Exercise 10Gram Stain

Gram staining, a differential staining technique, is one of the most important methods used by microbiologist. The technique allows us to separate bacteria into two fundamental groups; Gram positive and Gram negative. The differences between these two groups rests in the chemistry of their cell wall. Gram positive cells have a cell wall composed of a very thick layer of peptidoglycan, a uniquely bacterial molecule. While, Gram negative bacteria have a cell wall composed of a thin layer of peptidoglycan surrounded by an outer membrane.

Hans Christian Gram, developed the method in 1884 while trying to detect bacterial cells in mammalian tissue. By exposing the tissues to crystal violet (a basic stain) and then rinsing with a solvent, Gram, found the bacteria retained the purple color of the crystal violet while the tissues did not. He then flooded the tissue with safranin (a basic stain) as a counterstain to dye the cells that had been decolorized by the solvent. Safranin, a mild red color, is of much lower intensity then crystal violet, therefore, it does not change the color of the purple stained cells but it will stain those that are clear a light red. Gram soon learned that not all bacterial cells retained the crystal violet after the solvent rinse. Some bacterial cells would be rinsed clear only to be stained red by the counterstain. These bacteria are called Gram negative.

When bacteria are flooded with crystal violet the stain binds to cell wall molecules of all bacteria, like any simple stain will, staining them all purple. Because Gram positive bacteria have a thick peptidoglycan layer (30 layers or more) the stain penetrates deep into the meshwork of these molecules. Gram negative bacteria only have one or a few layers of peptidoglycan molecules. When iodine is added it acts as a mordant (fixes dye), complexing with the crystal violet to form a molecule too big to escape the cross-bridging of

the peptidogycan layers. When solvent is rinsed over the cells the Gram positive cells will retain the crystal violet-iodine complex while the Gram negative cells will have the crystal violet-iodine complex washed away. Because Gram negative bacteria do not have the peptidoglycan cross-bridging to trap the crystal violet-iodine complex it is easily removed by the solvent. Gram negative bacteria are unstained at this point. Using safranin as a counterstain you can flood the slide and stain those cells that were washed clear by the solvent. At this point Gram positive bacteria will be purple and Gram negative bacteria will be light red or pink.

Gram staining is a technique fraught with many potential problems. The most critical step is the decolorizing step. Here it can be easy to over rinse with the solvent (over decolorize) and wash away the crystal violet-iodine complex from Gram positive cells. Doing this would cause the Gram positive cells to stain red from the safranin counterstain and give you a false negative. It is also possible to under decolorize, leaving Gram negative bacteria looking purple.

It is important to use a young culture when Gram staining. Less then 24 hours is best. Older cultures can lose their ability to retain the Crystal violet-iodine complex and appear Gram variable or Gram negative.

Additionally, it is important, when making your heat-fixed smear, it not be too thick. A thick smear can interfere with the decolorizing step leading to erroneous results. Thin smears work best.

Assignment

Gram staining requires practice to be able to consistently produce reliable results. For that reason, today's lab will actually span two lab periods and should have you doing a minimum of three Gram stains. The first will be of your teeth scrapings for oral bacteria. Because you should have both Gram positive and Gram negative bacteria in your mouth this sample will serve well as a means of evaluating your technique. You will also run a Gram stain on two known microbes; one Gram positive and one Gram negative. You will then establish a fresh culture and run a Gram stain on it next lab session.

Materials glass slide Gram staining kit staining tray wash bottle Bunsen burner inoculating loop slide forceps

ProcedureBegin with a clean slide and make a heat-fixed smear of your culture or teeth scrapings. (For oral bacteria run the toothpick along your gum line and then transfer cells by rolling the toothpick in the drop of water on your slide.)

Place your smear on the rack in your staining tray and flood with crystal violet. Let stand for 60 seconds.

Holding your slide with slide forceps gently rinse off the crystal violet with your wash bottle.

Flood your slide with iodine and let stand for 60 seconds. Iodine will act as a mordant, complexing with the crystal violet that is bound to the cells.

Again, gently rinse off the excess iodine.

Hold the slide with your slide forceps so the run off is directed into your tray and rinse the slide with the decolorizing solvent (ethanol or ethanol/acetone mixture) three or four times until it flows clear. This can be tricky and is the most critical step to successfully mastering the Gram

stain.

Immediately, rinse off the solvent with distilled water from your wash bottle. Solvent left on the slide will continue to decolorize.

Finish by counterstaining with safranin. Flood the slide for 60 seconds then rinse with distilled water. Blot dry and observe using oil immersion.

Figure 1: Gram staining sequence: crystal violet, rinse; iodine, rinse; decolorize, rinse; safranin, rinse.

Questions1. What color are Gram negative cells after decolorizing with ethanol.

2. What is the best age for your culture when performing a Gram stain? Why?

3. Why is Gram staining considered a differential staining process?

4. What is the purpose for using a counterstain?

Figure 2: Gram + cocci, Gram - rods, oral epithelial cells with G+ and G- bacteria.