FST 3201- 3 (BASIC FOOD MICROBIOLOGY)

EXPERIMENT TITLE :
MICROBIAL STAINING METHOD

EXPERIMENT DATE:
12 APRIL 2023

LECTURER’S NAME:
PROF. MADYA. DR. FARINAZLEEN BINTI MOHAMAD GHAZALI
PROF. MADYA. DR YAYA RUKAYADI

MEMBERS OF SUBGROUP 6

NAME MATRIX NUMBER

NOR SYUHADA BINTI BAHARUDIN 210374

NURUL AISYAH BINTI ABDUL RAHMAN 209903

NOR SYAZANA BINTI ZAINUDDIN 210216

FARAH SOFEA BINTI SUHAIMEE 209799

MARKING BOX:
ABSTRACT

This experiment discussed the principles of different microbial staining methods. This

experiment involved simple staining, gram staining and structure staining. The three different

staining methods started with heat fixation which is the spreading of microbial culture to the

slide and passed the slide through the flame to heat-fix the bacteria. Simple staining used a

single stain dyes while gram staining or differential staining consist of four fundamental steps

which are primary stain, followed by mordant, decolourisation and counterstain. Structure

staining needed strong dyes and vigorous staining conditions such as heat. Simple staining

tells us about the shape and arrangement of the cell while gram staining shows obvious

differences between gram-positive and gram-negative bacteria. Escherichia coli is a

gram-negative bacteria while Staphylococcus aureus and Bacillus cereus are gram-positive

bacteria.
INTRODUCTION

In the laboratory, bacterial and fungi morphology may be examined in two ways

which are observing living unstained microorganisms or observing killed stained

microorganisms. In order to perform microbial staining, fixing, and staining are crucial. The

goal of making a smear is to keep the germs on the slide and to keep the sample from being

lost during the staining step. A smear can be formed using either a solid medium or broth.

There are various steps required in creating a bacterial smear such as fixing and staining

methods. The production of the smear requires attention to several aspects that help avoid

contamination of the culture and assure the preparer's safety.

Fixing is done to keep the bacteria from being washed off the slide during staining

operations. Heat fixation is the most common method of fixing. The microbial smear is

heat-fixed during heat fixation by rapidly passing the slide through the flame of a Bunsen

burner. The heat coagulates the microbial proteins, causing them to adhere to the slide.

Next, simple staining is used to recognize the basic shapes of bacterial cells by

creating contrast which use basic dyes which are positively charged then interact with the

slightly negatively charged bacterial cell wall thus lending the color of the dye to the cell wall.

Differential staining which involves Gram-positive bacteria have thick layers of

peptidoglycan in their cell walls, which is a material found in the cell walls of many bacteria.

In gram-positive bacteria, peptidoglycan accounts for around 90% of the cell wall. This

allows them to appear blue to purple when stained with a Gramme dye while Gram-negative

bacteria have cell walls made up of thin layers of peptidoglycan and a high lipid (fatty acid)

content. This allows them to appear red to pink when stained with a Gramm dye.
 Lastly,endospore-producing bacteria require a physiologically favorable environment

for biosynthesis in order to create endospores. However, once formed, these endospores

can withstand a variety of harsh environments. Endospore production (sporulation) is the

result of a complicated set of actions. Each vegetative bacterium produces one endospore.

The vegetative component of the bacteria is degraded once the endospore is created, and

the dormant endospore is released. Therefore strong dyes and vigorous staining are

needed.

OBJECTIVE

The objective of this experiment was to master microbial staining methods as well as to

describe the concept of different techniques and how to carry out the correct microbial

staining methods. Next, this experiment was also about to assemble, analyse and assess

the experimental data. Lastly, to work together with the companion students in both

experiments along with the documentation of the experiment.

MATERIAL AND METHOD

A) SIMPLE STAINING

Materials: Stain solutions, crystal violet, bacterial cultures, blotting paper and clean glass

slides.

Methods:

Firstly, a smear from either an agar plate or broth was prepared using the fixing

method described in the earlier section. Then, simple stains were added by the drop of

Crystal violet for 10 seconds.The excess stain was rinsed off under a gentle stream of water

and dried the slide with blotting paper. For the next step, the slide was then viewed under a

microscope using the oil immersion lens and observation was recorded.

