GSt Gram Staining
Learning Objectives
The student will
Use aseptic techniques in the safe inoculation of various forms of media.
Follow oral and written instructions and manage time in the lab efficiently.
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
Differential staining distinguishes organisms based on their interactions with multiple stains. In
other words, two organisms may appear to be different colors. Differential staining techniques
commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining,
flagella staining, and capsule staining. This link to the OpenStax Microbiology text provides more detail
on these differential staining techniques. (OpenStax CNX, 2018)
The Gram stain is a
differential staining procedure that
involves multiple steps. It was
developed by Danish microbiologist
Hans Christian Gram in 1884 as an
effective method to distinguish
between bacteria containing the two
most common types of cell walls.
(OpenStax CNX, 2018) One type
consists of an inner plasma
membrane and a thick outer layer of
peptidoglycan. The other type
consists of a double phospholipid
bilayer with a thin layer of
peptidoglycan between the two. The
Gram Staining technique remains one of the most frequently used staining techniques.
The steps of the Gram stain procedure are listed below and illustrated in Figure. (OpenStax CNX,
2018)
1. First, crystal violet, the primary stain, is
applied to a heat-fixed smear, giving all of
the cells a purple color. You will recall that
crystal violet is a basic stain (excess OH-
ions). It adheres to the cell because the
positively charged chromogen is attracted
to the negatively charged cell as described
in the Simple Staining exercise. (See
figures 2 and 3.) This step is chemically
Figure 1 Simplified structures of Gram negative cells (left) and Gram positive
cells (right)
Figure 2 Crystal violet, a simple basic stain, is added.
