ABSTRACT

Staining is a technique used in microscopic techniques to enhance the clarity of the

microscopic image and in this experiment, we are using Bacillus sp. and unknown bacteria.
In gram staining we have to determine the gram of the bacteria either it is gram-positive or
gram-negative. The smear first was flooded with crystal violet and excess dye was washed
with tap water and let it dry. After that, the smear was exposed to iodine, 95% alcohol and
lastly 0.25% safranin. The smear must be washed with tap water and let it dry before putting
the next reagent. Then, the smear was observed under the microscope and we get for Bacillus
sp. it is gram-positive because it shows purple color and the shape is bacillus. While for
unknown bacteria is gram-negative because it shows red color and the shape is coccus.
INTRODUCTION
Stains and dyes are widely used in the scientific field to highlight the structure
of the biological specimens, cells and tissue. The most widely used staining procedure in
microbiology is the Gram stain, discovered by the Danish scientist and physician Hans
Christian Joachim Gram in 1884. Gram staining is a differential staining technique that
differentiates bacteria into two groups: gram-positives and gram-negatives. The procedure is
based on the ability of microorganisms to retain color of the stains used during the gram stain
reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the
primary stain, purple. Gram-positive bacteria are not decolorized by alcohol and will remain
as purple. After decolourizations step, a counterstain is used to impart a pink color to the
decolorized gram-negative organisms (Tan, 2013).
The Gram stain is a very important preliminary step in the initial characterization and
classification of bacteria. It is also a key procedure in the identification of bacteria based on
staining characteristics, enabling the bacteria to be examined using a light microscope. The
bacteria present in an unstained smear are invisible when viewed using a light microscope.
Once stained, the morphology and arrangement of the bacteria may be observed as well.
AIMS
1. To develop the skill in preparing slides.
2. To expose the students with the method for differentiation of bacteria types.

THEORY
The Gram stain is a differential stain which allows most bacteria to be divided into
two groups that are gram-positive and gram-negative. The technique is based on the fact that
the gram-positive cell wall has a stronger attraction for crystal violet when Grams iodine is
applied than does the gram-negative cell wall. Grams iodine is known as a mordant. It is able
to form a complex with crystal violet that is attached more tightly to the gram-positive cell
wall than to the gram-negative cell wall. This complex can easily be washed away from the
gram-negative cell wall with alcohol. Gram-positive bacteria, however are able to retain the
crystal violet and therefore will remain purple after decolorizing with alcohol. Since gram-
negative bacteria will be colorless after decolorizing with alcohol, counterstaining with
safranin will make them appear pink (Benjamin, 2013).
The peptidoglycan that present in the cell wall is the main part in order to determine
the gram-positive and gram-negative. Because even though peptidoglycan present in both
bacteria but the arrangement of the peptidoglycan contribute to the classification of the gram-
positive and gram-negative of the bacteria. The gram-positive has a thick peptidoglycan
while the gram-negative has a thin peptidoglycan.

Figure 1 : The cell wall structure of Gram-positive and Gram-negative
Bacteria is vary in their morphology features the most common morphologies are
coccus, rod-shaped bacteria that is generally occur singly and lastly is spirillum that is spiral-
shaped bacteria. Bacillus sp. is known has a rod-shaped.

Figure 2: The morphology of bacteria
APPRATUS AND MATERIALS
1. Glass slide 9. Iodine
2. Dropper 10. Crystal violet
3. Microscope 11. Cloth or tissue
4. Bunsen burner 12. 95% alcohol
5. Bacillus sp.
6. Unknown bacteria
7. 0.25% Safranin
8. Distilled water

PROCEDURE
A. Preparation of Cell Smear
1. The slide was cleaned for the preparation of smeared with water or scouring powder.
2. After the cleaning of slide, it rinsed using water and 95% of alcohol.
3. Slide was dried and placed on laboratory towels.
4. The smear was prepared using dropper. Avoid thick and dense smear because this will
reduce the amount of light.
5. The slide was heat using Bunsen burner, by forming rapid passage two or three times.

