VIETNAM NATIONAL UNIVERSITY HCMC

INTERNATIONAL UNIVERSITY
SCHOOL OF BIO-TECHNOLOGY
REPORT 2
EXPERIMENT 1: STAINING, BACTERIAL
COLONY AND CELL MORPHOLOGY

Subject: Cell Culturing Lab.

Date lab performed: 17/11/2016.
Students Name:
Trng Anh Th
Students ID: BTBCIU14046
Lab partner:
Nguyn Thy Dung Thi
Trnh Ngc Anh Th
Instructor: MSc. Tran Le Hong Ngoc
I. ABSTRACT
Stains are solutions that contain a solute called a chromophore dissolved in a
solvent. A chromophore is the color possessing portion of the solution and is
therefore responsible for the stains color. When the stain is applied, there is an
attraction between the negatively charged cell surface and the positively
charged chromophore, leading to the surface of the cell taking on the color of
the stain. In this experiment, we will prepare a smear and stain three species of
bacteria, which is E- Coli, Staphylococcus Aureus, and Salmonella by doing
gram stain reaction and using KOH as an indicator of Gran Stain reaction.

II. INTRODUCTION
The Gram stain is important and widely used differential stain for bacteria. On
the basic of their reaction to Gram stain, bacteria can be divided into 2 groups:
Gram positive and Gram negative. Gram-positive Bacteria have cell walls that
contain thick layers of peptidoglycan and no lipopolysaccharides. These stain
purple. Gram-negative Bacteria have walls with thin layers of peptidoglycan, an
outer cell wall containing lipopolysaccharides and high lipid content. In the
Gram staining procedure, the primary stain is crystal violet, and all cells take up
the purple crystal violet stain. Grams iodine is applied to bacteria smears. The
iodine acts as a mordant, enhancing ability of stain to enter and bind to bacteria.
The iodine binds with crystal violet and locks it into peptidoglican of bacteria.
It is also intensified the purple color. A decolorizing agent, alcohol, is added
next. This is the crucial, differentiating step. The dehydrating effect of the
alcohol causes the peptidoglycan-rich cell wall of the Gram positive cells to
shrink, holding the crystal violet-iodine complexes in the cells. The alcohol
dissolves the lipid in the Gram-negative cell walls, leaving the walls porous.
Crystal violet-iodine complexes leak out of the Gram-negative cells leaving
them without color. At this point the Gram positive cells are purple and the
Gram negative cells are clear. A counterstain, safranin, is added to allow
visualization of the Gram negative cells.

III. MATERIALS AND METHOD

Materials
- Escherichia coli
 - Staphylococcus aureus
- Salmonella
- KOH 3% solution
- Sterile toothpicks
- Beaker
- Crystal violet
- Grams iodine solution
- Alcohol: acetone mixture
- Safranin
- Loop

Methods
- Smear preparation
Obtain a clean slide and divide it into 3 equal parts.
Place a drop of water in the middle of each part
Using aseptic technique, smear a loopful of bacterial sample
from a pure culture onto each part of the slide.
Smear the sample in a circle on each part of the slide
Let the preparation on the slide dry.
- Gram staining
Place slide in staining rack, flood with crystal violet. Stain
for 1 minutes
Gently rinse with water and drain.
Flood the slide with Gram's iodine. Let stand for 1 minute.
Rinse with water and drain.
Flood slide with decolorizing agent (alcohol : acetone)until
stain no longer washes from the slide when held at an angle.
Quickly rinse slide with water.
Flood slide with safranin. Allow stain to remain for 30
seconds.
Rinse with water and drain.
Blot slide within a pad of blotting paper. Do not rub. The
slide should be completely dry before microscopic
examination.
Observe bacterial cells at 4x,10x,100x, and 1000x
magnification.
 - Using KOH as an indicator of gram stain reaction
Obtain a clean slide and divide it into 3 equal parts.
Place a drop of 3% KOH in the middle of each part
Using aseptic technique, smear a loopful of bacterial sample
from the culture medium to the drop.
Mix the bacteria into the solution rapidly and in a circular
motion
Raise and lower the toothpick after 5-8 seconds to determine
the stringing effect. If there is stringing which means that
increasing the viscosity within 15 seconds, the bacteria are
considered to be Gram negative.
IV. RESULTS
1. E-coli
Gram is negative since after staining
with safranin, they obtain a pink
color. KOH test is opposite which is
positive.

2. Staphylococcus aureus
Gram is positive since after staining with safranin,
they obtain a purple color. KOH test is opposite
which is negative

3. Salmonella
 Gram is negative since after
staining with safranin, they
obtain a pink color. KOH is
opposite which is positive.

V. DISCUSSION
After Gram staining, Gram positive bacteria should appear a purple while Gram
negative bacteria should appear pink. However, we could not exactly identify
the bacteria as the Gram staining was not as effective because the bacteria were
not stained properly. This could have been effect of many factors. For instance,
it might have been caused from a poor technique used while gram staining. In
KOH, the result is opposite. This also because this test requires a large amount
of cells