Method

Besides Gram's stain, there is a wide range of staining methods available. The procedures for these other methods follow quite closely those of Gram's stain. By using appropriate dyes, different parts of the cell structure such as capsules, flagella, granules, or spores can be stained. Staining techniques are widely used to visualize those components that are otherwise too difficult to see under an ordinary light microscope either because of the lack of color contrast between the object under examination and the background or because of the limited resolving power of the light microscope. In addition, staining techniques are useful in detecting the presence or absence of certain cell components, thus allowing a relatively simple scheme of differentiation or identification of microorganisms. In this respect, Gram's stain ranks among the most important diagnostic tools in biological science.

In Gram's method, which is based on the ability of a cell in retaining the crystal violet dye during solvent treatment, it is the difference in the microbial cell wall that is amplified. The cell walls for gram-negative microorganisms have a higher lipid content than gram-positive cells. Originally, both kinds of cells are penetrated by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed too easily. This step is commonly referred to as fixing the dye. However, the subsequent treatment with the decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker gram-positive cell wall, closing the pores as the cell wall shrinks during dehydration. As a result, the the diffusion of the stain-iodine complex out from the cell is obstructed, and the cells remain stained. The actual mechanism of decolorization is currently not well understood and remains controversial. At any rate, if the decolorizer treatment is properly timed, there exists a period during which the crystal violet-iodine complex is effectively removed from the gram-negative cells but still retained in the gram-positive ones. Thus, the length of the decolorizer treatment is critical in clearly differentiating the gram-positive cells from the gram-negative cells. A prolonged exposure to the decolorizing agent will remove all the stain from both types of cells. The student is cautioned that some gram-positive cells lose the stain easily and therefore may appear gram negative.

Finally, although not essential, a counterstain of safranin is applied to the smear to dye the decolorized gram-negative cells with a pink color. Thus, the size and shape of both types of cells can be more easily observed under a microscope. At the same time, they can be differentiated by the imparted color. If desired, the slides can be permanently mounted and preserved for record keeping.

A mixture of S. cerevisiae and E. coli will be examined in this experiment, although Gram's stain is usually applied to differentiate bacteria, which are usually too small to be seen clearly under a light microscope. S. cerevisiae cells will be stained by the crystal violet-iodine complex and should appear purple-brown in color. In contrast, the much smaller E. coli, cells should appear pink, the color of safranin. Repeat the entire process until a satisfactory slide is prepared and properly focused in a microscope for the approval of the instructor. Note the improvement in cell visualization as compared to a plain unstained slide.

List of Reagents and Instruments

A. Equipment

• Bunsen burner
• Microscope
• Slide
• Cloth pin
• Water bottle

B. Reagents

• Crystal violet
• Methylene blue
• Ethanol, 95%
• Acetone
• Ammonium oxalate
• Iodine
• Potassium iodide
• Sodium bicarbonate
• Safranin O

Procedures

1. Prepare Reagents:
   • Gram Crystal Violet Solution: Dissolve 20 g of crystal violet, 95 % dye content, in 100 ml of ethanol to make a crystal violet stock solution. Similarly, dissolve 1 g of ammonium oxalate in 100 ml of water to make an oxalate stock solution. Working solution is obtained by mixing 1 ml of the crystal violet stock solution with 10 ml of water and 40 ml of the oxalate stock solution. Store the working solution in a drop bottle.
   • Methylene Blue Solution: Dissolve 1 g of methylene blue, 90 % dye content, in 100 ml of ethanol; this is Solution A. Mix 0.03 g of KOH in 300 ml of water; this is Solution B. Mixing Solutions A and B yields the working solution.
   • Gram Iodine Solution: Dissolve 1 g of iodine, 2 g of potassium iodide, and 3 g of sodium bicarbonate in 300 ml of water.
   • Gram Decolorizer Solution: Mix equal volumes of 95 % ethanol and acetone.
   • Gram Safranin Solution: Dissolve 2.5 g of safranin O in 100 ml of 95 % ethanol to make a stock solution. Working solution is obtained by diluting one part of the stock solution with five parts of water.
2. Prepare a Slide Smear:
   • Transfer a drop of the suspended culture to be examined on a slide with an inoculation loop. If the culture is to be taken from a Petri dish or a slant culture tube, first add a drop or a few loopful of water on the slide and aseptically transfer a minute amount of a colony from the Petri dish. Note that only a very small amount of culture is needed; a visual detection of the culture on an inoculation loop already indicates that too much is taken.
   • Spread the culture with an inoculation loop to an even thin film over a circle of 1.5 cm in diameter, approximately the size of a dime. Thus, a typical slide can simultaneously accommodate 3 to 4 small smears if more than one culture is to be examined.
   • Hold the slide with a cloth pin. Air-dry the culture and fix it or over a gentle flame, while moving the slide in a circular fashion to avoid localized overheating. The applied heat helps the cell adhesion on the glass slide to make possible the subsequent rinsing of the smear with water without a significant loss of the culture. Heat can also be applied to facilitate drying the the smear. However, ring patterns can form if heating is not uniform, e.g. taking the slide in and out of the flame.
3. Gram Staining:
   • Add about 5 drops of crystal violet stain over the fixed culture. Let stand for 60 seconds. Note that a clothes pin is used to hold the slide during the staining procedure to avoid staining one's hand.
   • Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle. Note that the objective of this step is to wash off the stain, not the fixed culture.
   • Add about 5 drops of the iodine solution on the smear, enough to cover the fixed culture. Let stand for 30 seconds.
   • Pour off the iodine solution and rinse the slide with running water. Shake off the excess water from the surface.
   • Add a few drops of decolorizer so the solution trickles down the slide. Rinse it off with water after 5 seconds. The exact time to stop is when the solvent is no longer colored as it flows over the slide. Further delay will cause excess decolorization in the gram-positive cells, and the purpose of staining will be defeated.
   • Counterstain with 5 drops of the safranin solution for 20 seconds.
   • Wash off the red safranin solution with water. Blot with bibulous paper to remove the excess water. Alternatively, the slide may shaken to remove most of the water and air-dried.
4. Liberally wash off any spilled stain immediately with water to avoid leaving permanent marks in the sink, lab bench, or glassware.
5. Examine the finished slide under a microscope.

References

1. Bartholomew, J. W. and Finkelstein, H., Relationship of cell wall staining to Gram differentiation, J. Bacteriol., 75, 77, 1958.
2. Syndney M. Finegold and William J. Martin, Diagnostic Microbiology, Chapt. 3, Mosby Co., St. Louis, 1982.
3. Salton, M. R. J., The Bacterial Cell Wall, Elsevier Press, Amsterdam, 1964.
4. Gregersen, T., Rapid method for distinction of gram-negative from gram-positive bacteria, Eur. J. Appl. Microbiol. Biotechnol., 5, 123, 1978.