2.2.1: Specimen Preparation and Simple Staining

Bacterial samples must be placed on microscope slides for staining. Specimen preparation begins as a sample is taken from a culture and then introduced to the microscope slide and spread into a thin film. This thin film of bacterial sample is known as a smear. As the specimen preparation is carried out it is important that those cells be attached or “fixed” to the slide, so they are not rinsed off the slide during the staining process. The process known as fixation, is done to attach the cells to the slide, but also to prevent distortion of cells that would alter their morphology. Fixation also results in the death of the cells, thereby making the sample safer to handle. There are two types of fixation, chemical fixation and heat fixation.

Chemical fixation is accomplished by applying a chemical reagent such as methyl alcohol or formaldehyde to the sample. Heat fixation is done by passing the air-dried smear back and forth over the flame of a Bunsen burner smear side up.

Once the smear has been fixed the sample may then be stained. Samples are stained in order to add contrast to the cells, allowing them to be viewed using the microscope.

Staining techniques employ dyes in solution (stain) that will chemically interact with the cells in the smear. There are two types of dyes employed in the lab, acidic dyes and basic dyes. Acidic dyes, also known as anionic dyes, are negatively charged and therefore are attracted to positively charged structures. Basic dyes, also known as cationic dyes, are positively charged and thus will be attracted to negatively charged structures. It is important to understand that cells are filled with molecules, such as proteins and nucleic acids that are negatively charged, and thus the cell itself will carry an overall negative charge internally. When an anionic dye is applied to a cell it is repelled and thus would stain the surrounding background around the cell. A basic dye, however, would be attracted to the negatively charged molecules inside the cell and therefore ionically bond to those structures, staining the cell itself. Examples of acidic dyes include India ink, nigrosine, and eosin. Examples of basic dyes include methylene blue, crystal violet, and safranin.

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