slices of microorganism cells.
 2 Nutritional Determination of chemical substances and special physical conditions (temperature, light, and gas) needed to support the growth of microorganisms.
 3 Culture Determination of the shape of the growth colonies of microorganisms in various laboratory mediums, both liquid and solid.
 4 Metabolic Identification and measurement of chemical changes by microorganisms. Some tests are easy to do, but only to find out whether microorganisms cause chemical changes in a particular substance or not. Example of converting carbohydrates to acids. In other tests, there is a method that identifies most of the chemical compounds that are seen in the metabolic process, this allows to reconstruct step by step how the microorganisms cause changes.
 5 Chemical composition Determination of chemical composition as a component of cells. Various techniques to break cells and isolate cell components specifically from the mixture obtained for example cell wall fragments, nucleus changes, and membranes. There are also laboratory procedures to determine the arrangement of each component.
 6 Arrangement of antigens Characterization of microorganisms, especially in bacteria and viruses, by studying antigens, chemical substances that exist on its surface. An antigen is the chemical substance of a microorganism which when injected with animals will form a chemical substance (antibody) that can be identified by laboratory procedures. Antigens and antibodies are part of a complex immunological system.
 7 Pathogenic Determination of the potential of a microorganism culture. To cause disease is done by inoculating animals or plants with pure culture of the relevant microorganisms.

Coloration of Microorganisms

Usually to be able to see the shape and species of microorganisms two common techniques are used. Two techniques that are commonly used in preparing material or specimens are:
  1. Suspension of organisms in a liquid, and
  2. Use a thin layer or spread a specimen that is dried, fixed, and stained.

Wet accretion techniques and hanging drip techniques

Wet preparations or hanging drops preparations make it possible to examine living organisms that are suspended in flow. Wet preparations are obtained by placing a drop of a substance containing an organism on the slide and covering it with a very thin glass called a cover glass. To reduce the rate of evaporation and eliminate the flow of air on these droplets, usually next to the glass object circled with petroleum jelly. Special slide with deep concave area for hanging drops. Hanging drops preparations are especially useful if they want to identify microorganisms but the morphology of the microorganisms examined will be damaged due to chemical or heat treatment, and is difficult to stain (Figure).
Image: hanging drop technique
Image: hanging drop technique

Coloring technique

The advantage of this coloring technique is that it can:
  1. better observing the morphology of microorganisms roughly;
  2. identifying the structural parts of cell microorganisms;
  3. help identify and or distinguish similar organisms.

The main steps in coloring techniques are:
  1. placement of spreads or thin layers of specimens, on the slide;
  2. fixation of the spread on the slide by heating, that is by passing it on fire several times, so that the microorganism can be attached to the slide;
  3. stained with a single coloring (simple coloring) or a series of coloring solutions (differential coloring).

Various colors

Simple coloring

Giving color to bacteria or other microorganisms by using a single solution of a dye on a thin layer or spread that has been fixed, called a simple coloring. The layer was put in a coloring solution for a period of time, then the solution was washed with water and the glass was dried with suction paper. Cells will be colored evenly, except certain parts will appear darker colored.

Differential Coloring

Coloring procedures that can produce differences between microorganism cells or parts of microorganism cells are called differential coloring techniques. This technique uses more than one dye solution.

Gram staining

One of the most important and most widely used differential staining techniques for bacteria is Gram staining. In this process a smear of fixed bacteria is subjected to purple crystal solutions, iodine solution, alcohol (as a pale agent), and safranin or some other suitable coloring agent. The results of staining bacteria with the Gram method produces two groups of bacteria, namely:

  1. Gram positive bacteria are bacteria that retain the purple crystalline dye and therefore appear dark purple.
  2. The other group is Gram negative bacteria, which are bacteria that will lose purple crystals when washed with alcohol, and when given a counter dye with safranin red, appear red.

The difference in color of bacteria to purple and red is caused by differences in the chemical structure of these bacteria. This Gram staining technique was first published in 1884 by a Danish Bacteriologist, Christian Gram.