Lisa Ta February 29, 2012. Biology 240 General Microbiology MW 3:45-5:00
Gram Stain Report Introduction
The purpose of this experiment is to determine the shape and Gram stain of the bacteria under a microscope. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. The Gram stain is a type of differential stain that allows a (micro)biologist to identify the differences between organisms and/or differences within the same organism.
Gram staining bacteria requires the use of aseptic technique to
ensure the sterility of the experiment. The purpose of the Gram stain is to show whether the bacteria are Gram positive (purple-colored), Gram negative (pinkcolored), or both.
Materials and Methods
Aseptic technique was used throughout throughout the experiment. A drop of deionized water was placed on a microscope slide and bacteria from unknown number 20 were also placed on the slide. Then the slide had to be air dried before heat fixing it, so that the Gram staining may begin. The first step in Gram staining was to place the primary stain, Crystal Violet or Methylene Blue, on the slide where the bacteria were heat fixed. The dye had had to stay on the slide for about one minute before rinsing with deionized water and blotted dry. Then the Mordant Iodine (which is the Gram stain dye) was placed on the slide for another minute and a half.
After the minute and a half was up, the slide was rinsed with
deionized water water and blotted blotted dry. The next step was to use 95% Ethanol to wash wash off the dyes from the Gram-negati Gram-negative ve bacteria. To distinguish when to stop using ethanol, the first drop of non-colored solution that slides off the slide was the
indicator.
Again the slide was rinsed with deionized water and blotted dry.
Lastly, the saffarin dye was placed on the slide to counterstain the Gramnegative bacteria for one minute, rinsed with deionized water, and blotted dry.
Methods could also be referred to the lab manual pages 159-163.
Microscope E1 was used to view unknown number 20 to determine whether the bacteria were Gram-positive or Gram-negative and cocci or bacillus.
Results
Through the process of the experiment, unknown number 20 which was on a Nutrient Agar slant medium which was determined to be cocci (sphere) shaped appeared to be Gram-negative.
Discussion
What determines Gram-positive and Gram-negative bacteria is due to difference in cell wall composition. Because the cell walls walls of Gram-negative cells have a higher content of lipids and a thinner layer of peptidoglycan, the alcohol used in the decolorizing step made the Gram-negative cells incapable of retaining the methylene blue-iodine complex. On the other hand, Gram-positive Gram-positiv e cells have a thicker peptidoglycan that traps the methylene blue-iodine complex, making it less vulnerable to decolorization.
The Gram stain technique was was used on unknown unknown number 20. The unknown appeared to be Gram negative and cocci shaped. Since this is an unknown, the literature literatur e value is unknown.
Possibilities of difference in literature and
experimental value could be due to excess primary and mordant iodine dyes being flushed out entirely by the 95% Ethanol, or that the mordant iodine dye was not left on long enough.
Reference
Bauman, R.W. “Microbiology with Diseases by Body System”, 2 nd ed. Pearson Education, Inc. S.F. 2009. p.107-109.
Leboffe, M.J., Pierce, B.E. “Microbiology Laboratory Theory and Application”, Brief ed. Moron Publishing Co. CO. 2008. p. 159-163.
