The following modification of the Wirtz method, described below, has been effective and trouble free in our experience. You are cautioned, though, that the color may fade after a few days. The result is a delicate combination of green and red that is readily recognized provided lighting is optimum. ***CAUTION*** Malachite green dye is toxic, and breathing of fumes or contact with skin can be hazardous. This procedure should be carried out in a fume hood. The stand, burner, dyes and pipets will be available in the hoods. The procedure is messy - wear a lab coat or old clothes.
• A smear is prepared and fixed with 20 passes through a flame.
• The smear is placed on a screen well above a moderate flame from a bunsen burner.
• A generous amount of saturated aqueous malachite green is applied to the slide and allowed to steam off for 10 to 20 minutes. Ideally, the heat is maintained so that the dye barely steams, that is, whitish vapors are barely visible. Dye must be added so that the liquid does not dry up.
• After cooling the slide is rinsed with tap water to remove excess stain.
• The slide is then counterstained for a minute in 0.25% aqueous safranin. The slide is then rinsed, blotted, and dried.
Spores stain a light green,while the rest of the cell stains pink. Spores are best seen with oil immersion microscopy. Often, the colors are not very strong, so it is necessary to have the microscope in good alignment with optimum contrast and lighting. Make color notes right away, as the green may fade after a few days.
Observations on living bacteria
Sometimes assay results are compromised because a contaminating organism grows in the medium instead of the intended bacterial isolate. For a quick check to verify that cell morphology is consistent with the culture from which the inoculum was taken, a wet mount can be prepared and examined in dark field and/or phase contrast. If present, endospores are often evident in phase contrast, allowing one to avoid having to do a spore stain.
Very often, identification of an unknown organism requires knowledge of its motility, that is, its capability for translational movement. The results of motility agar incubations can be difficult to interpret, partically for aerobic bacteria that don't grow well deep into the agar. A good quick check for motility is to examine a very young culture using the hanging drop method. A young culture would be a broth culture inoculated the night before, or a broth culture that was diluted 10 fold or so in the morning, incubated, and examined in the afternoon. A hanging drop culture is prepared by placing a very small drop of medium on a coverslip, then inverting the coverslip over a depression slide so that the bottom of the drop does not make contact with the slide itself. Vaseline can be used if necessary, to make a sealed chamber.
Hanging drops can be examined using all objective lenses, although to be able to look throughout the depth of the drop the limit may be 100x. The curved depression slide will distort the effects of phase contrast, but dark field may work and will be sufficient to detect movement. All live bacteria move by Brownian (molecular) motion, at a vibration rate that is inversely proportional to the size of the cell. Rapid Brownian movement is a common characteristic of non-motile cocci such as Staphylococcus, Streptococcus, or Micrococcus. However some bacteria are flagellated, and exhibit translational movement as well. Truly motile organisms will zip across the microscope field. Look for definite directional motion, tumbling, and movement against currents.
Dispose of wet mounts carefully, since the bacteria will be viable.