Lisa Ta

February 29, 2012.

Biology 240
General Microbiology
MW 3:45-5:00

Gram Stain Report

Introduction

The purpose of this experiment is to determine the shape and Gram stain of the
bacteria under a microscope. The reason for staining bacteria is due to the fact
that most bacteria are transparent and cannot be seen through the microscope.
The Gram stain is a type of differential stain that allows a (micro)biologist to
identify the differences between organisms and/or differences within the same
organism. Gram staining bacteria requires the use of aseptic technique to
ensure the sterility of the experiment. The purpose of the Gram stain is to show
whether the bacteria are Gram positive (purple-colored), Gram negative (pink-
colored), or both.

Materials and Methods

Aseptic technique was used throughout the experiment. A drop of deionized

water was placed on a microscope slide and bacteria from unknown number 20
were also placed on the slide. Then the slide had to be air dried before heat
fixing it, so that the Gram staining may begin. The first step in Gram staining was
to place the primary stain, Crystal Violet or Methylene Blue, on the slide where
the bacteria were heat fixed. The dye had to stay on the slide for about one
minute before rinsing with deionized water and blotted dry. Then the Mordant
Iodine (which is the Gram stain dye) was placed on the slide for another minute
and a half. After the minute and a half was up, the slide was rinsed with
deionized water and blotted dry. The next step was to use 95% Ethanol to wash
off the dyes from the Gram-negative bacteria. To distinguish when to stop using
ethanol, the first drop of non-colored solution that slides off the slide was the
indicator. Again the slide was rinsed with deionized water and blotted dry.
Lastly, the saffarin dye was placed on the slide to counterstain the Gram-
negative bacteria for one minute, rinsed with deionized water, and blotted dry.

Methods could also be referred to the lab manual pages 159-163.

Microscope E1 was used to view unknown number 20 to determine whether the

bacteria were Gram-positive or Gram-negative and cocci or bacillus.

Results

Through the process of the experiment, unknown number 20 which was on a

Nutrient Agar slant medium which was determined to be cocci (sphere) shaped
appeared to be Gram-negative.

Discussion

What determines Gram-positive and Gram-negative bacteria is due to difference

in cell wall composition. Because the cell walls of Gram-negative cells have a
higher content of lipids and a thinner layer of peptidoglycan, the alcohol used in
the decolorizing step made the Gram-negative cells incapable of retaining the
methylene blue-iodine complex. On the other hand, Gram-positive cells have a
thicker peptidoglycan that traps the methylene blue-iodine complex, making it
less vulnerable to decolorization.

The Gram stain technique was used on unknown number 20. The unknown
appeared to be Gram negative and cocci shaped. Since this is an unknown, the
literature value is unknown. Possibilities of difference in literature and
experimental value could be due to excess primary and mordant iodine dyes
being flushed out entirely by the 95% Ethanol, or that the mordant iodine dye was
not left on long enough.
Reference

Bauman, R.W. Microbiology with Diseases by Body System, 2nd ed. Pearson
Education, Inc. S.F. 2009. p.107-109.

Leboffe, M.J., Pierce, B.E. Microbiology Laboratory Theory and Application,

Brief ed. Moron Publishing Co. CO. 2008. p. 159-163.