B) DIFFERENTIAL STAINING

Materials: Stain solutions, hucker’s crystal violet, gram’s iodine, 95% ethyl alcohol, safranin

red, bacterial cultures, blotting paper and clean glass slides.

Methods:

Firstly, a smear was prepared. Then, the slide was flooded with crystal violet stains

and left for 1 min. After that, crystal violet stain was poured off, washed with water, and

flooded the slide with gram’s iodine solution for 1 min. The slide was then rinsed with water

from a squirt bottle and blot dried to remove excess water with a Kimwipe. The next step

was holding the slide on a slant, the slide then flooded with the combination 95% alcohol,

until the dye washed from the cells. As most of the crystal violet dye stops leached from the

cells, the slide was then washed in water to stop the decolourisation process and the slide

was then counterstained with safranin red for 1 min. Next, the excess stain was then rinsed

off under a gentle stream of water and blotted the slide dry with blotting paper. Lastly, the

slide was then viewed under a microscope using the oil immersion lens and observation

was recorded.
B) STRUCTURE STAINING

Materials: Endospore stain solutions,5% malachite green solution, 0.5% safranin red,

blotting paper, clean glass slides and bacterial cultures.

Methods:

Firstly, a smear was prepared as described earlier. Then, saturated with malachite

green.Then the malachite green left sits on the slide for 1 min and the slide was then

carefully heated with forceps over the flame of a Bunsen burner until the stain began to

steam. For the next step, the slide was removed from the heat as the steaming stopped then

gently reheated and proceeded for 5 min. Next, the excess stain was washed off the slide

with water after 5 min. Any dye that got onto the bottom of the slide was washed and the

slide then blotted until dry with blotting paper. After that, the slide was smeared with safranin

red for 1 min. and the excess stain was rinsed off under a gentle stream of water and blotted

the slide dry with blotting paper. Lastly, the slide was then viewed under a microscope using

the oil immersion lens and observation was recorded. With this endospore staining

procedure, endospores will stain green while vegetative bacteria will stain red.

.
RESULTS

BACTERIA OBSERVATIONS

Escherichia coli

Rod-shaped, seen in single and pairs

Bacillus Cereus

Rod-shaped, seen in pairs and chains

Streptococcus aureus

Round shaped, seen in pairs and short chains

Table 1.0 was about the observations of simple staining of (E. coli), (B. cereus) and
(S. aureus)
 BACTERIA OBSERVATIONS

Escherichia coli

Rod shaped,
gram-negative bacillus as it appear in pink colour

Bacillus Cereus

Rod shaped,
Gram-positive bacillus as it appears in purple colour.

Streptococcus aureus

Round shaped,
Gram-positive coccus as it appears in purple colour.
Table 1.1 was about the observations of differential staining of (E. coli), (B. cereus) and
(S. aureus)

BACTERIA OBSERVATIONS

Bacillus Cereus

Rod shaped, the spore appear in green colour

Table 1.2 was about structure staining of (B. cereus)
DISCUSSION

Based on the results of the experiment, staining is carried out to help us distinguish
between the microbe and its surroundings, particularly the ability to see the cell wall,
vacuoles, and nuclear bodies. The result of staining happened due to the reaction between
cell components and dyes. There are few staining techniques which are simple staining,
differential staining and special staining. Each technique carries a different procedure and
outcome following on the result we would like to obtain. In simple staining single basic dye
such as safranin and methylene blue is used to see the cellular shape. The method is also
known as indirect staining since the objective is to have a better understanding of bacterial
size, shapes, and groups. It works as the positive-ion dye combines with the negative-ion
bacterial cytoplasm, the cell will be coloured immediately. Next, in terms of differential
staining, it is used to exploit the basic differences in the outer layers of bacteria. Differential
staining requires four steps to obtain the final result which are primary staining by crystal
violet, mordanting using Gram’s iodine solution decolourization using ethyl alcohol followed
by acetone and counterstaining with safranin red. Gram stains mainly provide two
information which are gram positive and gram negative due to the difference between the
structure of the bacterial cell wall particularly peptidoglycan thickness (FST3201 Laboratory
Week 5 manual, 2023). A positive result will likely show in the bacteria turning purple,
indicating gram-positive bacteria, whilst negative results in the microorganisms turning red
pinkish, indicating gram-negative bacteria. In this situation, Escherichia coli was negative,
whereas Staphylococcus aureus and Bacillus cereus were both positive. Furthermore,
malachite green solution and safranin red are used to determine the presence of
endospores in bacteria which is structure staining. When counter-stained with safranin, the
vegetative cells take the color of safranin and appear red or pink, in contrast to the
endospores that appear green (Tankeshwar. A, 2022).

