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PRACTICAL 2: GRAM STAINING

OBJECTIVES:
1. To acquire basic skill of smear technique and staining of bacteria.
2. To determine the morphology and gram stain of given bacteria under a microscope.

RESULTS:
BACTERIA STAINING RESULTS
Methylene blue stain
A

100x oil immersion objective

Shape: Rod or bacillus

Gram stain

100x oil immersion objective

Stain : Red dye


B Methylene blue stain

100x oil immersion objective

Shape : Rod or bacillus

Gram stain

100x oil immersion objective

Stain : Red

C Methylene blue stain

100x oil immersion objective


Shape : Spherical or coccus

Gram stain

100x oil immersion objective

Stain : Purple

D Methylene blue stain

100x oil immersion objective

Shape : Spherical or coccus

Gram stain
100x oil immersion objective

Stain : Purple

Table 1 : Result of bacteria and respective staining.

OBSERVATION

The morphology of the bacteria can be determined by methylene blue stain and the
result of gram-stain of bacteria can be observed after gram staining procedure. From the
experiment, both bacteria A and B are gram-negative bacillus. They appear red and have rod-
shaped under observation of microscope with total magnification of 1000x.

While for both bacteria C and D , the cell are gram-positive coccus. They appear purple
and have spherical-shaped under observation of microscope with total magnification of 1000x.

QUESTION

1. What is the function of mordant in Gram staining?

The mordant is referring to Gram’s iodine. Mordant is used to form the crystal violet-
iodine complex so that the dye cannot be removed easily. Mordant causes the violet dye to
form large crystal that get trapped by the meshwork of the cell wall. The layer of cell wall
in gram-positive cells is thicker, the entrapment of the dye is more extensive compared to
gram-negative cells.
2. What is the function of Safranin?

Safranin is used as a red dye counterstain. After application of iodine-acetone as


decolorizer, it dissolves lipids in the outer membrane of the gram-negative thus removes
the dye from them and gram-negative bacteria become colorless. By applying counterstain,
the gram-negative bacteria can be determined by the presence of staining the red dye.

3. Why don’t gram-positive bacteria get stained by Safranin?

After applying iodine-acetone as decolorizer, the crystal of dye still tightly embedded
in the gram-positive bacteria. The crystal of dye is inaccessible and resistant to removal
and the gram-positive will still appear violet even though safranin is applied. Besides,
safranin is much lighter color compared to crystal violet and will not show up against the
crystal violet.

4. Which step in the Gram stain is most likely to cause poor results if done incorrectly?

Decolorization step is the most critical phase of Gram stain procedure, this is because
over-decolorizing will result in the loss of the primary stain which is crystal violet. Thus
causing gram-positive bacteria to appear gram-negative. Besides, under-decolorizing will
not completely remove the crystal violet-iodine complex, causing gram-negative bacteria
to appear gram-positive.

5. Why must fresh bacterial cultures be used in a Gram stain?

Fresh bacterial cultures should be used in a Gram stain in order to prevent Gram stains
error. This is because older cultures of some gram-positive bacteria are subject to autolysis
and the breakdown of cell wall by enzyme produced the bacteria can occur, thus causing
older gram-positive bacteria to give a false gram-negative reaction.

6. How crystal violet reacts with peptidoglycan?

Crystal violet carry a positively charged chromophore and are attracted to negatively
charged cell components. In aqueous solution, crystal violet dissociates into CV+ ion. This
ion penetrate through the cell wall (peptidoglycan) of both gram-positive and gram-
negative bacteria. The CV+ interacts with negatively charged cell components (nucleic acid
and protein)of both gram-positive and gram-negative bacteria, staining the cells purple.

7. What is the purpose of doing decolourization with iodine-acetone?

The application of iodine-acetone is to dissolve lipids in the outer membrane of gram-


negative cells, thus leaving the thin layers of peptidoglycan exposed. Then, gram-negative
cell walls become leaky and allow the crystal violet-iodine complex to be washed from the
cell resulting in removal of dye (crystal violet-iodine complex).

