FT 112 General Microbiology

Name: __________________________ section: ______________

Date submitted: ________

Simple staining

Ft 112 Laboratory Exercise 2

Introduction

Most microorganisms, including bacteria, appear colorless when viewed

through a light microscope. Therefore, in order for them to be successfully
observed, bacteria need to be prepared. One of the techniques for viewing
bacteria is to stain the specimen. The stain is a dye that will color the
microorganisms and will emphasize certain structures. However, before they are
stained, the bacteria will need to be fixed to the microscope slide. Fixing the
bacteria will, both, kill the microorganisms and attach them to the slide. In order
for a specimen to be fixed, a thin film of material containing the microorganism
is spread over the surface of the slide, called a smear (tortora, funke, case,
2013).

Objectives

1. Define simple stain.

2. Tell the purpose of simple staining and what bacterial feature(s) can and
cannot be ascertained when using a simple stain.

3. Prepare a bacterial smear and properly heat fix the smear. Tell the
importance of fixation in preparing a bacterial sample for staining.

4. Prepare a simple stain.

5. Examine the bacterial smear with a microscope and make an accurate

illustration of the bacterial cells.
 6. Use the microscope to identify bacterial features (cell shape and cell
arrangements). Identify bacterial cell shapes and cell arrangements given
examples.

Materials

1. Staining kit

2. Glass slide

3. Check cell sample

4. Alcohol lamp

5. Cover slip

6. Microscope

Method

1. Collection of sample

2. Heat fix the slide. Allow the slide to cool.

3. Stain with crystal violet for 1 minute by flooding the slide with stain.
Rinse with water.

4. Apply iodine solution for 1 minute by flooding the slide with iodine.
Rinse with water

5. Carefully, decolorize for 3 seconds with gram stain decolorizer by

flooding the slide with decolorizer. Immediately, rinse with water.

6. Counterstain with safranin for 30 seconds by flooding the slide with stain.
Rinse with water.

7. Dry the slide by putting it between the pages of a book of bibulous paper.
Remove slide and view organisms using the oil immersion objective of
your microscope.
Examining the Gram Stain

1.The laboratory scientist then categorizes any bacteria that may be present by
color and shape during the microscopic evaluation:

• Color: typically, bacteria that are gram-positive appear purple to blue, and
bacteria that are gram-negative appear pink to red.

• Shape: the most common shapes include round (cocci) or rod-shaped (bacilli).

2.The laboratory scientist also looks for additional characteristics of the sample
by observing the groupings of the bacteria on the slide. Examples include:

• Cocci that are present singly, in pairs, in groups of four, in clusters or in

chains.

• Bacilli that are thick, thin, short, long or have enlarged spores on one end.

• If bacteria are present within white blood cells.

Questions to answer:

1.What are gram-positive and gram-negative bacteria?

Gram-positive and Gram-negative bacteria are two major groups of

bacteria distinguished by their cell wall structure, specifically their response to a
staining technique called the Gram stain. Gram-positive bacteria have a thick
peptidoglycan layer in their cell walls, which retains the crystal violet stain used
in the Gram staining process. They appear purple or blue under a microscope
after Gram staining. Many Gram-positive bacteria are known for causing
various human diseases, including skin infections, pneumonia, and food
poisoning. (Silhavy & Khane et.al May 2010).
 Gram-negative bacteria have a thinner peptidoglycan layer in their cell
walls, surrounded by an outer membrane composed of lipopolysaccharides
(LPS).
They do not retain the crystal violet stain well and appear pink or red after
Gram staining. Gram-negative bacteria often possess greater resistance to
antibiotics due to the protective outer membrane, making them a significant
concern in healthcare settings.
The classification into Gram-positive and Gram-negative groups is based
on differences in cell wall structure and has implications for bacterial
identification and treatment, as well as our understanding of their biology and
pathogenicity. ( 2023, BYJU’S).
On the other hand, In my own understanding about the gram-positive and
gram negative bacteria are those bacteria that was classified into different color
and shape due to its type and content that are usually came from our body.

2.What is a Gram stain?

Gram stain, is a method of staining used to classify and determine the

bacterial species into two large groups and these are gram-positive bacteria and
gram-negative bacteria.

3.What are gram-positive and gram-negative bacteria, give at least 5 examples.

Some examples of gram-positive bacteria include Staphylococcus,

Streptococcus, and Clostridium species. While the examples of gram-negative
bacteria include Escherichia coli (E. coli), Salmonella, and Pseudomonas
species.

