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 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.
3 microscope slides (precleaned)
SMEAR PREPARATIONS: Remember to label the slides.
1. S. epidermidis and P. aeruginosa mixture:
Prepare smear using aseptic technique.SEE PINK EXERCISE for SMEAR PREPARATION!! THE FIRST LOOPFUL OF ORGANISM IS NOT SMEARED OUT UNTIL THE SECOND ORGANISM HAS BEEN ADDED. The two organisms are then smeared out together. After air drying and heat fixing the Gram staining procedure is followed.
2. B. licheniformis:
3. M. smegmatis:
Prepare smears using aseptic technique. These organisms are growing on TSA slants. A loopful of distilled water is first placed on each slide. Bacteria are obtained from the slants using a sterile needle and proper aseptic technique. SEE PINK EXERCISE for SMEAR PREPARATION!! After air drying and heat fixing the Gram staining procedure is followed.
GRAM STAINING PROCEDURE:
1. Cover with CRYSTAL VIOLET for 20 seconds.
2. Gently rinse off the stain with water and shake off
3. Cover with GRAM'S IODINE for one minute.
4. Pour off the Gram's iodine.
5. Run 95% ETHYL ALCOHOL down the slide until the
solvent runs clear (about 10-20 seconds). THIS STEP
IS CRITICAL! THICK SMEARS REQUIRE MORE TIME
THAN THIN ONES. (DECOLORIZING AGENT)
6. Rinse with water to stop the action of the alcohol.
7. Cover with SAFRANIN for 20 seconds.
8. Gently rinse off the stain with water. Blot with
bibulous paper and clean off the bottom of the slide
with 95% alcohol.
a) DO NOT make your smears too thick!
b) Be very careful when you decolorize.
c) Be sure your cultures are young, preferably less
than 24 hours old. Older cultures tend to lose the
ability to retain stains.
Observe your smears in the microscope under oil immersion. Draw a few representative organisms from each smear in your lab report.
Pseudomonas Gram-negative rod
Bacillus Gram-positive, spore-forming rod
Mycobacterium Gram-positive, acid-fast rod