EXPERIMENT 5

Differential Staining Technique

Simple staining depends on the fact that bacterial cells differ
chemically from their surroundings and thus can be stained to
contrasts with their surroundings. Microorganisms also differ
from one another chemically physically, and thus may react
differently to a given staining procedure. This is the basic
principal upon which differential staining depends. Thus, we
may find differential staining as a method of distinguished
between types of bacteria.

A. The Gram Stain

The Gram Stain, the most useful staining procedure in
bacteriology, is a differential stain. In this procedure bacteria
are divided into two groups. The first of these groups is
stained purple by the gram stain, while the second group is
stained pink. The organisms stained purple was called grampositive; the organisms stained pink is the gram-negative.
Crystal violet is first applied, followed by the mordant iodine,
which fixes the stain. Then the slide is washed with alcohol,
and the Grampositive bacteria retain the crystalviolet
iodine stain; however, the Gramnegative bacteria lose the
stain. The Gramnegative bacteria subsequently stain with
the safranin dye, the counterstain, used next. These bacteria
appear red under the oilimmersion lens, while Grampositive
bacteria appear blue or purple, reflecting the crystal violet
retained during the washing step.

Another differential stain technique is the acidfast

technique. This technique differentiates species
of Mycobacterium from other bacteria. Heat or a lipid solvent
is used to carry the first stain, carbol fuchsin, into the cells.

Then the cells are washed with a dilute acidalcohol

solution. Mycobacterium species resist the effect of the acid
alcohol and retain the carbol fuchsin stain (bright red). Other
bacteria lose the stain and take on the subsequent
methylene blue stain (blue). Thus, the acidfast bacteria
appear bright red, while the nonacidfast bacteria appear
blue when observed under oilimmersion microscopy.

OBJECTIVE
To learn the gram staining method and to observe the
characteristic of gram-positive bacteria and gram-negative
bacteria.

MATERIALS
1. 24 hours nutrients broth cultures of the following
microorganisms for each group of student:
(a) E.Coli
(b) Staphylococcus aureus
(c) Bacillus subtillis
2. Unknown cultures
3. Microscope slides
4. Gram stain reagent sets
5. Immersion oil
PROCEDURE
1. A separate thin smears of E.Coli, Staphylococcus aureus and
Bacillus subtilis was prepared. It was air dried and heat fixed.
2. The smears were covered with crystal violet for one minute.
3. In a slow running tap water, the smears were rinsed slowly to
remove the crystal violet for five seconds.
4. Next, the smears were rinsed with the iodine reagent and
then the same reagent was applied for one minute.
5. The iodine reagent was rinsed as in step three.
6. The alcohol reagent was applied slowly. The decolourizer was
added until dyes does not run off from the smears.

7. The alcohol was rinsed immediately as in step three.

8. The smears were covered with safranin reagent for 30
seconds.
9. It was rinsed again and blotted dry with a piece of blotting
paper.
10. Each smear was observed under the oil-immersion
objective and the results were recorded.
RESULTS

Bacillus
subtilis

Staphylococcus
aureus

Unknown Culture
A

E.Coli 40x

E.Coli 100x

Unknown B
40x

Unknown B 100x

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 decolourization. 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.

CONCLUSION
Gram staining is used to determine the gram positive bacteria
will stain to a purple colour while the gram negative bacteria
stain to be in pinkish colour.

QUESTIONS
1. Are there any chemical differences between the cell wall of
gram positive and gram- negative bacteria, which might
explain differences in the rate of decolourization?
The cell wall in bacteria contains peptidoglycan, a
polymer of N-acetyl glucosamine, N-acetyl muramic
acid and amino acid. Gram positive cell walls contain a
thick layer of peptidoglycan layer that encircles the
cells. While the gram negative cell walls contain a thin
layer of peptidoglycan between the
cytoplasmicmembrane and the outer membrane.
Gram-negative bacteria stained with crystal violet are
decolorized by 95% alcohol within 2 min, whereas
Gram-positive bacteria require at least 3 min treatment.
Aqueous solutions of safranin, neutral red, and carbol
fuchsine replace crystal violet from stained Grampositive bacteria more quickly than alcohol alone, and
alcoholic solutions of these counterstains are in most
cases still more effective. Treatment of crystal violetstained organisms with alcoholic safranin for 15 sec will
distinguish Gram-positive bacteria (violet) from Gramnegative bacteria (pink). Alcohol containing very low
concentrations of iodine generally decolorizes crystal
violet-stained Gram-positive bacteria more quickly than

alcohol alone. Increasing concentrations of iodine in

alcohol reduce the rate of decolorization of stained
bacteria, but stained Gram-negative bacteria are still
readily decolorized. The addition of iodine to alcohol
increases the rate of extraction of crystal violet by
alcohol from Gram-negative organisms, but delays
extraction of dye from Gram-positive organisms, and
this applies when counterstain is also present. A twosolution modification of Gram staining is described in
which crystal violet-stained bacteria are treated with an
alcoholic solution of safranin, carbol fuchsin, and iodine.
2. (a) Does the age of the cultures effect the gram stain?
Yes
(b) Why?
Because old culture of gram positive cells may not
retain stain as well as younger cultures and could give
false negative results.
3. (a) Based on your experience in the laboratory do you feel
that the gram stain is a simple procedure?
I feel that the gram stain is not a simple procedure
(b) Why?
For gram stain, we use more than one stain such as
crystal violet, iodine, alcohol and safranin to our
smears. Unlike the simple staining, we stained either
crystal violet, methylene blue or carbol fuchsine to our
smears. Furthermore, the cell wall of the bacteria that
react in gram stain has thicker or thinner cell wall to
know which bacteria is a gram positive or gram
negative.
4. What is the influence of pH on the gram stain reaction?
pH affects the uptake of crystal violet by the bacteria.
Crystal violet at low pH and safranin do not stain the
walls of bacteria. When organisms of a gram positive
are stained with crystal violet at a high pH and then are
washed at a low pH, the dye disappears from the walls.
Organisms with stained walls resist both decolorizer and
counterstain after treatment with iodine and thus
appear gram positive.

REFERENCE
http://www.cliffsnotes.com/sciences/biology/microbiology/mi
croscopy/staining-techniques
http://openstudy.com/updates/5013e013e4b0fa24673074cd
https://answers.yahoo.com/question/index?
qid=20111230094139AAgjUuv
https://answers.yahoo.com/question/index?
qid=20090213082458AA78pye
http://www.ncbi.nlm.nih.gov/pubmed/52916