Micro 223-FG Group 5

09-28-10 Maya Popbozhikova

Pamela Taylor
Latifa Bouhbel
Kenneth Ting
Jodi Henson
Hong Ngo

Laboratory Experiment # 2

Gram Staining

1. Introduction:

The Gram stain technique was discovered in 1884 by Hans Christian Gram, and
remains as one of the most common differential stains used in the microbiology laboratory The
Gram stain takes advantage of differences in the chemical composition of the cell wall of the two
types of bacteria. Both Gram-positive and Gram-negative cells are stained by the primary stain,
Crystal Violet. A mordant, Gram’s iodine is added to the cells and forms the crystal violet-iodine
complex that readily enters the cells and gives both types a purple appearance. Gram-positive
bacteria have a thicker peptidoglycan cell wall which allows the complex to remain within the
cells but is washed out of the thin layer of peptioglycan and lipopolysaccharide of Gram-negative
bacteria during decolorization by ethyl alcohol. After this step, gram-positive cells are purple but
the Gram-negative cells are colorless and must be stained to be seen. A counterstain, safranin is
applied which stains the colorless Gram-negative cells pink. The addition of safranin does not
affect the appearance of Gram-positive cells as they are heavily stained with the more intense
crystal violet. Thus, Gram positive cells appear purple and Gram-negative appear pink.

2. Aim of the Experiment:

Gram staining is a differential staining procedure that simply identifies the two main
groups of bacteria - Gram positive, and Gram negative. The aim of the Gram Stain experiment is
to differentiate between Gram-positive and Gram-negative organisms, while simultaneously
learning about the cellular morphology and arrangement.

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 3. Materials:

Smear Preparation

 Microscope slides;
 Inoculating loop
 Bunsen burner;
 Wire loop
 Sharpie marking pen;
 Slide holder.

Gram Staining:

 Crystal violet stain

 Gram’s iodine
 Safranin stain
 Acetone - alcohol
 Bibulous paper

4. Procedure:

- Prepare a smear from one of the bacteria slants;

- Cover the smear with Crystal Violet and allow to stand for 1 minute;
- Gently wash off stain with deionized water;
- Cover smear with Iodine and allow to stand 1 minute
- Rinse with Ethanol until liquid runs clear. This should take no longer than 20 seconds;
- Quickly rinse with water to stop the decolarization process;
- Cover smear with Safranin and allow to stand for 1 minute;
- Gently wash off stain with water;
- Carefully blot the water drops from the slide with bibulous paper;

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 - Examine all stained slides under 100x oil immersion lens;

5. Results :

1. Observations of cells using ubiquitous plates from Laboratory # 1

Gram stain
Type of Cellular result
Source of Sample Stain used Colonial morphology
culture morphology

Round, white colonies,

1.Body: Ken’s Gram
Streak plate Gram stain medium size and Staphylococci
hand positive
numerous.
( Purple)

Circular, small and Gram

2. Environment:
Streak plate Gram stain numerous colonies, yellow Streptobacilli Negative
Sink
in color ( Pink )

Round small and medium Gram

3. Air Streak plate Gram stain colonies, light yellow in Streptobacilli positive
color. ( Purple)

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 1. Observations from slant culture of Escherichia coli, Staphylococcus
aureus, and Rhodospirillum rubrum

Type of Colonial Gram stain

Source of Sample Stain used Cellular morphology
culture morphology result

Gram negative
1. Escherichia coli Slant Gram stain ________ Diplobacilli
( Pink )

2. Staphylococcus Gram positive

Slant Gram stain ________ Staphylococci
aureus ( Purple )

3. Rhodospirillum Gram negative

Slant Gram stain ________ Spirilla
rubum ( Pink )

Gram positive
4. Bacillus cereus Slant Gram stain _________ Streptobacilli
( Pink )

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 6. Questions and Discussion:

