Gram Staining, Including Simple Staining Method {Lab 3 - May 14, 2015}

      For this experiment we try our first staining. This means we were coloring our bacteria in order to view them under the microscope. The Gram staining would tell us whether our unknown is gram-positive or gram-negative.  A gram-positive bacteria’s cell membrane is thicker than gram-negative and gram-negative has two layers with lipopolysaccharides on the outermost layer.

Our Stained specimen

Materials:

• Inoculating loop
• Bunsen burner
• Slide
• Crystal violet
• Gram’s iodine
• 95% ethanol
• Safranin
• DI water
• Bibulous paper
• Staining rack, clamp

Procedure:

      First, we placed a small drop of DI water in the center of a slide. Then using aseptic techniques Clare smeared our unknown onto her slide, and I smeared our environmental sample onto mine. After the water dried on our slides we picked up the slides with our clamps and quickly passed the slides through a flame 3 or 4 times to fix the bacteria on the slide. Then, we place the slides on staining racks across a sink and covered the fixed smear with crystal violet for 20 seconds and rinsed. This first part of the procedure is the simple-staining method.

Simple Stain of Unknown
(above and top left)


Simple Stain of Environmental
(bottom left)

   

      Next, the slide was covered with the Gram’s iodine for a minute. After that, we decolorized the fixed smear with 95% ethanol by holding the slide at a 45 degree angle and washing the slide with ethanol until the color washed away. After rinsing the ethanol off, the last stain was added, safranin. When it had been remove after a minute and the slide blotted with the bibulous paper the smears where ready for the microscope.

Results/application:

     The environmental was purple in color, which means it is gram-positive. The shape of the bacteria was cocci. The first unknown stain did not come out clear, so we prepared a second gram stain. After viewing this stain we could see it was red, signifying it is gram-negative. The shape of the unknown could be seen as bascille or rod shaped.

   

     Gram Stain of Environmental - Positive

 (above)

Gram Stain of Unknown - Negative

(left)

     Determining whether a bacteria is gram-postive or negative will determine what kind of treatment or medication to give to a patient.

c