Gram Stain Lab (Keystone Anchor Bio.A.1.2.

1)

PA Standards:
PA S. T. & E.:
3.1.B.A1. Compare and contrast the cellular structure and degrees of complexity of prokaryotic and
eukaryotic organisms.
PDE Keystone Anchors:
Bio.A.1.2.1: Compare cellular structures and their functions in prokaryotic and eukaryotic cells.

Introduction:

In 1884, Hans Christian Gram, a Danish doctor working in Berlin, accidentally stumbled on a
method which still forms the basis for the identification of bacteria. Individual bacterial cells are
hard to see, partly because they are small, but also because they are almost transparent; staining
the cells help make them visible. The Gram stain is a more complex staining technique and is
used as an aid in identifying unknown species of bacteria. Although the bacteria used in this lab
are nonpathogenic, in clinical settings this procedure aids in diagnosis, and is routinely
performed on patients with respiratory or other infections.
The technique uses three stains: crystal violet, Gram’s iodine, and safranin. Crystal violet
stains bacterial cells purple; Gram’s iodine is a mordant, which means that it helps fix the crystal
violet stain and makes it difficult for the crystal violet to be removed by decolorization; safranin
stain is applied last and only appears if cells lose crystal violet in the decolorization process
So how does it work? Gram didn't know - he simply worked empirically. We now know that
the Gram reaction is based on the structure of the bacterial cell wall. In Gram-positive bacteria,
the dark purple crystal violet stain is retained by the thick layer of peptidoglycan which
forms the outer layer of the cell. In Gram-negative bacteria, the thin peptidoglycan layer in the
periplasm does not retain the dark stain, and the pink safranin counterstain stains the
peptidoglycan layer.
After staining you will also be able to identify the shapes of the bacteria being studied.
The three basic shapes are round, called coccus (cocci); rod (bacillus); and spiral or corkscrew
(spirilla). Bacteria may exist as single cells or in pairs (diplo-), clusters (staphylo-), or chains
(strepto-). Gram stain results and shape are used to help identify the bacteria cultures.

Light your Bunsen burner! Test yogurt for contamination in a virtual lab, and judge
whether the bacteria you find are harmful or beneficial.
In this Virtual Labs interactive food science modules train high school and college students in
basic laboratory techniques.

Learn how to use Gram staining to differentiate beneficial bacteria from dangerous bacteria in
food samples. Working in the virtual lab of a dairy processing plant, you analyze yogurt samples
and follow step-by-step lab procedures to test for Salmonella and E. coli. Sterilize your
inoculation loop, and prepare your slide, then view the sample under a microscope and observe
the differences in appearance between beneficial bacteria and harmful bacteria. Virtual Labs:
Gram Staining familiarizes the user with food science lab equipment and teaches standard
techniques for this specific procedure. The interactive initially guides the user through each step
of the lab process; then users prepare Gram stain slides on their own to find out for certain
whether this batch of yogurt is safe.

Go to the following website to conduct a Gram Stain to see if the yogurt is safe to eat.
https://virtuallabs.nmsu.edu/stain.php

Questions:

1. Why do Gram + bacteria retain the purple crystal violet stain? – The stains used in Gram
staining react with components of the cell wall of the bacteria. The cell walls of Gram-
positive bacteria have a think layer of peptidoglycan, which binds with crystal violet stain.
Gram-negative bacteria contain very little peptidoglycan, so the crystal violet stain does not
securely bind to their cell walls.

2. Why does the crystal violet stain “wash out” of Gram - bacteria when they are decolorized in
ethanol? – Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell
wall) and lose the crystal violet-iodine complex during decolorization with the alcohol rinse,
but retain the counter stain Safranin, thus appearing reddish or pink.

3. What is the function of a mordant? – A mordant is a substance that increases the affinity of
the cell wall for a stain by binding to the primary stain, thus forming an insoluble complex
which gets trapped in the cell wall.

4. Why is timing so critical during the decolorizing step of the Gram stain
procedure? – The decolorization step must be performed carefully, otherwise over-
decolorization may occur. This step is critical and must be timed correctly otherwise the
crystal violet stain will be removed from the Gram-positive cells. If the decolorizing agent is
applied on the cell for too long time, the Gram-positive organisms to appear Gram-negative.
Under-decolorization occurs when the alcohol is not left on long enough to wash out the CV-
I complex from the Gram-negative cells, resulting in Gram-negative bacteria to appear
Gram-positive.
 5. Why does the counter stain safranin appear in the Gram – bacteria? – The decolorized
Gram-negative cells can be rendered visible with a suitable counterstain, which is usually
positively charged safranin, which stains them pink. Pink color which adheres to the Gram-
positive bacteria is masked by the purple of the crystal violet.

6. Why is this procedure important in the clinical setting? – A gram stain is an important
procedure in the clinical setting because it helps a doctor learn if their patient has a
bacterial infection, and it determines what type of bacteria are causing it. Furthermore, it
can also help the doctor determine an effective treatment plan.

7. Is the yogurt safe to eat? – The yogurt is not safe to eat, as there were various Gram-
negative bacteria. The further investigation includes determining how the yogurt became
contaminated and identifying the exact strain of bacteria that contaminated the yogurt.