1) Which of the three differential stains would likely be the first used when
identifying an unknown bacterium? Explain.
Almost all bacteria can be differentiated by Gram stain into the tow groups – gram-
positive or gram-negative, because only a very small portion of bacteria species are either spore
formers or acid-fast, so the Gram stain would be the first used stain to identify an unknown
bacterium. Gram stain also emphasizes the cellular structure.
2) What is the function of a mordant?
The function of a mordant is to cause the primary stain to attach better or to increase the
affinity of a stain for a biological specimen, so the specimen is not removed during
decolorization.
3) For differential staining, how does a counterstain differ from a primary stain?
The counterstain is different color than the primary stain to benefit the differentiation of
gram-positive and gram-negative bacteria.
4) How do gram-positive and gram-negative bacteria differ in cellular structure?
How does this contribute to their differential staining properties?
Gram-positive bacteria have a thicker peptidoglycan cell wall than gram-negative bacteria
composed of a layer of lipopolysaccharide as part of their cell wall. These structural differences
in their cell walls affect the retention or escape of the crystal violet-iodine complex. And for that
reason Gram-positive cell wall absorbs the crystal-iodine complex better in the presence of the
decolorizer opposed to Gram-negative cell.
5) Which is the most critical step in the Gram – stain procedure? Why? If this
procedure is done incorrectly, how might that affect the final results?
The decolorizer step is the most critical, because it is the step in which the cells become
differentiated (Gram – positive cells are purple and Gram – negative cells are colorless). If too
much decolorizer is used, Gram – positive cells lose the primary stain and are counterstained

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pink, which leads to erroneous results. If too little decolorizer is used, Gram – negative cells will
not lose the primary stain and will remain purple after counterstaining.

6) How does culture age affect the results is Gram – stain?

There is possibility that old cultures of Gram – positive cells will not inhibit the stain as
well as younger cultures and could give wrong results. A Gram-positive culture that is older than
18 hours can convert to gram variable or gram-negative and give erroneous results.
7) How does culture age affect the results of a spore stain?
An older culture will give good results of a spore stain, because when bacteria exhaust all
the nutrients available in the medium, they will produce endospores as a form of survival and
resistance.
8) Why must smear thickness be considered before performing a Gram-stain?
Smear thickness must be considered before performing a gram-stain, because thick
smears don’t allow the observation of cells’ arrangement. Also, a thick smear can entrap the
primary stain which is not removed by alcohol or acetone and the cells in the entrapped stain can
appear Gram-positive leading to erroneous results.

9) What color are bacteria endospores after a Gram- stain is performed? What
does this tell you about the physical properties of endospores?
Basic dyes do not penetrate spores, so Gram staining will result in colorless spores. This
tells us that spores are very invulnerable structures.

10) Bacillus anthracis, the causative agent of antrax, is an endospore-foremr. Why

does this trait enhance its capabilities as a biological weapon?
Bacillus anthracis enhance its capabilities as a biological weapon, because it’s easy to be
produced and it can survive tough environmental conditions such as high temperatures, UV light,
and desiccation.

11) What makes Mycobacterium particularly resistant to staining? How are the
bacteria in this genus grouped in terms of Gram classification?

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 Mycobacterium is particularly resistant to staining because it has a peptidoglycan layer
furnished with mycolic acids that make the cell wall waxy and impenetrable to stains. The
bacteria in this genus are grouped Gram-positive, because of cell wall thickness and also because
if some similarities in the genetic material.
12) How do you think the acid-fast nature of Mycobacetrium contributes to its
virulence?
The waxy cell wall of Mycobacterium protects the bacterium against phagocytosis and
some antibiotics while in the host, so the pathogen has greater opportunity to cause disease.
Matching Question:
1. Staphylococcus aureus before primary stain is added.
No color
2. Pseudomonas aeruginosa after primary stain is added.
Purple
3. Bacillus megaterium after the mordant is added.
Purple
4. Staphylococcus aureus cells after the decolirizer is added.
Purple
5. Moraxella ( Branhamella ) catarrhalis after the decolorizer is used.
No color
6. Bacillus megaterium after the counterstain is added.
Purple
7. Pseudomonas aeruginosa after the counterstain is added.
Pink

Differential Stain

SPORE STAIN ACID-FAST STAIN

GRAM-STAIN
(Schaffer-Fulton) ( Kinyoun )

Primary Stain Crystal Violet Malachitegreen Carbolfuchisin

Mordant Iodine Heat Heat
Decolorizer Alcohol-acetone Water Acid-alcohol

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Counterstain Safranin Safranin Methylene blue

Cell type = color

Gram-positive = Purple Soprangium = Green Acid-fast bacteria = Red
after competition of
Gram-negative= Pink Vegetativecell =Pink Non=acid-fast bacteria = Blue
stain

Multiple Choice Questions

1. Bacteria cell wall is composed of:
Peptidoglycan
2. The exosprium, or endospore coat, is composed of:
Proteins
3. Endospores are produced by bacteria in the genus:
Bacillus and Mycobacterium
4. Acid-fast staining is useful for identifying the causative agent of:
Leprosy and Tuberculosis

7. Conclusion: