Simple and Gram Staining of Bacteria

And Aseptic transfer of microorganisms and proper handling of bacteria

By

Bright Belinda Afra

3/04/2010

Microbiology lab: 303

Thursdays 8:00-10:50

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Introduction

The process and understanding of simple gram staining has been introduced in this lab
experiment. Simple stains use basic dyes which are positively charged. These positive dyes
interact with the slightly negatively charged bacterial cell wall therefore lending the color of the
dye to the cell wall. Gram staining is one of the most important techniques in microbiology that
helps identify bacteria. The Gram stain is a technique used to classify bacteria in which a
bacterial specimen is first stained with crystal violet, then treated with and iodine solution,
decolorized with alcohol and counterstained with safranin. Bacteria are classified as gram
positive and gram negative based on their cell wall properties. Gram positive cells have a thick
peptidoglycan cell wall which is able to retain the crystal violet iodine complex that occurs
during chemical staining. However, gram negative have a thin peptiodoglycan cell wall, which is
covered with the outer membrane. The results in the experiment will differentiate the correct
name of the bacteria and show which bacteria is gram positive and gram negative.

In the Aseptic transfer of microorganisms and proper handling of bacteria this particular
laboratory bacteria, must be cultured in order to facilitate identification and to examine their
growth and metabolism. In order to keep bacteria alive they must be inoculated into various
forms of culture media. When doing inoculations, unwanted microbes should not be introduced
or contaminated, into the media. Aseptic technique is used to prevent contamination. In
transferring of bacteria all cultures should be sterilized such as broth. Broth cultures provides
large numbers of bacteria in a small space and are easily transported (Transfer of Bacteria
Aseptic Technique worksheet). Agar slants are test tubes containing solid culture media that
provides a solid growth surface. Agar deep is often used to grow bacteria that prefer less oxygen
than is present on the surface of the medium. Transferring and inoculation are usually performed
with a sterile, heat- resistant. When a wire is bent at the end into a loop it’s called inoculating
loop; when straight it is an inoculating needle (streak plate technique).

Objectives

In the experiment, we will prepare a bacterial smear, and heat fixation to understand
bacterial morphology by doing the simple staining procedures and also gram staining. These two
procedures will give us the ability to distinguish bacteria based on the gram reaction, size, shape
and arrangements.

Materials and Methods

Burner
Loop
Microscope
Bacterial Cultures: Escherichia coli (agar slant tube); and Bacillus cereus (agar plate)

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Clean glass slide
Permanent marker
Crystal violet
Gram’s iodine
95% ethyl alcohol
Safranin
Bibulous paper

In performing the experiment for Aseptic technique was performed and two bacteria
cultures were given A and B. Tube A was Escherichia coli and tube B was Bacillus cereus. One
was aseptically introduced to the agar plate (A) by zig zag and the other bacterium was
introduced by the agar plate (B) by a four quadrant streaking technique. The plate and the tube
was both inoculated threw incubation at 37 degrees Celsius for 18-24 hours for growth. The
plates were then kept refrigerated at 4 degrees Celsius so the bacteria can stay alive without
growth. The growth was then observed on the agar plate and agar tube
In performing the experiment for simple and gram staining, a clean glass slide was
obtained, waving the slide over the open flame of a burner a couple times so the remaining dust
and stains were removed which is called heat fixation. A marker was used to draw two dime
sized circles on the back of the glass slide and labeled “A” and “B” for each specimen. Two
loopfuls of phosphate buffered saline was added to each circle. The Bacillus cereus was spread
evenly in the circles to make then smears (on the opposite side of the circles). The loop was
flame, and then we waited until it cooled down. Escherichia coli culture from its agar slant was
spread in circle B. The smears was air dried and went through a process called heat fixation,
which consist of flaming the slides 2-3 times.
After the smears and heat fixation, simple staining was conducted. A drop of crystal
violet was added and then spread on both slides for 60 seconds. The CV was carefully rinsed off
with running tap water. The bibulous paper was used to absorb the remaining water on the slide.
The slide was air dried, after the slide was dried we examined the slide under a microscope in
10X, 40X and 100X (oil immersion) lens to identify the arrangement and shape of the
specimens.
The gram staining procedures were conducted by repeating the process of smear
preparation and heat fixation. The CV was used to cover both specimens A and B and used as the
primary stain to bind the cell membrane. After CV was rinsed off, a drop of Gram’s Iodine was
added over the CV smears and stayed for 20 seconds. The iodine was then rinsed off with
running tap water. The smear was submerged with 95% ethyl alcohol for 10 seconds. The
alcohol was rinsed off quickly and then a drop of safranin was added to each specimen for 45
seconds. The safranin was rinsed off with running tap water and then dried with bibulous paper.
The slides were examined under the microscope to determine the gram reaction of the gram
negative and gram positive bacteria.

Discussion

I have 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

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structure which is known as cocci and a crystal/violet purple pigment. In the gram staining of
Escherichia coli and Bacillus cereus bacteria, slide 1 Bacillus cereus had a round shape
structure and a pigment of pinkish purple, pink or light pink all resulting in a gram negative
bacterium. Slide 2 Bacillus cereus, had a rod shape (bacillus) structure and a purple or purplish
red pigment which resulted in a positive gram stain. Gram reaction help demonstrate the
structural differences of gram negative and gram positive bacteria.
Gram negative bacteria are the bacteria that do not retain crystal violet dye. In the gram stain test
gram negative bacteria has a red or pink color. Gram positive bacteria are those that are stained
violet, which can retain the crystal violet stain due to the high amount of peptidoglycan in the
cell wall.

Results

Bacillus cereus- gram positive, rod shape bacterium (purple color)

Escherichia coli- gram negative, rod shape bacterium (pink color)

See data attached