1.

0 ABSTRACT

The experiment is prepared to identify the characteristics of bacteria whether it is gram

negative or gram positive by using the gram staining method. The materials that should be
added into bacteria are crystal violet that shows purple colour, safranine that shows pink
colour, iodine and alcohol too. Bacteria that classified in gram positive will show purple
colour while bacteria that shows pink colour are in gram negative. This is because gram
positive bacteria have many characteristics, one of that is it have thick peptidoglycan and it
cause the cell to absorb the colour of crystal violet and it will remain purple even though it
has been rinsed by the colourizer which is the alcohol followed by adding safranine. On the
other hand, gram negative have thin peptidoglycan, which the colour of crystal violet is easy
to remove by decolourizer and if safranine is added on it the cell will easily absorbed
safranine colour which is pink. Bacteria that we used in this experiment are E.coli and
Lactobacillus. Therefore, the result that we got is E.coli shows pink colour and it is classified
into gram negative while Lactobacillus shows purple colour under microscope and it is
classified under gram negative group.

2.0 INTRODUCTION

Staining is a technique used to create great contrast in microscopic image. It is able to

highlight structure of biological tissues for viewing under microscope. Dye is used in staining
technique to qualify or quantify the presence of specific compound. There are three types of
staining techniques which are simple, differential, and special staining.

Simple stain is applied to highlight the entire microorganism to make us easy to observe the
cell shape and the basic structure of the microorganism. Unlike simple stain, differential stain
is used to differentiate bacteria based on their major group. Differential stain reacts
differently with different kind of bacteria. Gram stain and acid-fast stain are frequently used
in this kind of staining technique. Then, special stains are used to color and isolate the
specific parts of microorganism such as flagella. It is also used to show the presence of
capsules of the microorganism.

In this experiment, we used gram stain method which is part of differential stain technique.
Gram stain used to differentiate two major types of bacteria which are gram-positive and
gram-negative bacteria. The gram-positive bacteria that was used in this experiment is
Lactobacillus sp. while gram-negative bacteria is E.coli. On the other hands, to make this
experiment success, we need some reagents like crystal violet, iodine, 95% ethanol, and
safranin.

Different kinds of bacteria react differently to gram stain because they have different
structure of cell wall which may affect the retention of combination of crystal violet and
iodine. The cell wall of gram-negative bacteria have thin layer of peptidoglycan and contain
lipopolysaccharide whereas the cell wall of gram-positive bacteria contain thick
peptidoglycan and no lipopolysaccharide.

When crystal violet and then iodine were applied to gram-positive and gram-negative bacteria
cells, it will combine to form crystal violet-iodine complex which is larger than crystal violet
molecule that entered the cell. Due to the size, it is hard to wash it out by alcohol when it is
applied in gram-positive bacteria that contain thick layer of peptidoglycan. However, it is
easy to wash it in gram-negative bacteria because alcohol will disrupt the outer
lipopolysaccharide layer and crystal violet-iodine complex is washed out through the thin
layer of peptidoglycan and make the cell become colorless.

Therefore, at the end of the experiment, we can see that the gram-positive bacteria retain the
color of crystal violet dye, while gram-negative bacteria will turn to pink after applied the red
dye which is known as safranin. (Tortora, et.al, 2014)

3.0 OBJECTIVES
1. The objective of this experiment is to improve the student’s skills in preparing slides.
2. Besides that, it also handled to expose to the students about how to differentiate the
bacteria according to their types.

4.0 THEORY

Gram staining is a common technique used to differentiate two large groups of bacteria based
on their cell wall constituents. The Gram stain procedure differentiates between Gram
positive and Gram negative bacteria by staining the cell wall red or violet. Gram positive
bacteria which possess a thicker peptidoglycan layer, maintains the crystal violet stain during
the decolourization and counterstaining process. Meanwhile, Gram negative bacteria loses the
crystal violet stain and are instead stained by the safranine during counterstaining process.
Overall, Gram positive bacteria will appear purple, while Gram negative bacteria will appear
red or pink. Gram staining consists of three steps which is staining, decolourization and
counterstaining (Bruckner, 2012).

4.1 Staining

Firstly, bacteria are stained with crystal violet dye. This will stained them purple. Next, a
Gram's iodine solution which consist of iodine and potassium iodide is added to form a
complex between the crystal violet and iodine. Usually, it is called as crystal-violet-iodine
(CV-I) complex. This complex is a larger molecule than the original crystal violet stain and
iodine and is insoluble in water. It is function to intensify the colour of stain. Thus, the
bacteria will appear dark purple in colour.