Upon applying iodine-acetone onto gram-positive, the highly cross-linked and multi-
layered peptidoglycan becomes dehydrated and traps the crystal violet-iodine complex
within the cell. The crystal violet-iodine complex still tightly embedded and relatively
inaccessible and resistant to removal.

After decolorization, he gram-negative bacteria loses the purple color and stains red
dye from the positively charged dye, safranin, whereas the gram-postive bacteria remains
purple in color.

8. What will happen if we used iodine first and just after that we used crystal violet to do
staining?

The cells will stain yellow upon applying the iodine solution. Moreover, the
crystal violet dye will not form large and insoluble crystal (crystal violet-iodine
complex) because formation of crystal violet-iodine complex will only occur when
iodine is applied after the application of crystal violet.

Decolorization of gram-positive bacteria probably will happen, resulting in false


gram-negative reaction.

DISCUSSION

During preparation of smear, the glass slide is allowed to fix by passing it rapidly
through the Bunsen flame three times before start staining. The fixation of bacteria is done to
kill the bacteria and to fix them to the slide so that the bacteria will not washed out during
process of staining.

Methylene blue is commonly used in staining in order to observe the morphology of


the bacteria. The blue color of methylene blue helps biologist to observe the morphology of
bacteria well.

In the first step of gram staining, crystal violet stains cells producing all the same purple
color. The second step is the mordant, which is Lugol’s iodine. This step is the key to
differentiate between gram-positive and gram-negative bacteria. The mordant causes the
crystal violet to form insoluble crystal violet-iodine complex that get trapped by the meshwork
of the cell wall (peptidoglycan). For gram-positive bacteria, it has thicker layer of
peptidoglycan, causing the entrapment of the dye is much more extensive compared to gram-
negative bacteria.

Application of iodine-acetone in the third step dissolves lipids in the outer membrane of
the gram-negative bacteria, which removes the dye from them. In contrast, the crystal violet-
iodine complex tightly embedded in the gram-positive and it is inaccessible and resistant for
removal. Due to gram-negative are colorless after decolorization, it stains red dye of safranin.
Gram-positive bacteria stain purple even after applying safranin, this is because safranin is
much lighter color compared to crystal violet and it will not show up against the crystal violet.

There are few mistakes had been done during the staining this is because the result obtained
for bacteria B and D are contradict with the expected result. Bacteria B should stains purple
upon completing the staining, while bacteria D should stains red upon completing the staining.

Few mistakes probably had been done during the staining are over-decolorizing and under-
decolorizing of iodine-acetone. Over-decolorizing with iodine-acetone occur to bacteria B
resulting in red stain outcome instead of purple stain, while under-decolorizing with iodine-
acetone occur to bacteria D resulting in purple stain outcome instead of red stain. Besides, the
culture is not well spread on the glass slide during preparation of smear resulting in overlapping
bacteria that can be seen through the result of bacteria B.

Thus to prevent false result of staining, proper staining procedure should be practiced by
decolorizing adequately and washing it off immediately to prevent over-decolourizing and
under-decolourizing. During preparation of smear, the culture should be evenly spread in order
to give better results that can ease in observing the morphology of the bacteria.
SUMMARY

Gram staining method is an important diagnostic staining technique for bacteria in


Microbiology. It allows ready differentiation of major categories based upon the color reaction
of the cells: gram-positive, which are stained purple, and gram-negative, which are stained red.
The difference in staining outcomes is due to structural variation found in the cell walls of
bacteria.

Besides, gram staining can also be a practical aid in diagnosing infection and in guiding drug
treatment.

REFERENCES

Differential Staining: The Gram Stain. (1884). Retrieved March 10, 2018, from
https://www.hccfl.edu/media/568172/9-exercise%20vi.pdf

Gram Staining. (2011). Retrieved March 10, 2018, from


http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=1

Talaro, K. P., & Chess, B. (2008). Foundations in Microbiology.

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