Gram Staining Bacteria
From Your Mouth
Name __________________
Introduction:
Bacteria are transparent and microscopic (clear, like glass). It is very difficult to
observe bacteria without adding color to them. Staining is the process by which color is
added to bacteria so they can be viewed under the light microscopes in our classroom.
Gram-staining is a specific staining technique invented in 1844 by the Danish
bacteriologist, Hans Christian Gram. Gram-staining is one of the most important
staining techniques in microbiology. It is almost always the first test performed for the
identification of bacteria.
Two stains are used in the Gram-Staining method. The first stain is called the
Primary Stain. The second stain used is called the Counter Stain.
The primary stain of the Gram-staining method is Crystal Violet. Methylene
Blue is sometimes substituted for Crystal Violet and is equally effective. The bacteria
that are stained by the crystal violet appear purple and are referred to as Gram-Positive.
A counter stain is the stain that is used to stain all of the bacteria that remain
unstained after treatment with the crystal violet. The most common counter stain used to
stain the remaining unstained bacteria is safranin red or basic fuchsin. These bacteria
will appear pink or red and are referred to a gram-negative.
A mordant is a chemical used to “fix” or seal the stain into the bacteria. A
mordant is used so the stain cannot be easily removed. Gram’s Iodine is the mordant
used in the Gram-Staining Method.
A decolorizer is a chemical used to remove excess stain during a staining
procedure. Ethyl alcohol is the decolorizing agent used in the Gram-Staining Method.
Heat fixing is the process of passing the slide containing the specimen through a
flame 2-3 times. This dries the specimen and makes it “stick” to the glass slide. This is
necessary because the stains are liquid and would wash the specimen from the slide if it
were not fixed to the slide. Caution must be taken when heat fixing bacteria. If you
leave the specimen containing bacteria in the heat too long, you will boil the cytoplasm.
Boiling the cytoplasm will result in the destruction of the bacteria cells, making staining
and identification impossible.
The lengths of time the stains, mordant and decolorizing agent are left on the
specimen are critical during the Gram-Staining Method. Leaving a stain on the specimen
too long can result in bacteria that are stained too dark. Leaving a stain on too briefly
may result in a stain that is too light. Leaving the decolorizing agent on too long or too
briefly will result in too much or too little removal of stain. Staining can also be affected
by the quality and age of the chemicals used. Fresh chemicals may not need to be left on
the sample as long as older chemicals. Chemicals may lose their effectiveness over time.
The development of chemical stains and staining procedures has been a valuable
tool in providing health care to patients. Gram-staining is used every day in medical
laboratories across the world. Identification of bacteria is critical for the proper diagnosis
and treatment of illnesses. A Medical Doctor needs this information so he or she can
decide which antibiotic to prescribe to the patient.
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Directions:
A. Answer the following questions about Gram-Staining. Find your answers in
the Introduction and in the results of your Internet Search.
1. Who invented the Gram-Staining Method and when?
2. What organism is stained in this method?
3. When looking at a stained specimen, how do you tell the difference
between gram-positive bacteria and gram-negative bacteria?
4. What is the most common primary stain used in Gram-Staining?
5. What is the most common counter stain used in Gram-Staining?
6. What is the purpose of using a mordant in Gram-Staining?
7. What is the purpose of using a decolorizer in Gram-Staining?
8. What might happen if you forget to heat fix your specimen to the
microscope slide?
9. How is Gram-Staining used in health care today?
10. What are the structural differences in the cells of Gram-Positive and
Gram-Negative Bacteria that result in them accepting different stains?
11. Describe what a well-stained specimen looks like as opposed to a
poorly stained specimen. Analyze the conditions that may have
caused a poorly stained specimen and explain what may have
happened to create that result.
B. Perform an Internet Search on the Gram Staining Method. You are looking
for:
1. The step-by-step process of the Gram-Staining Method.
2. The differences in the structure of Gram-Positive and Gram-Negative
bacteria.
3. Print out this information and staple it to this assignment.
4. You will use these steps to Gram-Stain a specimen of bacteria from the
teeth and gums in your mouth.
C. Use the Gram Staining kits in the classroom to perform the Gram-Staining
Method on a bacterial specimen from your mouth. Use a cotton swab to
retrieve a bacteria specimen from the teeth and gums of the back of your
mouth.
D. Ask your teacher for the laboratory supplies and equipment required to
perform a Gram-Stain.
E. When you have completed the Gram-Staining of the sample, let it dry and
view it. You will view it at the highest magnification available on the
classroom microscopes, which is 1000x with the oil immersion objective.
F. Sketch the stained sample inside the circle on the next page. Color the
bacteria the color. Label the bacteria you have sketched and colored as:
gram-positive, gram-negative, correct shape, correct arrangement.
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Sketch:
gram-stained mouth bacteria
Magnification: 1000x with oil immersion objective
Labeled:
gram-positive, gram-negative, shape,
arrangement
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Data Analysis: You have stained and observed bacteria. Now it is time to compile your
information into the following Data Table.
Gram-Stain
Result
Bacteria Shape
Bacteria
Arrangement
Bacteria Count (Oil
Objective)
Gram Positive
(Purple)
Gram Negative
(Pink)
Calculate the Following Statistical Information for all the class data:
Data Set: Arrange your bacteria count class information from smallest to highest.
Statistic
Gram (+)
Gram (-)
Mean (average)
Median (middle)
Mode (most)
Range (highest – lowest)
Stem and Leaf Plots:
Gram (+) Data Set
Gram (-) Data Set
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