4.2 Decolourization

A decolourizer such as the 95% ethyl alcohol is added to the sample. For Gram positive
bacteria, the alcohol dehydrates the peptidoglycan layer. This cause the pores of the cell wall
to shrink and tighten. The small pores causes the large crystal-violet-iodine complex unable
to be removed from the peptidoglycan layer. Thus, it remains purple. Meanwhile, for Gram
negative bacteria, the outer layer of the cell wall is degraded and the porosity of the cell wall
is increased. The thinner peptidoglycan layer of Gram negative cells helps the crystal-violet-
iodine complex to be easily removed. Thus, Gram negative bacteria appear colourless. In this
process, only Gram negative bacteria are decolourized.

4.3 Counterstaining

This final step uses safranine as the counterstain reagent. The function of safranine is to stain
the sample red. Since Gram negative bacteria were decolourized, it will absorb the safranine.
Hence, Gram negative bacteria will appear red or sometimes pink. Meanwhile, since Gram
positive bacteria is already stained purple, it does not absorb the safranine (Cappuccino &
Sherman, 2008).
5.0 PROCEDURE
1. The glass slide is sterilized by passing it through a burning flame.
2. The surface of sample A is scraped gently by using a wooden cotton bud.
3. After that, sample A from the wooden cotton bud is then transferred on the sterile glass
slide.
4. A few drops of crystal violet are dropped onto the sample A and after 30 seconds, excess
crystal violet is rinsed off with distilled water.
5. Then, a few drops of iodine are dropped onto the sample A and again after waiting for 30
seconds, the excess iodine is rinsed off with distilled water.
6. Generous amount of alcohol are sprayed onto sample A and after waiting for 30 seconds
rinsed it off with distilled water.
7. A few drops of safranine are dropped onto sample A and this time after waiting for 1
minute, the excess safranine is rinsed with distilled water.
8. The sample A was let to dry for a few minutes, later cover slide was placed onto the
sample.
9. The glass slide is then placed on the light microscope to be observed.
10. All observation was later been recorded.
11. Step 1 until step 11 was then repeated with sample B.

6.0 APPARATUS

Bunsen burner, glass slide, cover slip, light microscope, safranin, iodine solution, lugol’s
solution, distilled water, sterile cotton swab.
7.0 RESULTS

Lactobacillus sp. E.coli

Gram staining

Cell morphology (shape) Rod shape Coccus (spherical shape)

Cell morphology Diplobacillus Streptococcus

(arrangement)

Cell color Purple Pink

Gram reaction Gram-positive bacteria Gram-negative bacteria

8.0 DISCUSSION

Gram staining technique is used to color the bacteria sample so that we can differentiate
between gram positive and gram negative bacteria. We will find out the differences between
gram positive and gram negative bacteria after the experiment being held. Gram positive will
show it colour was purple in colour and gram negative will show pinkish in colour. The
difference in colour is due to the cell wall constituent of the bacteria. For gram positive
bacteria, it has a very thick cell wall that consist many layer of peptidoglycan that will traps
the violet dye in the cytoplasm. Peptidoglycan will form a thick and rigid structure of the
bacteria. Peptidoglycan is mainly a polysaccharide. Furthermore, gram positive bacteria
consist of Lipoteichoic acid (LTA) and technoic acid which made up another major
constituent of Gram-positive bacteria cell wall. LTA embedded in the thick peptidoglycan
layer. Gram-negative bacteria has thin layer of peptidoglycan that bonded to lipoprotein and
periplasm. Due to the lack of peptidoglycan, violet dye are easily to rinse and the cell will
appear in pinkish in colour after safranin is added.

Firstly, the bacteria was stained with the Crystal Violet (CV). When CV is dilute in the
aqueous solution, it will dissociate into two component which are CV+ and Cl-. Those
positive and negative charge will penetrate the cell wall of the bacteria. CV+ will react with
the cell wall of the bacteria that is negatively charged and will change the colour of the
bacteria into purple. Secondly, the bacteria was stained with the Iodine. The function of the
iodine are to increase the affinity of cell towards CV-I stain. By the adding of the Iodine, the
bacteria remain purple in colour. Next, the third substance that added was 95% alcohol. The
decolorization of the CV-I was occurred. This happened to the gram negative bacteria. The
high dehydrating effect of the alcohol has remove the colour of the CV-I that being traps in
the cytoplasm. Lack of peptidoglycan has causing the CV-I complex less binding to
peptidoglycan and easily to be decolourized.

