Gram Staining: A Sterile Technique

By TT -

Introduction


Gram staining is a method used to identify bacteria strains and potential contamination.  The bacteria strains are categorized as gram-positive (G+) and gram-negative (G-).  Bacteria have a membrane made up of phospholipids and other materials.  Within that layer, in some bacteria, there is a large area containing peptidoglycan.  Other bacteria have a smaller area containing peptidoglycan.  Bacteria with more peptidoglycan are gram-positive and bacteria with less peptidoglycan are gram-negative. Gram-positive strains are purple and gram-negative strains are red.  

Materials & Procedures


There are several reagents used in the gram-staining process.  The stains include crystal violet, which is the purple-colored stain, and safranin, the red-colored stain.  The other reagents used are Gram's iodine, .95 ethanol, and deionized water.  Other materials used include an inoculating loop, cell culture, a glass slide, a bunsen burner, a striker, bibulous paper, a microscope, and a clothespin. 

First, after turning on the gas to the connected bunsen burner, the striker is used to ignite a flame. Then the flask with the bacteria culture in it is sterilized.  To do so, first, gently lift the foil at the outer edges, and quickly roll the foil around within the flame.  Next, remove the foil and roll the lip of the flask in the flame. Continuing the sterilizing portion of this procedure, sterilize the inoculation loop by placing the circular tip in the flame until it is red, also sterilize any part of the inoculation loop that may enter the flask, using the flame, when using it to retrieve the cultured cells. 

Once the inoculation loop is sterilized, wait about 30 seconds to allow it to cool, this will protect the cells from being killed by the introduction of heat. Once the loop is cooled, lift the foil and place the inoculation loop into the flask, ensuring that the circular tip is under the surface of the substance and swirl around.  Remove the loop from the flask and transfer the cells onto a clean glass slide. Place the foil back over the top of the flask and sterilize the loop again.  After waiting about 30 seconds, place the loop back into the flask to retrieve more cells and transfer them onto the glass slide.  Place the glass slide aside to allow cell culture to dry.  Once the slide is dry, attach a clothespin to the end of the slide, to heat fix the cultured cells. To heat fix, slowly wave the glass slide, bottom-side down, back and forth through the flame, 3 times. Heat fixing denatures the proteins in the cells, allowing them to stick to the slide when the dying process occurs.   

To begin staining, the primary stain, crystal violet, is used.  Use the dropper to add enough drops to cover the area containing the added cells. Allow the stain to sit for 60 seconds. Once the 60 seconds have passed, use the deionized water (D.I. water) to rinse the stain off of the slide. The next step is called mordant.  In this step, Gram's iodine is used to ensure that the Gram-positive strains are able to absorb the crystal violet stain. Allow Gram's iodine to sit for 45-60 seconds. Again, once this time has passed, use the D.I. water to rinse the slide.  Next, .95 ethanol (alcohol) is used to rinse the Gram-negative strains of the crystal violet stain. Only allow the alcohol to sit for 10 seconds, we want to avoid killing the cells. Rinse the slide with D.I. water.  Finally, add the safranin stain to the slide and allow it to sit for 60 seconds. Rinse the slide with D.I. water.  Dry the slide using bibulous paper by placing the slide in between the sheets of the paper and gently pressing down to pat dry. To observe the results of the Gram-staining procedure, use a light microscope. 

Results & Conclusions




The cultured cells of the Deinococcus radiodurans were purple indicating that they are Gram-negative.  There were no red streaks or stains on the slide, similar to those in the image above, indicating that there were no other strains of bacteria present.  These results also denote that Deinococcus radiodurans is a bacteria strain with a greater amount of peptidoglycan which may be useful information in future observations of this strain. 



Comments

  1. EricaFebruary 11, 2022 at 10:50 AM

    Antonyque,

    Very nice work with your gram staining and summery. Good luck in the lab!

    ReplyDelete

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