Exercise 2: Postlab Outline

Introduction:

Staining is a technique used to enhance contrast in samples, generally at the microscopic level.
The simple stain can be used to determine cell shape, size, and arrangement. True to its name, the
simple stain is a very simple staining procedure involving only one stain. Gram staining is a common
technique used to differentiate two large groups of bacteria based on their different cell wall
constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups
by coloring these cells red or violet. Gram staining involves four processes: staining with a water-soluble
dye called crystal violet, putting mordant such as Gram’s Iodine, decolorization with Acid Alcohol, and
counterstaining, usually with safanin. Due to differences in the thickness of a peptidoglycan layer in the
cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a
thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram
negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining
process.
Staphylococcus aureus is a Gram-positive, round-shaped bacterium that is a member of the
Firmicutes, and it is a usual member of the microbiota of the body, frequently found in the upper
respiratory tract and on the skin. It is often positive for catalase and nitrate reduction and is a facultative
anaerobe that can grow without the need for oxygen. Although S. aureus usually acts as a commensal
of the human micro biota it can also become an opportunistic pathogen, being a common cause of skin
infections including abscesses, respiratory infections such as sinusitis, and food poisoning.

Objectives

At the end of the exercise, the students shall have

1. Prepare a bacterial smear

2. Have knowledge on how to properly stain a bacteria
3. Distinguish Gram positive from Gram negative bacteria
Simple Stain of Staphylococcus aureus

Methylene Blue Malachite Green

Gram Stain

Low power objective High power objective

ANSWERS TO QUESTIONS
1. In the preparation of a bacterial smear, what is the purpose of fixing the bacteria to the slide?
The purpose of fixing bacteria to the slide is that it denatures bacterial enzymes and prevents
them from digesting cell parts which causes the cell to break. It also enhances the adherence
of bacterial cells to the slide.
2. What are the different ways of fixing the bacteria?
The different ways of fixing the bacteria are heat fix bacterial smear and chemical fixation. Heat
fix bacterial smear is used for the overall morphology of the bacteria but not structures within cells.
Chemical fixation used to protect fine cellular structures & the morphology of larger, more delicate
microorganisms.
3. Enumerate the advantages derived from staining bacteria.
 It gives quick results when examining infections.
 It is simple and cost-effective.
 It helps with determining appropriate treatments for infection.
 It allows for various methods of testing.
 It is basically a key procedure in identifying bacteria.
4. In the Gram stain method, what are the uses of each of the reagent?
In the Gram stain method, there are 4 reagents used. First, the crystal violet, a primary stain,
that imparts color to the microorganism. Second is iodine which is a mordant that intensifies the color of
the primary stain. Third is acid alcohol that acts as a decolorizer that removes the color of the two stains
from the microorganism. The last one is safranin which is a secondary stain that provides a contrasting
stain to the primary stain.
5. Based on the results of your Gram staining method which organisms are Gram positive and what is
their color? Which is Gram negative and what is their color?
Based from the experiment, there are two microorganisms that are Gram stained. These are
Staphylococcus aureus and Escherichia coli. S.aureus is Gram positive because its color in the
microscope is blue while E. coli is Gram negative because its color in the microscope is red.
6. What is the reason for the differences in staining reactions of the above organisms?
There is a difference in the staining reactions of the organisms above because of the
peptidoglycan layer that is present in their cell wall. Gram positive organisms have thick peptidoglycan
layer which is why it appears blue/purple in the microscope since the color of the primary stain sticks to
its peptidoglycan layer up until to the last process of Gram staining. On the other hand, Gram negative
organisms have thin peptidoglycan layer in which the color of the primary stain was decolorized and the
color of the safranin will stick to it causing it to appear as color red.
7. List other differential stains used in microbiology.
The other differential stain used is the Acid Fast or the Ziel-Neelsen Method. This is used for
waxy bacteria or Mycobacterium which produces the waxy substance called mycolic acid when they
construct their cell walls. The end appearance of the organism is color pink or violet in a dark
background.
Conclusion

Staining technique involve the application of dye in a sample, some require only one dye to be
applied others also require more than one dye. There are two staining techniques used in the
experiment the simple staining and the gram staining. Simple staining used a single dye to emphasize
certain structures in the specimen. Gram staining is a differential staining used to identify whether a
bacteria is gram-positive, purple/violet appearance, or gram-negative, red appearance. With the use of
gram staining technique we were able to identify S. aureus as a gram positive bacterium.

Staphylococcus aureus (S. aureus) is a gram positive bacterium. It has a thick peptidoglycan layer
with techoic acid and lipotechoic acid, wherein the dyes used in staining can adhere to. Dyes are used to
color certain features of a specimen before examining. It contains salts made up of positive or negative
ions. Basic dyes have positively charged chromophore, an example of basic dye is Malachite green, the
chromophores of malachite green will be absorbed by the cells or organism causing the coloration of the
sample which make them stand out during viewing. Acidic dyes have negatively charge chromophore,
one example of acidic dye is eosin, the chromophore of eosin will be absorbed by the background not by
the cells or organism which produces a silhouette or outline against a colorful background. The dyes
used in S. aureus are methylene blue and malachite green which are basic, the reason why the cell is
observable in the specimen.