SSt Simple Staining
Learning Objectives
The student will
Use aseptic techniques.
Follow oral and written instructions and manage time in the lab efficiently.
Apply correct terminology regarding microbiological techniques, instruments when making
observations.
Use the bright field light microscope to view microbes under oil immersion, make accurate
observations and appropriate interpretations and store the microscope according to lab
procedures.
Properly prepare a bacterial smear for accurate staining and describe the chemical basis for
simple staining and negative staining.
Background/Theory
A brightfield microscope creates an image by directing light from the illuminator at the
specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures.
(OpenStax CNX, 2018) This alteration of light as it passes through the specimen and into the lens system
of the microscope creates the image. Bacterial cells are so small and thin that they alter the light very
little and, consequentially, are virtually transparent or invisible. In other words, the cell lacks contrast
with the background. Staining the specimen will artificially create contrast so that cells may be visible.
Along with magnification and resolution, contrast is the third element that contributes to the amount of
detail that can be observed.
Some staining techniques involve the
application of only one stain to the sample;
others require more than one.
In simple staining, a single stain or dye is used
to emphasize particular structures in the
specimen. A simple stain will generally make all
of the organisms in a sample appear to be the
same color, even if the sample contains more
than one type of organism. (OpenStax CNX,
2018)
Stains, or dyes, aqueous slat solutions.
They contain a positive ion and a negative ion
one of which imparts the color. The chromogen
(sometimes called the chromophore) is the
colored ion. If the chromogen is positively
charged, the counter ion is the negatively
charged hydroxide ion OH- making this type of
stain a basic stain. (Here the term basic does
not mean elementary. Instead, basic refers to
chemical property of having excess hydroxide
ions.) Because most cell structures have a
negative charge, the positively charged, colored
Figure 1 A basic stain has a positive chromogen and the negative
hydroxide ion. The colorless cell has a net negative charge.
