In medical research and disease diagnosis, the knowledge of micro-organisms is very important. Though not all micro-organisms are harmful, some cause various diseases in animals and humans. The study of these micro-organisms is known as microbiology. Normally they are invisible to the naked eye hence the term micro. This means that in their analysis, microscopes have to be used to enable visibility. Some of the procedures used in the determination of these micro-organisms are discussed in this essay;
Staining in microbiology is a procedure used in the laboratory for micro-organism identification. In most cases micro-organisms have transparent cytoplasm making them invisible even under the light microscope. Staining gives them color so that they can be viewed. This procedure is applicable in the study of micro-organisms or the identification of the sample is unknown. There are different staining techniques available for use; the simple stain techniques involve the use of basic positively charged dyes which cling to the negatively charged cytoplasm elements giving it the color of the dye used. This is known as the simple stain procedure.
Alternatively, the negative stain procedure can be used. Negatively charged dyes are used and they act by forming a layer around the cytoplasmic cells due to repulsion since they are also negatively charged. This causes the microorganism to be viewed as it is left unstained and surrounded by stained cells (Madigan & Martinko, 2005).
Secondly, we have the differential stain techniques which differentiate two types of microorganisms. Firstly, the gram stain technique is used to distinguish gram-positive and gram-negative bacteria, and the acid-fast technique is used to differentiate mycobacterium from other types of bacteria. In the Gram stain technique, crystal violet is applied to the sample and then mordant iodine is applied. Alcohol is then used to wash the slide containing the sample. The gram-positive bacteria retain the iodine stain while the gram-negative bacteria lose it. Safranin dye is applied next and the gram-negative bacteria retain it making them appear red when observed under the oil immersion lens while the gram-positive ones appear blue or purple under the same lens (Bancroft & Gamble, 2002).
In the acid-fast technique, carbofuchsin is applied to the sample under heat or using a lipid solvent and then washed with a dilute solution of acid and alcohol. Mycobacterium retains the stain and appears bright red while other bacteria lose the stain and take the methylene blue stain and thus appear blue (Black, 1993).
Bacteria from different genera exhibit different anatomical characteristics and this is the basis on which they are differentiated in the laboratory. Given a sputum sample from a patient suspected to be infected with bacteria from Bacillus, Escherichia, or Mycoplasma genera, a series of procedures are undertaken to determine which one is present in the sample. As a starting point, we need to know that Escherichia and Bacillus are both rod-shaped however bacillus is Gram-positive while Escherichia is gram-negative. Mycoplasmas are also Gram-negative but they lack a cell wall (Ryan & Ray, 2004).
We start by use of simple staining and observing under the microscope. Both Bacillus and Escherichia are rod-shaped a feature we can use to rule out both or rule out Mycoplasmas. If we rule out Mycoplasmas, we then use the Gram stain to determine whether it is Bacillus which is Gram-positive or Escherichia which is Gram-negative. Under the microscope after the Gram stain procedure, Bacillus will appear blue or purple while Escherichia will appear red.
References
Bancroft, J., & Gamble, M. (eds). (2002). Theory and Practice of Histological Techniques. (5th ed). London. Churchill-Livingstone.
Black, J. 1993. Microbiology. NY. Prentice-Hall.
Madigan, M., & Martinko, J. (editors). (2005). Brock Biology of Microorganisms (11th ed.). NJ. Prentice-Hall.
Ryan, K. & Ray, C. (editors) (2004). Sherris Medical Microbiology (4th ed.). NY. McGraw Hill.