Brief Description:  

This method is to find whether a bacteria is gram negative (thin peptidoglycan layer in the cell wall) or positive (Thick peptidoglycan layer in the cell wall).

“The procedure for a gram stain test is to make a smear of the bacteria that one wishes to test and one of each control for positive and negative on a slide. Typically the controls are E. Coli for gram negative and Staphyylococcus Epidermidis for the positive control. Let these air dry and then heat fix on the slide by passing over a flame two to three times. Place the slide on a staining rack over a sink and flood with a Crystal Violet Stain and leave alone for one minute. At the end of the minute tip slide and rinse with DI water into sink.  Next flood slide with Iodine and leave for another minute. Again at the end rinse off slide into sink with DI water. Next, while holding slide at an angle, add ten drops of Ethanol to the slide and let drain into the sink. Immediately after this rinse with DI water. Lastly, flood the slide with Safarin and let sit for another minute. At the end rinse with DI water and finally dry by blotting the slide dry. Now look at the smears under the microscope under oil immersion. The E. colishould look pink and the Staphylococcus epidermidis should look purple. Check the bacteria in question to see what color its cells look and compare to the controls before making a final judgment.”; (Mann 2009).

Some things to avoid when preparing smear are using to much of a colony and making the smear too thick, it makes the stain difficult to read accurately, and the heat fixing should be quick passes and not prolonged exposure. (Fugelsang 1997)

Application in Wine Microbiology:

The use for this in winemaking is its assistance in making identifications of bacteria in wine. The easiest use for this is its help in distinguishing lactic acid bacteria (gram positive) and acetic acid bacteria (typically gram negative) (Delfini and Formica 2001). This can also be used on an unknown bacterial agent in wine in cases of spoilage.


  • Delfini, Claudio and Joseph V. Formica. 2001. Wine Microbiology: Science and Technology. Marcel Dekker Inc. New York.
  • Fugelsang, K. C. 1997. Wine Microbiology. Chapman & Hall. New York.
  • Mann, Eric. Fall 2009.  UC Davis Microbiology 101 Laboratory Manual.