1. International Medical School
Shah Alam Campus
Medical Microbiology
Laboratory Manual & Practical Experiments Work Record
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Practical Laboratory
Experiment (2)
Simple and Gram Staining
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2. Gram Staining
• Introduction
• Theory
• Materials
• Staining Procedures
• Microscopy
• Interpretation
Introduction:
The Gram stain is a differential stain which allows most bacteria to be divided into two groups,
Gram-positive bacteria and Gram-negative bacteria. The technique is based on the fact that the
Gram positive cell wall has a stronger attraction for crystal violet when Gram's iodine is applied
than does the Gram negative cell wall. Gram's iodine is known as a mordant. It is able to form a
complex with the crystal violet that is attached more tightly to the Gram-positive cell wall than to
the Gram-negative cell wall. This complex can easily be washed away from the Gram-negative cell
wall with ethyl alcohol. Gram-positive bacteria, however, are able to retain the crystal violet and
therefore will remain purple after decolorizing with alcohol. Since Gram-negative bacteria will be
colorless after decolorizing with alcohol, counterstaining with safranin will make them appear
pink. The procedure was discovered by Christian Gram in 1884. The chemical basis was not
understood by Gram and is still not fully understood today. It is known, however, that the two
groups of bacteria have very different cell walls and that the type of cell wall dictates the way a
bacterium responds to the Gram stain.
The Gram stain is probably the most commonly used staining procedure in microbiology. It is
extremely useful in identifying bacteria. It is important that you understand the color changes that
occur at each step in the Gram stain. It is also important that you understand the function of each
reagent used in this procedure. It takes some practice and patience to be able to reliably Gram
stain.
You will first stain a mixture of a Gram-positive coccus and a Gram-negative rods. This will allow
you to check your technique. If you have pink rods and purple cocci, you have stained correctly. If
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3. you have pink rods and cocci you have over decolorized. If you have purple rods and cocci, you
have underdecolorized.
Your second smear will be a spore-forming Gram-positive rod, Bacillus. Endospores do not "take"
the Gram stain because of their thick spore coat. After Gram-staining, you will see purple rods and
some of them will contain clear (white) ovals or circles. You will find it useful in the future to be
able to identify endospores in a Gram stain.
Your third smear will be an "acid-fast" rod, Mycobacterium. This group of genus is Gram-positive
but is surrounded by a large amount of waxy lipid. This makes it somewhat impermeable to normal
basic stains. Therefore this organism usually stains a light purple in color.
Applications of Gram staining:
1) Differentiation of bacteria into Gram positive and Gram negative is the first step towards
classification of bacteria.
2) It also the first step towards identification of bacteria in cultures.
3) Observation of bacteria in clinical specimens provides a vital clue in the diagnosis of
infectious diseases.
4) Useful in estimation of total count of bacteria.
5) Empirical choice of antibiotics can be made on the basis of Gram stain’s report.
6) Choice of culture media for inoculation can be made empirically based on Gram’s stain
report.
Miscellanea:
1. Although Gram stain is useful in staining bacteria, certain fungi such as Candida and
Cryptococcus are observed as Gram positive yeasts.
Materials:
1. Microscope slides (pre-cleaned)
2. Loops or sterile swaps
3. Heat source or heater at 42ºC.
4. Gram stain reagents (crystal violet, Gram's iodine, 95% ethyl alcohol, and safranin)
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4. 5. Agar plates of 24 hr NA or TSB cultures of Stapylococcus epidermidis, Pseudomonas
aeruginosa or nay G+ve cocci or bacilli.
6. 24 hr TSA slant of Bacillus licheniformis or G-ve bacilli.
7. 48 hr TSA slant of Mycobacterium smegmatis (non-pathogenic and used for research)
Gram Stain Procedure
1. Wash your hands with soap and water and make it dry before putting on your gloves
2. Wear latex gloves before beginning this lab procedure if necessary.
3. Let us know if you have a latex allergy before putting on the gloves.
Smear preparing:
1. Make a specimen smear by placing a small amount of bacteria on a toothpick, and gently
smearing it onto a clean glass slide.
2. Take another slide and use its edge to scrape (smear) the specimen into a very thin film of
material.
3. Let the specimen on the slide air dry, and then heat fix it by having Dr. Saleh or by the lab
technician, pass the slide through a candle flame 3-4 times. The slide should not get too hot
to touch, and it should never stop as it passes through the flame.
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5. Helpful suggestions:
a) DO NOT make your smears too thick!
b) b) Be very careful when you decolorize.
c) c) Be sure your cultures are young, preferably less than 24 hours old. Older cultures
tend to lose the ability to retain stains.
Gram staining procedure:
1. Cover the specimen with 1-2 drops of Crystal Violet stain for 60 seconds, and then
gently wash it off with very slow running water from the tap. If the water is running too
fast, the specimen will be washed off!
2. Cover the specimen with a few drops of Gram’s Iodine for 60 seconds, and then gently
wash the specimen again as in step 3.
3. Use Ethyl Alcohol as a solvent. Tilt the slide slightly and apply Ethyl Alcohol drop by
drop onto the slide above the specimen, so that the alcohol runs down the entire
specimen. Stop applying the Ethyl Alcohol when the fluid flowing off the edge of the
slideis no longer colored. The thinnest part of the smear should be colorless. This will
take approximately 5 seconds. Then, gently wash the slide again with very slow
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6. running water. Note: the Gram Positive cells will retain some of the violet coloring, but
the majority of the stain will be rinsed away by the solvent.
4. Cover the specimen with a few drops of Safranin stain for 60 seconds, and then gently
wash with very slow running water.
5. Gently blot the slide with a paper towel, but do not rub the specimen smear.
6. Put a coverslip over the specimen smear.
7. Examine the specimen slide under a microscope at each magnification level.
-Cells that are purple or violet are Gram Positive Bacteria
-Cells that are pink or red are Gram Negative Bacteria
Pictures to help you for smear preparation and gram stain methods:
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1-Label the slide with the patient’s initials
or the specimen number.
2-Choose an isolated colony off of the agar plate
and obtain bacteria with a sterile swab.
3- Place the swab on the microscope slide and
spread the colonies in a circular motion.
4- Heat to fix the microorganisms on the slide
by placing the bottom of the slide to heat for
approximately 30 seconds. Your smear is ready
for gram staining.
5- Place slide on staining tray or hold with
forceps above the sink.
6- Flood the surface of the slide with Crystal
Violet stain and let sit for one minute.

