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
gram-positive; 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 Gram‐positive
bacteria retain the crystal‐violet iodine stain; however, the Gram‐negative
bacteria lose the stain. The Gram‐negative bacteria subsequently stain with
the safranin dye, the counterstain, used next. These bacteria appear red
under the oil‐immersion lens, while Gram‐positive bacteria appear blue or
purple, reflecting the crystal violet retained during the washing step.

Another differential stain technique is the acid‐fast 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 acid‐alcohol
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 acid‐fast
bacteria appear bright red, while the nonacid‐fast bacteria appear blue
when observed under oil‐immersion 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 Unknown Culture A

Staphylococcus
aureus
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 Gram-positive
bacteria more quickly than alcohol alone, and alcoholic solutions of
these counterstains are in most cases still more effective. Treatment
of crystal violet-stained organisms with alcoholic safranin for 15 sec
will distinguish Gram-positive bacteria (violet) from Gram-negative
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 two-solution 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/microscopy/st
aining-techniques
 http://openstudy.com/updates/5013e013e4b0fa24673074cd
 https://answers.yahoo.com/question/index?qid=20111230094139AAgjUuv
 https://answers.yahoo.com/question/index?qid=20090213082458AA78py
e
 http://www.ncbi.nlm.nih.gov/pubmed/52916