Simple, Negative and Gram Stains
Simple Stain SlideToday we created three different types of stains: Simple Stain, Negative Stain and Gram Stain (gram positive).
Before we stained, we learned how to fix bacteria on a microscope slide. First place a small drop of water on a microscope slide. Then using a sterile loop smear a small amount of bacteria in the drop of water across the slide. Allow the water to evaporate then pass the slide through a flame three times to heat fix it. Next, add 95% methanol to the dried smear for one minute and finally rise off methanol.
Simple Stain: Begin with a fixed slide. Next, cover the slide with crystal violet stain for 20-30 seconds. Rinse off excess stain with water then blot drop with bibulous paper. Finally examine the smear under oil immersion lens.
From observing our unknown bacteria under a simple stain we learn the bacteria is coccus.
Before we stained, we learned how to fix bacteria on a microscope slide. First place a small drop of water on a microscope slide. Then using a sterile loop smear a small amount of bacteria in the drop of water across the slide. Allow the water to evaporate then pass the slide through a flame three times to heat fix it. Next, add 95% methanol to the dried smear for one minute and finally rise off methanol.
Simple Stain: Begin with a fixed slide. Next, cover the slide with crystal violet stain for 20-30 seconds. Rinse off excess stain with water then blot drop with bibulous paper. Finally examine the smear under oil immersion lens.
From observing our unknown bacteria under a simple stain we learn the bacteria is coccus.
Gram Stain Slide
We next prepared a Gram Stain. Begin with a fixed smear slide. Cover the slide with crystal violet stain for 20-30 seconds. Rinse slide from excess stain with water then cover smear with Gram’s iodine stain for one minute. Rise again to remove excess iodine then decolorize the slide with 95% ethanol. Holding slide at 450 angle drop by drop add the decolorizing ethanol until the color stops running. Immediately rinse the slide with water to remove the decolorizing agent. Finally cover the smear with safranin for one minute, rise with water to remove excess and blot with bibulous paper. Examine slide under oil immersion lens.
From the Gram Stain we learned that our unknown bacteria was gram positive due to the purple color.
From the Gram Stain we learned that our unknown bacteria was gram positive due to the purple color.
Negative Stain Slide
Finally we prepared a Negative Stain. Being by placing a small drop of nigrosin near end of slide. Using aseptic technique and an inoculating loop, place small amount of bacteria smearing it within the diameter of the nigrosin drop. Touch the short end of another clean slide at a 45-degree angle spreading it so there is a feathered end. Allow the smear to air dry completely then observe slide under oil immersion lens.