B. Simple Staining
1. The slide was placed on the staining tray and the smear was flooded with one
indicated stains, with appropriate exposure time for each : Carbon fuchsin 15-30
seconds, crystal violet 20-60 seconds, methylene blue 1-2 minutes.
2. Smear slide was gently washed with tap water to remove excess stain. The slide was
hold parallel to the stream of water in order to reduce the loss of organisms from the
preparation.
3. The smear was bolted using bibulous but not to wipe the slide.
4. The slide was examined using oil immersion microscope.
C. Gram Stain
Slant Cultures
1. The slide was smear and undergo heat fixation.
2. Crystal violet was flooded for one minute.
3. Excess dye was washed gently with tap water and dried the slide using paper towel.
4. The smear was exposed to grams iodine for one minute by washing with iodine, then
adding more iodine and leaving it on smear until one minute is over.
5. The smear was washed with tap water and drained carefully.
6. Then, smear was washed with 95% of alcohol for 30 seconds.
7. After that, the smear was washed with tap water at the end of the 30 seconds to stop
the decolorization and drained.
8. The smear was counterstain with 0.25% safranin for 30 seconds.
9. The smear was washed, drained, blotted and examined under oil.
10. The cell morphology, group and relative sizes was drew.
Broth Cultures
1. The ring was drew under open slide to mark the area for the smear of the broth since
broth cultures was nearly visible to naked eye.
2. Do not add drop of water to the borth.
3. The smear was heat fixed and gram stain the smear using the above procedure.
4. Slide was examined.

RESULT
A. Gram Staining
Bacillus sp. UNKNOWN


Shape : Bacillus
Arrangement : Cluster
Cell Color : Purple
Gram-positive
Shape : Coccus
Arrangement : Chain
Cell Color : Red
Gram-negative
DISCUSSION
In this experiment we did not do the simple staining because the morphology of the
bacteria can be seen from the gram staining method. From the experiment, it shows that the
Bacillus sp. has a bacillus shape and the arrangement is cluster. The colour of the cell is
purple and this indicate that it is gram-positive. While for the unknown bacteria it shows that
the shape is coccus and the arrangement is chain. The colour of the cell is red and it indicate
that the cell is gram-negative. The gram bacteria shows different color due to the arrangement
of the peptidoglycan in the cell wall. The peptidoglycan that present in the cell wall is the
main part in order to determine the gram-positive and gram-negative.
Crystal violet is the first dye use is gram staining. It forms a complex with iodine and
get fixed permanently in gram-positive bacteria. The dye iodine complex is not washed out
from these organisms and not decolorized. This is because it contain a thick peptidoglycan
while the gram-negative have a thin peptidoglycan. Therefore, the dye is not fixed
permanently in gram-negative bacteria. The gram-negative bacteria will lose the dye and
become colorless while the gram-positive bacteria retain the color of the crystal violet. So
when gram-negative bacteria is stained with 0.25% Safranin, the red color will appear while
for gram-positive will still retain the violet color.
The crystal violet is the primary stain, which stains everything in the smear blue. The
Gram's iodine acts as a mordant and expose it around 1 minute. It is able to form a complex
with crystal violet that is attached more tightly to the gram-positive cell wall than to the
gram-negative cell wall. This complex can easily be washed away from the gram-negative
cell wall with alcohol and in this experiment we are using 95% alcohol. Gram-positive
bacteria, however are able to retain the crystal violet and therefore will remain purple after
decolorizing with alcohol. Since gram-negative bacteria will be colorless after decolorizing
with alcohol, counterstaining with Safranin will make them appear pink. Before putting the
next dye, the smear must first washed with tap water and dried it using paper towel to avoid
the dye is mix with one another.
CONCLUSIONS
In conclusion, the Bacillus sp. is a gram positive bacteria and has a bacillus shape.
While the unknown is a gram-negative bacteria and has a coccus shape. The graam of the
bacteria is determine from the color of the bacteria. The bacillus sp. shows purple color while
the unknown shows red color.
RECOMMENDATIONS
1. Every time dye is use, make sure each dye have appropriate time of exposure to make
sure that there bind with the bacteria cell wall before wash it away.
2. When wash the smear, make sure the slide is parallel with the tap water to avoid the
smear watch out.
3. Dry the slide carefully and lightly with paper towel to prevent from wiping the smear.
REFERENCES

Benjamin, M. (2013). Biologycorner Corporation. Retrieved from Biologycorner web site:
www.biologycorner.com
Tan, S. (2013). Amrita Virtual Lab Collaborative Platform. Retrieved from NME ICT initiative
of MHRD: http://amrita.vlab.co.in/?sub=3&brch=73&sim=208&cnt=1