Moreover, the fourth staining sources is the safranin. Safranin will cause the bacteria
to turn to pinkish in colour. This only happen to the gram negative bacteria. In this
experiment, methylene blue solution was used to stain the bacterial smears. Why is
methylene blue used to stain the bacterial cells in this experiment? Since methylene blue is a
basic dye, it has a high affinity for acidic substances. The presence of negatively charged
molecules in the cells causes it to be attracted to the positively charged dye.

Due to the staining procedure, the morphology of the bacterial cell will be clearly seen when
examined under the oil immersion. From the observation, we can infer that both Escherichia
coli and Bacillus subtilis are rod shaped bacteria. However, we can note the difference in
their arrangment. Escherichia coli is arranged in an individual manner with noticeable space
between each cell, whereas for Bacillus subtilis, each rod appears to be longer than of
Escherichia coli. The bacteria Staphylococcus aureus appears oval shaped, like a bunched
grape like clusters. It is also very small in size compared to the other three bacteria

When staining process occur, we will started the staining with the Crystal Violet. Crystal
violet will absorb in the thick and thin layer of peptidoglycan which will colorize the bacteria.
At this point, we still cannot differentiate the type of bacteria. So we proceed the staining
with the Iodine where it will combine with the crystal violet to form crystal violet-iodine
complex. At this point bacteria will look dark purple color. Then we decolorize the bacteria
with the 95% alcohol. This point is very important as we start to distinguish the type of
bacteria. Gram negative bacteria will decolorize due to the thin layer of peptidoglycan. The
CV-I complex will be washed away and causing the bacteria become colorless. The thick
peptidoglycan will retain the dark purple color of the gram positive bacteria. Then proceed
the staining with the safranin as the counter stain. At this point safranin will colorized the
colorless gram negative bacteria to pink color.

9.0 CONCLUSION

Based on the experiment that has been conducted, it can be concluded that the experiment
was successfully accomplished. This is because the result was matched with theory that has
been stated which is E.coli is a gram-negative bacteria while Lactobacillus is a gram-positive
bacteria. It can be proven by observing the colour changes of the cell after gram staining
process. All the objectives were also successfully achieved. The first objective is to
differentiate between gram-positive and gram-negative bacteria, so the objective was
achieved when those two samples of bacteria can be classified into two major groups which
are gram-positive and gram-negative bacteria. Second objective is to observed morphological
characteristic of these bacteria. From this experiment, we can identify that E.coli has coccus
shape, has thick wall of peptidoglycan and give purple colour in gram staining. Lactobacillus
has rod/bacillus shape, has thin wall of peptidoglycan and give pink colour in gram staining.
Third objective is to make sure that we are familiar in preparing bacteria smear. So, in this
experiment there are a few steps in smear bacteria process that are listed in the procedure.

10.0 RECOMMENDATION

In this experiment, there are some precautions steps recommended in order to improve the
quality of the results. First, for the step of decolourization, make sure that this step is not
exceeds the time limit to avoid the smear lost its colour. Secondly, make sure to do not let the
tap water fall directly on the smear while washing the slide after staining to avoid the smear
from disrupted. Thirdly, adjust the flame on the Bunsen burner properly to make sure that the
flame was not so strong that will directly kill the microorganism. Fourthly, wave the slides
and loop that we are going to use to make sure that there are no other microorganisms on the
slides. Next, wash the apparatus that we going to use with detergent and distilled water
cleanly before start the experiment to clean up the dirty things that stick on the apparatus.
Lastly, always prefer to observe the slides under 10× magnifications first to get the best area
for observation.

11.0 REFERENCES
1. Bruckner, M. (2012, May 29). Gram Staining. Northfield, MN: Carleton College.
2. Cappuccino J., & Sherman N. (2008). Microbiology: A Laboratory Manual. San
Francisco, CA: Pearson Education, Inc., publishing.
3. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. (2014). Observing
Microorganisms Through a Microscope. Microbiology: An Introduction. (11th edition),
Essex: Pearson Education.

12.0 APPENDICES
CONTENT

CONTENT PAGE
1.0 ABSTRACT 1

2.0 INTRODUCTION 1-2

3.0 OBJECTIVE 2

4.0 THEORY 2-3

4.1 Staining
4.2 Decolourization
4.3 Counterstaining
5.0 PROCEDURE 4

6.0 APPARATUS 4

7.0 RESULT 5

8.0 DISCUSSION 5-6

9.0 CONCLUSION 7

10.0 RECOMMENDATION 7

11.0 REFERENCES 8

12.0 APPENDICES 8