Staining Microorganisms for Microscopic Examination

Exercise 2

Gelliza Joy E. Diozon

0419 – 1162

October 24, 2019

A scientific paper submitted in partial fulfilment of the requirements in Microbiology and

Parasitology laboratory under Ms. Jamie Louise S. Batalon, 1st semester 2019
 Abstract

The Gram Stain, or gram staining is one of the different kinds of Staining, it allows one
to distinguish between Gram-positive and Gram-negative bacteria on the basis of
differential staining with a crystal violet-iodine complex and a safranin counterstain. The
cell walls of Gram-positive organisms retain this complex after treatment with alcohol
and appear purple, whereas gram-negative organisms decolorize following such treatment
and appear pink. There is a precise and accurate conclusion on our experiment by
following the general protocol and procedure of the Gram Staining. Escherichia coli,
Staphylococcus aureus and Lactobacillus, cheek cells stain, teeth cells stain are the
bacteria used for staining. Escherichia coli, teeth cells stain are the Gram Negative
bacteria, while Staphylococcus aureus and Lactobacillus, cheek cells stain are the Gram
positive bacteria. There are two other different kind of Staining; Simple Staining and
Differential Staining. Teeth Scraping as an example of Simple Staining; while Cheek
Scraping is an example of Differential Staining an example of this is Cheek Scraping.
Bacteria can see using the microscope with a high power objective.

Introduction

In microbiology, differential staining techniques are used more often than simple stains as a
means of gathering information about bacteria. Differential staining methods, which typically
require more than one stain and several steps, are referred to as such because they permit the
differentiation of cell types or cell structures. The most important of these is the Gram stain.
Gram’s staining techniques is use to aid in the identification of bacteria, beginning with a

preliminary classification into one of two groups: Gram positive or Gram negative. The main

difference between these two types of bacterial cells is their cell walls. Gram negative cell
walls have an outer membrane (also called the envelope) that dissolves during the alcohol
wash. This permits the crystal violet dye to escape. Only the decolorized cells take up the
pink dye safranin, which explains the difference in color between the two types of cells.

At the Gram stain procedure, Gram positive cells appear purple, and Gram negative
cells appear pink. The differential nature of the Gram stain is based on the ability of some
bacterial cells to retain a primary stain (crystal violet) by resisting a decolorization process.
Gram staining involves four steps. First cells are stained with crystal violet, followed by the
addition of a setting agent for the stain (iodine). Then alcohol is applied, which selectively
removes the stain from only the Gram negative cells. Finally, a secondary stain, safranin, is
added, which counterstains the decolorized cells pink.
Materials and Method

In simple staining (Teeth Scraping) stained specimen for 3-5 mins with methylene blue,
washed with water and air dry.

In Differential Staining (Cheek Scraping) stained with methylene blue for 5 seconds. Washed
with water, counterstain with carbol fuchin for 3 seconds. Washed with water and dry.

In Gram Staining

A. Staining of Escherichia coli

Escherichia coli is the bacteria were used for staining. The first thing to do is
the general protocol before starting the experiment. After doing the general
protocol, we start our experiment by staining the specimen with crystal violet
for 1 minute then washed it gently with tap water, not directly on the smear.
Covered the smear with iodine solution for 1 minute and washed again with
tap water, and the excess moisture shake off. For 15 to 20 seconds decolorize
the smear with 95 % of alcohol, and washed with water. The safranin solution
applied for 30 seconds to counterstain the smear then washed again with tap of
water and blot dry.

B. Staining of Staphylococcus aureus

Staphylococcus aureus is the one of the bacteria were used for staining. The
first thing to do is the general protocol before starting the experiment. The
smear was stained with crystal violet for 1 minute then washed it with tap
water not directly on the smear. Covered the smear with iodine solution for 1
minute, washed with tap water and shakes off the excess moisture. For 15 to
20 seconds the 95 % alcohol applied to decolorized the smear, again washed
with tap water. The smear was counterstain with safranin solution for 30
seconds it was washed with tap water and blot dry.

C. Staining of Lactobacillus
 Lactobacillus this is the last bacteria were used for staining. Before starting
the experiment the general protocol was done, crystal violet added to the
smear for 1 minute the washed with water, not directly on the smear. Covered
the iodine solution for 1 minute and washed again with water, the excess
moisture was shake off. The smear was decolorized of 95 % alcohol for 15 to
20 seconds, then washed again with tap water. The smear was counterstain
with safranin solution for 30 seconds it was washed with water and blot dry.

Results and Discussion

 Escherichia coli, Staphylococcus aureus, Lactobacillus bacteria are stained by the

crystal violet. Iodine solution is used to cover the smear so that the dye cannot be removed
easily. This step is commonly referred to as fixing the dye. We use safranin solution to
counterstain all the smear for us to distinguish the result easily. By using the high power
objective of the microscope we seen the color of the bacteria we experiment, and based on
the results of our experiment Escherichia coli resulting that this bacteria is a Gram- Negative
because of this characteristic, Escherichia coli color is pink viewing using microscope, while

Staphylococcus aureus and Lactobacillus resulting that this bacteria is a Gram – Positive
because of it color purple mixed with light blue we saw this by using a high power objective
of microscope.

Cheek cells is a Gram- positive because of its color. In microscope, cheeks cell is
color purple-blue, and based on the description of Gram-positive the color turns to purple-
blue while teeth cell is a gram negative, in microscope teeth cell is color pink that’s why this
cells is a gram-negative.
Cheek Cells Stain Teeth Cells Stain

Lactobacillus Staphylococcus aureus

Escherichia coli
Summary and Conclusion

Gram positive bacteria have a distinctive purple appearance when observed under a
light microscope following Gram staining. This is due to retention of the purple crystal violet
stain in the thick peptidoglycan layer of the cell wall. Gram negative bacteria appear a pale
reddish color when observed under a light microscope following Gram staining. This is
because the structure of their cell wall is unable to retain the crystal violet stain so are colored
only by the safranin counterstain. Giving the results of our experiment Cheek Cells Stain,
Lactobacillus, Staphylococcus aureus is a Gram positive bacteria while Escherichia coli and
Teeth Cells Stain is a gram negative bacteria therefore I conclude, we can easily distinguish
the characteristics of bacteria by using a gram staining. We can identify the bacteria by its
color, if it’s Gram Positive or Gram negative bacteria.

References

https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/differential-staining-
techniques/

Microbiology: A Laboratory Experience by Holly Ahern is licensed under a Creative

Commons Attribution-Non Commercial-Share A like 4.0 International License

http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/Gram1.htm#targetText=Bacteria
%20cell%20walls%20are%20stained,to%20as%20fixing%20the%20dye.

-Web Page Written by George Xu,MD, Ph.D. 10/31/97

https://www.technologynetworks.com/immunology/articles/gram-positive-vs-gram-negative-323007
Karen Steward PhD

Science Writer