Microbial fixation is one of the methods necessary prior to staining for better results. The
fixing process is vital for preventing microorganisms from being washed away from the slide
during staining procedures. It is necessary to heat-fix the microbial smear by rapidly passing
the slide over a Bunsen burner flame to ensure that the heat coagulates the microbial
proteins, forcing them to attach to the slide (FST3201 Laboratory Week 5 manual, 2022).
One probable reason for the microorganism's absence is because it was not exposed to
sufficient heat from the Bunsen burner. As a result, the microbe washes off the plate during
staining. Besides, it is possible that the cultures on the agar were not scraped correctly.
Finally, as stated previously there are two types of gram bacteria which are gram-positive
and gram-negative, both will develop different reactions.
As for gram negative bacteria, they can release endotoxins if something disturbs their cell
wall while gram positive bacteria there are presence of toxins such as neurotoxin that can
cause numerous food-borne diseases. Aside from that, gram-negative bacteria have two
membranes, internally and externally whereas gram-positive bacteria lack a protective outer
membrane. Next, gram-negative bacteria own thinner peptidoglycan cell walls than
gram-positive bacteria which are placed between their two membranes. Additionally, as
mentioned above gram positive bacteria possess purple color while gram negative bacteria
possess red pinkish colour after procedure was done. Most of the shapes owned by
gram-negative bacteria are spherical, rod and spiral-shaped while gram-positive bacteria are
spherical, rod or branching filaments.

CONCLUSION

In a nutshell, we can comprehend the different method principles of microbial staining for
different observations. Besides, we also performed the microbial staining procedures
correctly. Next, we are also able to collect, analyse the shape and structures and evaluate
the experimental data. In this experiment we also applied the three methods we learnt
theoretically which are simple staining in observing shapes and cell arrangement, differential
staining in differentiating types of gram and finally structure staining in observing the
endospore structure.
QUESTIONS

1. What affect may heat fixation have on the characteristics of a Microorganism?

● Heat fixation prevents the microorganisms from being washed away during staining
and adheres them to the slide and by coagulating the microbial proteins thereby
causing it to stick onto the slide. The chemical and physical characteristics in the
microorganisms could change, as well as the shrinkage, swelling and hardening of
various components

2. What information can be obtained from a simple stain?

● Simple staining can be used to determine the cell shape, size, and arrangement of
bacteria.

3. What is the function of the iodine solution in Gram staining?

● iodine solution in the gram stain is to fix the dye on the slide in order to form
insoluble substance

4. What counter stain is used in Gram staining, and why is it necessary?

● Counter stain is used to directly stain the decolorized gram-negative bacteria. As the
gram-positive bacteria retain the crystal violet-iodine colour, the gram-negative
bacteria will turn pink.

5. What is the advantage of Gram-stain over simple stain?

● Gram staining highlights different bacteria types through the use of special dyes. It
aids in the diagnosis of a specific organism and tells the difference between gram
negative and gram positive bacteria.

6. What might cause a Gram variable reaction?

● The methods and techniques used, the age of culture and the bacterium itself.

7. What cellular structures can be seen with Gram-staining? Why?

● Cell wall. The cell walls of bacteria consist of numerous interconnecting layers of
peptidoglycan which is porous and lets small molecules through.
● Differences in cell wall thickness also make Gram staining possible. Gram staining is
used for the general identification of bacteria; bacteria with thick cell walls are
gram-positive, while bacteria with thinner cell walls are gram-negative.
8. When is it useful to perform a Gram-staining?
● When diagnosing and treating certain bacterial infections.

9. What would you expect from a Gram stain done on a slide that was heated
too hot during the heat-fixed smear?
● The cell wall would be damaged and may lead to inaccurate results, which may
cause Gram-positive bacteria to falsely appear as gram-negative bacteria.

10. Why did you view all experiments under an oil immersion lens?
● To achieve greater clarity of an image at high magnification. Oil prevents light from
bending and distorting the image of an object under study.
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