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7- Rinse the slide with distilled water. 8- Flood the slide with Gram’s Iodine and time
for one minute.
9- Rinse the slide with distilled water.
10- Flood the slide with Gram’s decolorizer and
time for 30 seconds.
11- Rinse the slide with distilled water.
12- Flood the slide with the counterstain,
Safranin, and let sit for one minute.
13- Rinse the slide with distilled water.
14- Dry the slide curfully by using paper or left
the slide on the rak for few minutes.

8. Gram staining summary
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9. Microscopy and Results:
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10. 1. Observe your smears under the microscope, use 10x, 40x and oil immersion.
2. Draw a few representative organisms from each smear in your lab report.
3. Let others (including Dr. Saleh, Dr. Durgadas or the technician to view your slide.
4. On a separate page in your laboratory report, draw one or more pictures of your
specimen, using colored pencils.
5. Write your name on the picture(s), and write down the location where the sample was
collected if possibly.
6. When done, place your slide in the bucket containing bleach or in a special container.
7. Clean the oil lens by special soft paper, clean your work area and discard your gloves in
the assigned waste basket.
8. Wash your hands thoroughly with soap and water following this experiment.
Draw your results here and interpret your focusing
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11. Low power 10x High power 40x
Oil immersion 100x
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Submit the report in the end of the laboratory before leaving the lab. Thank you for your collaboration.

12. Interpretation:
Once you have the slide in focus at 100X, here are some things to evaluate:
1. How good was your Gram stain?
2. What is the size of the cells you are viewing?
3. What is the morphology of the bacteria?
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