4.What is the difference between a Gram stain and a bacteria culture?

 In my own understanding gram stain was a test to determine if a person
contains a gram-positive or gram-negative bacteria. While a bacteria culture was
way of multiplying the bacteria.

Gram stains provide quicker results than bacteria cultures, in most cases,
a Gram stain can’t diagnose the type of bacterium. Bacterial culture is a method
that allows the multiplication of bacterial cells in or on a culture medium under
controlled laboratory conditions. (Karen Steward, PhD November 2021).
Observation
Results and Discussion (with picture)

IMAGE

ABALAYAN,
ELLIZA
AMOR F.

Gram-positive
Cocci

SARNO,
PRINCESS
LIA V.

Gram-positive
Gram-negative
Cocci with slightly
Bacilli

TABUÑAG,
HONEY LYN

Gram-negative
Cocci with slightly Bacilli
 Observation

After we collected the sample and perform the fixation. We proceed to

staining test. And my observation for that laboratory activity was if you have
collected a more specimen from your cheeks and dried it well using fixation as
well as if you perform the proper staining you can view the bacteria better
even you don’t have to set your microscope in a high level of total
magnification since the stained sample was attached well and fixed properly
from the glass slide.

Result and Discussion

 SARNO TABUÑAG ABALAYAN
The three different samples that came from Me, from Ms. Tabuñag and Ms.
Abalayan would obviously come up with different results since we are a different
person and we contain a different kinds of good and bad bacteria. The first picture
was mine and the bacteria that I have were Gram-positive and Gram-negative
because of its color which are pink and violet. My sample contains a combination
of Cocci and Bacilli since it showed up that it has a ocular or round shape and
spiral shape of bacteria. The total magnification that I set in order to view my
sample was only 715. I think I contain these kind of bacteria because lately I have
a mild cough. And according to ( 2023, BYJU’S) Most cocci bacteria are disease-
causing pathogens. Pneumococcus. They are anaerobic gram-positive members of
the Streptococcus genus. Streptococcus pneumonia (pneumococcus) colonises the
nasal cavity and the respiratory tract and is the main cause of many pneumococcal
infections like meningitis, pneumonia and sepsis.

The other sample that came from Ms. Tabuñag was having a result of only
Gram-negative type of bacteria since as we can see it was only a color red that
having a cocci but slightly bacilli shape the magnification that has been used upon
viewing her specimen was 725. While according in the (2023. TESTING.COM).
A negative Gram stain is often reported as “no organism seen.” This may mean
that there is no bacterial infection present or that there were not enough bacteria
present in the sample to be seen with the stain under a microscope. Positive Gram
stain results usually include a description of what was seen on the slide.
 The last picture was a stained sample of miss Abalayan and it shows that
after setting the microscope in 720 magnification she was having a result of
cocci gram-positive bacteria. According to (Medically reviewed by William C
Lloyd III, MD, FACS of 2023) Gram-positive bacteria are a type of bacteria that
have a thick cell wall. This cell wall helps protect the bacteria from antibiotics
and other substances that might damage them. Gram-positive bacteria can cause
a range of infections, from food poisoning to serious respiratory diseases.

Conclusion

At last, I conclude that, gram staining was one of the very useful, helpful
and convenient way in the laboratory to find out if a person has a bacterial
infection. If we do, the test will show if your infection is Gram-positive or
Gram-negative. A Gram stain may also be used to diagnose fungal infections.
Furthermore, the result will matter or can affect if the amount of stain was
exactly enough to view the results as well as if the staining process was properly
followed and performed. The source of the sample could also be one of the
factor to have a different results.
References

https://byjus.com/biology/difference-between-gram-positive-and-gram-
negative-bacteria/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857177/#:~:text=Gram
%2Dnegative%20bacteria%20are%20surrounded,found%20in%20the
%20Gram%2Dnegatives.Published in Advance April 14, 2010, doi:
10.1101/cshperspect.a000414 Cold Spring Harb. Perspect. Biol. 2010.
Copyright © 2010 Cold Spring Harbor Laboratory Press; all rights
reserved

https://www.sciencedirect.com/referencework/9780080552323/xpharm-the-
comprehensive-pharmacology-reference

https://www.technologynetworks.com/immunology/articles/an-introduction-to-
culturing-bacteria-355566

https://www.mayomedicallaboratories.com/test
catalog/Clinical+and+Interpretive/8078

https://www.medicalnewstoday.com/articles/gram-positive-
bacteria#characteristics