DISCUSSION

The Gram Staining Method

For Gram Staining Method, as usual before starting the experiment, it is crucial to
perform aseptic technique in order to minimize contamination. Firstly, smears of two
microorganisms (Escherichia coli and Bacillus cereus) are prepared. In theory, both of this
microorganisms are from two different groups and expected to observe two different result in
this gram staining experiment. After preparing the smears of the microorganisms, the smears
were air dried and heat fixed. Then, stained both smears using crystal violet dyes. Crystal
violet dyes are basic dyes that have positively charged particle that helps them to bind to
negatively charged molecule like teichoic acid at the cell wall of bacteria. This crystal violet
dye can dissociate into cv+ and cv- ions. These ions can penetrate deeps into the cell wall of
bacteria and interacts with the negatively component on the bacterial cell wall. 1 minute later,
the crystal violet was washed with tap water and then the slides are dried.

The next step is to add iodine onto each smears. Iodine was being added as a mordent
to form crystal violet-iodine complex, CVI complex. This complex enables the dyes to not be
easily being removed. Next, the iodine washed with tap water and dried off the excess water.
After that, 95% of ethyl alcohol was being added to acts as a decolorizing agent. It interacts
with the lipid membrane of both positive and negative bacteria, and this would cause the
gram-negative bacteria to lost their outer membrane and exposing the peptidoglycan. The
CVI complex are being washed from the outer membrane of gram-negative bacteria and
cause the purple colour to decolorize.

Meanwhile, for gram-positive bacteria, the addition of alcohol dehydrated the layer of
peptidoglycan which in turn would trap the CVI complex. This cause the gram positive
bacteria appeared to be purple colour as the CVI complex are being retained. The addition of
alcohol is not being more than 15 seconds as this would break the cell wall of the bacteria,
thus resulting in no stain to be observed. The slides are washed with water and dried off. In
the next step, safranin was used to counterstain both smears. This is to enables the gram
negative bacteria to be visualize easily as it can be stain in pink colour. The gram-positive
bacteria do not being stained pink when safranin was being introduced because the
peptidoglycan layer already have CVI complex. Then, the slides were washed using tap water
and dried off. Finally, we observed our specimen using microscope under oil-immersion
objective lenses. This particular lens has more mirrors inside and it requires the use of oil to
refract light rays towards the centre of the lenses.

Negative Staining Method

In the preparation of the simple stain, and most other bacterial stains, the slide is heat fixed.
This process causes protein to coagulate and can visually reduce the size of the bacteria so
that true size is less apparent. When comparing the size of the microorganisms observed in
this lab, for example, with microorganisms prepared by simple stain in the previous lab, the
microorganisms in this lab appeared almost twice as large. One concern is that some of these
organisms may also produce capsules. If so, then the presence of a capsule in the negative
stain would make the organism appear larger than its actual size. We will have to perform a
capsule stain with the organisms to determine if the size we obtained in the negative stain is
in fact the true size of the organism.

Simple Staining Method

We had concluded that the procedures were conducted properly and successful. In the simple
stain of Escherichia coli and Bacillus cereus bacteria, slide 1 and 2 had a spherical shape
structure which is known as cocci and a crystal / violet purple pigment

CONCLUSION

From the experiment that we have done, finally, we can conclude that gram staining is
the method of distinguishing between gram positive and gram negative bacteria. In this
experiment, we were provided some material to help us for reaching the aim of this
experiment such as Crystal violet, Grams iodine, 95 % ethyl alcohol, safranin, and
microscope slide. However, before doing the experiment, we absolutely need to pay attention
on the precautions. There are several procedures that we have to do in order to avoid the error
in this experiment, such as prepare smear from cultures of microorganism, heat fix the
smears, place the slides on a staining rack, and so on. By preparing negative slides that are
not heat fixed and that stain the area surrounding the organism rather than the organism itself,
we may be able to get a better idea of its true shape and size. Heat can significantly distort
the shape and size of an organism and may lead to false identification.

REFERENCE

Brown E. Alfred, Benson s Microbiological Applications, ninth edition, McGraw Hill

Publication
Cappucino G. James, Sherman Natalie, Microbiology A laboratory manual, seventh
edition, pearson education

YashRoy R C (1990) Lamellar dispersion and phase separation of chloroplast

membrane lipids by negative staining electron microscopy. Journal of Biosciences,
vol. 15 (2), pp 93-98.