P665
A student was given a microscope slide of scraped human skin cells as well as a petri dish containing a growth of a single bacterial culture isolated from those cells. The cells on the slide were stained for chromatin. The students’ role was to examine the skin cells for phases of mitosis as well as to identify the species of bacteria using two particular microbiological techniques, each of which address major differences in bacteria.
Technique 1: Gram Stain
A small amount of bacteria is placed on a microscope slide. The slide is briefly placed over a hot flame to heat-fix the bacteria, allowing it to adhere to the slide. Then, a few drops of crystal violet (a purple dye) solution are applied. Several drops of Gram’s iodine are added as a trapping agent, “”trapping”” the color in cells that absorbed it; Iodine complexes with crystal violet, resulting in a compound that is too large to leave bacteria with thick cell walls. This is followed by alcohol to wash away any excess crystal violet solution that remains outside the cells. Lastly, a counterstain such as safranin is added to visualize the bacteria with thinner cell walls, whose crystal violet washed away..
Gram-positive bacteria have a thicker peptidoglycan layer and a thin lipid layer, while gram-negative bacteria are the opposite. Crystal violet becomes trapped with iodine in the thick peptidoglycan layer of gram-positive bacteria, but not in the thin layer of gram-negative bacteria.
Technique 2: Catalase Reaction Test
Some bacteria contain the enzyme catalase, which catalyzes the conversion of hydrogen peroxide to water and oxygen gas. Bacteria are placed in a test tube with growth media and allowed to grow overnight. A few drops of hydrogen peroxide are added to the test tube and it is sealed for several hours. After the time passes, the test tube is observed for gas bubbles to indicate a positive reaction.
There were four possible bacterial cultures that were given to students in the class, the characteristics of which are shown in the table below:
| Gram Stain | Catalase Reaction Test | |
| Staphyloccocus aureus | + | + |
| Streptococcus pyogenes | + | – |
| Pseudomonas aeruginosa | – | + |
| Bacillus cereus | + | + |
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