Having a specimen cultured begins an investigation toward identifying if a disease causing bacteria is present in a patient. Identifying the primary variety and morphology of a bacteria present can provide important information to a physician regarding treatment. Whether a specimen is received on a culture swab, an aspiration of fluids or a body specimen, there may be tiny microorganisms present. These minuscule organisms can cause elevated temperatures, chills, fever or general malaise.
In order to provide proper treatment, a physician must know which organism is causing an illness. Since many bacteria exist on the skin and throughout the entire body, it is important to separate normal flora from a pathogen. The first step in doing so is to gram stain a properly collected specimen.
Gram stain reactions of bacteria provide a tremendous amount of information about the type of bacteria which are present. A laboratory technologist will prepare a slide with the specimen, heat fix it, stain it using crystaline violet, and then decolorize to view the bacteria.
Under the Microscope
Once a slide has been prepared it is ready to be examined. The slide is placed on a microscope and scanned first of all to see the overall scheme of what is present. Observation for white blood cells and epithelial cells provide information about the probability of a bacterial infection or whether the specimen is contaminated.
Then the search begins for microorganisms and the color of them. The gram stain will stain bacteria purple if they are gram positive and pink if they are gram negative. Purple bacteria retain crystal violet because their cell wall is thicker, containing significant amounts of peptidoglycan. Crystal violet stain is readily absorbed into the bacteria’s cell wall.
Crystal violet is then rinsed with water and iodine is added to cover the slide. Iodine is a mordant which helps set the crystal violet stain in a complex with the cell wall. A decolorizer is added which removes the color if it can. If in the case of gram positive bacteria the stain is set, then the counterstain, safranin cannot be absorbed into the cell wall, leaving the cell with the appearance of being purple.
If the microorganism is a gram negative bacteria, then the primary stain is not retained and the counterstain can be absorbed or pass through the cell wall. Gram negative bacteria have a much thinner cell wall with fewer peptidoglycan than gram positive bacteria.
Clinical Significance of Gram Stain Reactions
Gram stain reactions of bacteria will be demonstrated through the gram stain and give the physician enough general information to begin treatment with antibiotics known to act against gram positive or gram negative bacteria, depending on the type observed. Early treatment can make the difference between a short or long recovery time.