BIOL 2325L: FUNDAMENTALS OF MICROBIOLOGY
LABORATORY| LAB REPORT 4| 2024
Bacterial Structure, Simple Stains, Negative Stains, Gram and Acid-Fast Stains
Introduction:
Visualizing bacteria is difficult because of their small size and also because their refractive index is
close to their aqueous surroundings, making them nearly transparent. To effectively visualize bacteria, they
must be stained to enhance the contrast between the cells and their surroundings (Leboffe and Pierce,
2012). There are many different types of stains and staining procedures used: staining procedures along
with bright-field microscopy have become important tools in microbiology.
All staining procedures, whether simple or otherwise, employ dyes or stains. Chemically, a dye may
be defined as an organic compound containing a benzene ring plus a chromophore and an auxochrome
group. Benzene (C6H6) is an aromatic ring structure and is a colorless organic solvent. A chromophore is a
chemical group that imparts color to benzene. A chromogen is a colored, organic compound consisting of a
benzene and a chromophore. An auxochrome is a chemical compound that imparts the property of
ionization of the chromogen; ionization allows binding to fibers or tissues (Leboffe and Pierce, 2012).
Dyes or stains bind to cellular components, such as proteins or nucleic acids, depending upon the
electrical charge found on the chromogen portion and on the chemical properties of the chemical
component. In terms of charge, dyes can be acidic or basic (Leboffe and Pierce, 2012). Acidic dyes are
anionic, meaning that when ionized they have a negative charge. Acidic dyes have a strong affinity and will
bind to positively charged components of the cell. Basic dyes are cationic, meaning that when ionized they
have a positive charge. Basic dyes bind well to negatively charged cell components. Overall, cells have a
net negative charge, so there are many macromolecules to which basic dyes will bind: DNA, RNA,
phospholipids, many proteins and other molecules. Most staining procedures make use of basic dyes.
The simple stain “paints” the cells, using only one reagent. This enhances the contrast between the
cells and their surroundings. Simple stains allow visualization of morphological shape (cocci, bacilli, and
spirilla) and arrangement (strepto, staphylo, diplo, and tetrads). In contrast to simple stains, a differential
stain is a staining technique which allows the bacteria to be separated into groups based upon staining
reaction (color). Thus, a differential stain goes beyond a simple stain in that it not only provides contrast for
viewing, but also provides non-morphological information about the type of cell being viewed (Leboffe and
Pierce, 2012). Differential staining requires the use of at least three chemical reagents that are applied
sequentially to a heat fixed smear. The first reagent is called the primary stain. The purpose of the primary
stain is to color all the cells. In order to establish color contrast, the second reagent, the decolorizer, is used
to selectively remove dye from some cells based on certain characteristics of the cell. The final reagent, the
counterstain, has a contrasting color to the primary stain and is used to stain cells or components
selectively decolorized by the decolorizer. In this way, cell types can be distinguished from each other on
the basis of the stain that is retained.
, Two common differential stains are the Gram stain and the Acid Fast stains. The Gram staining
method is one of the most important staining techniques in microbiology. It is almost always the first test
performed for the identification of bacteria (Leboffe and Pierce, 2012). This staining procedure allows
bacteria to be divided into two major groups: Gram positive cells and Gram negative cells. The basis for
differential staining in the Gram stain is the structural differences in the cell walls of these two types of
cells. Gram staining is based on the ability of the bacterial cell wall to retain the Crystal Violet dye during
solvent treatment. The cell walls for Gram-positive (G+) microorganisms have a higher peptidoglycan and
lower lipid content than gram-negative (G-) bacteria. The Gram stain uses four different reagents: 1)Crystal
Violet (primary stain) stains all the cells purple, 2)Gram's Iodine (mordant) forms an insoluble complex (the
CV-I complex) with Crystal violet which binds to Mg-RNA complexes in the cell wall, 3) 95% ethanol
(decolorizer) serves a dual role as a lipid solvent and as a protein-dehydrating agent, and 4) Safranin
(counterstain) stains red those cells previously decolorized by the action of the alcohol (i.e., the gram-
negative cells). Gram-positive cells do not take up the counterstain because the binding sites for the dye
are already occupied by the CV-I complexes.
Like the Gram stain, the acid-fast stain is a differential stain, meaning it provides information other
than cell morphology and arrangement. The acid-fast stain is used to distinguish members of the genus
Mycobacterium from all other bacteria (Leboffe and Pierce, 2012). The two important Mycobacterium spp.
in humans are M. tuberculosis, which causes tuberculosis, and M. leprae, which causes leprosy or
Hansen’s disease. There are numerous Mycobacteria species found in animals and in soil, some of which
can cause disease in humans under certain circumstances. The acid-fast bacteria have an atypical Gram-
negative cell wall, which contains waxy substances (mycolic acid and others) in the outer membrane, which
give this structure an unusual permeability property. Because of this, these bacteria are particularly
difficult to treat with many antibiotics (Leboffe and Pierce, 2012). These mycloic acids are the basis for the
acid-fast reaction, and these bacteria are often referred to as being “acid-fast” because they resist rigorous
decolorization, even using acid alcohol as the decolorizer. Acid-fast bacteria retain the primary stain in the
acid-fast staining procedures and will appear maroon/dark red in color while non-acid-fast gram negative
and gram-positive bacteria will be decolorized by treatment with acidified alcohol and thus will accept the
counterstain. The acid fast stain uses three different reagents (the mordant is heat): 1) Carbolfuchsin
(primary stain) stains will stain all cells maroon/dark red, 2) Acid alcohol (decolorizer) removes the primary
stain from the non-acid-fast cells, leaving them colorless. The acid-fast organisms will retain the primary
stain, and 3) Brilliant Green. This counterstain is used to stain the non-acid-fast cells a contrasting color.
Guided Question (cite sources): Who was the Gram staining technique named for, and when was it
developed?
The Gram stain was named after Hans Christian Gram and he developed this method in 1884 (Gram
Staining, 2005).
