Activity 2 Gram Stain

DIVINA GRACIA VIBAL CIELO BSM-II
Activity # 2 THE GRAM STAIN
Instruction:
 Sketch the gram staining procedures.
 Make a colorful drawing of a specimen (bacteria) under a gram stain
procedure that shows characteristic morphology.
Guide Questions:
1. Why are the steps in gram staining so carefully standardized?
2. Why is mordant necessary for the staining of bacterial flagella?
3. Based on the procedure, do you feel that the gram procedure is a
simple one? Why? Why not?
4. A student completed the gram stain technique but neglected to use the
gram’s iodine. Describe the appearance of the cells.
5. What are the factors that will affect the staining affinity of bacteria?
6. What other techniques were used to evaluate bacterial gram staining
reaction? Discuss.
7. Why isn’t gram stain used on acid-fast bacteria? If you did gram stain
acid-fast bacteria, what would their gram reaction be? What is the gram
reaction be? What is the gram reaction of non-acid fast bacteria?
ANSWERS:
1. Gram's stain remains one of the most useful tools we have for the accurate
and rapid identification of isolates. Despite our long-standing familiarity with
this process, every step of the way-from reagent preparation and QC to

staining and interpretation-still warrants careful attention. One of the main
reasons would to avoid contamination.
The measures are systematic, as it is the first universal differentiation test.
If everyone was using different methods so you couldn't be 100 percent sure
that the outcomes of each were the same. The colors, highlights, times and all
are standardized so that the same results are obtained for everybody who
does it.
If some of the measures were out of place the staining would not work
properly.
The measures are in between, with water washings:
 Primary stain (violet crystal) directed to gram-positive bacteria
 Iodine to dye first
 Wash away unbound acetone dye
4) Counter-staining targeting all bacteria, but only isolating all
gram-negative unstained bacteria
If you do 2, 3, or 4 first, all will be stained "gram-negative." Crystal violet
stain would not be able to interact with gram-positive bacteria (or less
likely to).
2. Mordant is a chemical that is added to the solution to intensify the stain.
Function: to increase the affinity of a stain for a biological specimen and to
coat a structure (such as a flagellum) to make it thicker and easier to see after
it is stained with a dye.
3. For me the procedure is not simple because there are several factors that
may affect the result of gram staining like for example: If the smear is too
thick, proper decolorizing will not be possible. If the smear is overheated
during heat fixing, the cell walls will rupture. Concentration and freshness of
reagents may affect the quality of the stain. It is not a simple stain. A simple
stain is when you use just one dye; just one stain to colorize the cells.
4. Gram’s iodine acts as an astringent causing the crystal violet dye to
penetrate and stain gram-positive organisms. The gram-positive organisms
will not be decolourized as easily as gram-negative organisms by an acetone-
alcohol. Safranin is added as a “counter stain” which stains all non-gram-
positive organisms a pink/red colour. If Gram’s iodine is not used in this
process, the gram-positive organisms will not show up violet, they will instead
show up a pink/red colour.
5. The factors that will affect the staining affinity of bacteria are the following;
Concentration of the dye - The greater the concentration of the dye, the
more the dye is bound to tissue components. Temperature - An increase in
temperature increases the rate at which the dye diffuses throughout the tissue
sample. It can also alter tissue components so that they are more receptive to
dye penetration. pH of the staining solution - Cells and other tissue
elements often have an affinity for stains/dyes with specific pH ranges.
Therefore, the pH of the staining solution can have a direct impact on the
ability of a dye to bind with its intended tissue element. Tissue fixation -
Fixation alters and reorganizes certain molecular structures within the tissue
sample such that there is increased permeability for staining. Unfixed tissue
elements have limited binding sites for dyes. Mordants - Mordants are
chemicals that may be needed to bind dyes to certain tissue elements. They
can also be used to intensify the staining results.
6. Gram staining differentiates the bacteria from their cell walls by the
chemical and physical properties. Gram-positive cells in the cell wall have a
thick coating of peptidoglycan, which contains the primary dye, crystal violet.
Gram-negative cells have a thinner layer of peptidoglycan, which causes the
crystal violet to wash out when ethanol is applied. The counterstain, usually
safranin, or fuchsine, stains them pink or red. Upon addition of crystal violet,
Lugol's iodine solution is often applied to reinforce the stain 's bonds with the
cell membrane. Gram staining is almost always the first step in recognizing a
bacterial organism in advance. While Gram staining is a valuable diagnostic
tool in both clinical and research settings, not all bacteria can be definitively
classified by this technique. This gives rise to gram-variable and gram-
indeterminate groups.
7. The acid-fast stain is specifically used to identify bacteria that possess a
waxy lipid within the structure of their cell walls. Due to the presence of this
lipid, water-based stains, such as the gram stain, so not work well on acid-fast
organisms. If gram stain did in acid fast bacteria, nothing will happen. the
gram reaction of non-acid fast bacteria is gram positive.

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DIVINA GRACIA VIBAL CIELO BSM-II

Activity # 2 THE GRAM STAIN

 Sketch the gram staining procedures.

 Make a colorful drawing of a specimen (bacteria) under a gram stain

procedure that shows characteristic morphology.

1. Why are the steps in gram staining so carefully standardized?

2. Why is mordant necessary for the staining of bacterial flagella?

3. Based on the procedure, do you feel that the gram procedure is a

simple one? Why? Why not?

gram’s iodine. Describe the appearance of the cells.

6. What other techniques were used to evaluate bacterial gram staining

reaction be? What is the gram reaction of non-acid fast bacteria?

and rapid identification of isolates. Despite our long-standing familiarity with

this process, every step of the way-from reagent preparation and QC to

reasons would to avoid contamination.

The measures are systematic, as it is the first universal differentiation test.

The measures are in between, with water washings:

 Primary stain (violet crystal) directed to gram-positive bacteria

 Iodine to dye first

 Wash away unbound acetone dye

4) Counter-staining targeting all bacteria, but only isolating all

gram-negative unstained bacteria

If you do 2, 3, or 4 first, all will be stained "gram-negative." Crystal violet

2. Mordant is a chemical that is added to the solution to intensify the stain.

Function: to increase the affinity of a stain for a biological specimen and to

it is stained with a dye.

thick, proper decolorizing will not be possible. If the smear is overheated

4. Gram’s iodine acts as an astringent causing the crystal violet dye to

penetrate and stain gram-positive organisms. The gram-positive organisms

will not be decolourized as easily as gram-negative organisms by an acetone-

alcohol. Safranin is added as a “counter stain” which stains all non-gram-

positive organisms a pink/red colour. If Gram’s iodine is not used in this

show up a pink/red colour.

more the dye is bound to tissue components. Temperature - An increase in

dye penetration. pH of the staining solution - Cells and other tissue

elements often have an affinity for stains/dyes with specific pH ranges.

can also be used to intensify the staining results.

Gram-negative cells have a thinner layer of peptidoglycan, which causes the

bacterial organism in advance. While Gram staining is a valuable diagnostic

classified by this technique. This gives rise to gram-variable and gram-

7. The acid-fast stain is specifically used to identify bacteria that possess a

gram reaction of non-acid fast bacteria is gram positive.

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