UNIVERSITI TEKNOLOGI

MARA NEGERI SEMBILAN

BIO461: MICROBIOLOGY
LABORATORY REPORT
PRACTICAL : 2 (STAINING TECHNIQUES IN MICROBIOLOGY)

CLASS GROUP : AS2011B1

LECTURER : DR AMIRUL ADLI ABD AZIZ

GROUP MEMBERS :

NO. NAME STUDENT ID

DISCUSSION
For the first part of the experiment, simple staining by methylene blue was done. In simple staining
process, the only purpose is to dye the entire sample to observe the cellular shape and basic structures of
the bacteria. Methylene blue for staining the sample because the surface of most bacteria cells is
negatively charged. Meanwhile, methylene blue gives up the hydroxide ion and thus causes the stain to
become positive charged. This allows the stain to attach to the surface of the bacteria strongly and causes
the cell to become visible for observation. Heat fixation was also included during the process of simple
staining. The purpose of heat fixation is to kill the bacteria cell so that it will not be washed away during
staining and to preserve the size and shape of the bacteria cell. Based on Figure 1, a group of cocci,
spherical-shape bacterium in blue color was observed.

In addition, during Gram staining process, the smear was heat fixed to slide to kill the bacteria so
that the cells will not be washed away during staining process and to preserve the shape and size of the
cells. After the heat fixation process, the smear was stained by crystal violet for one minute. This is to
make sure all the cells can be able to absorb the stain from crystal violet. After that, iodine was dropped
on the glass slide because iodine acts as a mordant. A mordant increases the affinity of the cell wall with
the stain and causes the stain to be trapped in the cell wall. The color of the smear changed its color
becoming dark purple. Next, the glass slide was rinsed by alcohol for 20 seconds. This is to decolorize the
gram-negative bacteria. This is due to gram-negative thin layer of peptidoglycan and thick layer of lipids;
the solution of crystal violet and iodine get completely washed away during decolorizing. Lastly, drops of
safranin were dropped on the glass slide and caused gram-negative bacteria to become red in color. Based
on Figure 2, a group of cocci, spherical shaped bacterium in dark purple color while a group of bacilli in
red color was observed. Based on the observation, it can be concluded that the group of cocci are gram-
positive bacteria while the group of bacilli are gram-negative bacteria.

Furthermore, in endospore staining, malachite green was used as the primary stain for the
experiment. After the malachite green was dropped on the glass slide, the glass slide was steam for five
minutes. Bacterial endospores have a highly resistant structure produced by some bacteria as a defensive
mechanism. However, when the stain is steamed, the primary stain will be forced into the spore of the
cell. During decolorizing process, the glass slide was washed by tap water and vegetative cells were
decolorized. This is due to malachite green is water soluble and has low affinity with cellular material.
After that, safranin was dropped for counter stain any cells that have been decolorized. Based on Figure
3, A group of bacilli bacteria observed in red
 and green color. Based on the observation, it can be concluded that the cells in green color are endospore
while the cells in pink color are vegetative cells.

Lastly, for capsule staining, a negative staining process was used for the experiment. In negative
staining method, a translucent, dark colored background with stained cells will be formed. The
background was formed by Indian ink. However, the capsule was not stained by Indian ink. After that,
drops of safranin were dropped on the glass slide. The purpose of safranin is to counterstain the cells. By
counterstaining the cells, the cell wall of bacteria will absorb the stain while the capsule of the cell
appears colorless. Based on Figure 4, a dark red background with colorless capsules of bacteria can be
observed.

1. Why do you have to do heat fix before doing simple staining and Gram staining?

During the process of simple staining and Gram staining, heat fixation of the sample is a must procedure
because the purpose of heat fixation is to kill the bacteria in the smear. This will cause the sample to be
attached to the glass slide so that the microbes in the sample will not be washed off during the staining
process. It will also make the bacteria to easily take up the stain. Other than that, heat fixation also
preserved the shape and size of the microbes and allowed the observation of the sample to become much
more easily.

2. Before doing capsule staining, do you have to do a heat fixed smear? Explain your
answer.

Heat fixation is not included during capsule staining process. This is because most bacterial capsules
consist of semi-rigid polysaccharide layer, and it is very moist. If they undergo heat fixation, it will
destroy the capsule and cause the bacteria to shrink. Thus, the smear will not be stained and cause the
observation of the sample to become impossible.

3. In Gram staining, what would happen if you accidentally use safranin as the primary
stain and crystal violet as the counter stain?

In Gram staining, crystal violet is used as the primary stain while the safranin is used as the counter stain.
If the process were reversed with safranin as the primary solution and crystal violet as the counter stain,
this would cause all the bacteria to become red when safranin was added to
the smear. When the crystal later was added later, it may cause the gram-negative bacteria to
change its color from red and become violet in color while gram positive bacteria remain red in
color. Thus, this will give a false result because crystal violet is used to dye the gram-positive
bacteria purple.

7.0 CONCLUSION
In conclusion, a bacterial smear is a thin layer of bacteria that is spread on a slide and the smear
can be prepared from either a solid or broth media. Heat must be applied to the bacterial smear to
keep it adhered to the slide and prevent the sample from being lost during the staining technique.
However, most types of cells lack significant natural pigment, thus it is difficult to observe under a
light microscope unless they are stained. To make bacterial cells more visible, several types of
staining techniques can be used such as simple staining and gram staining. In addition, special
staining techniques can also be used to determine specific external or internal structures that are
not found in all bacterial cells such as capsule staining and endospore staining.