This layer makes the cells less susceptible to decolorization by Ethyl Alcohol. Using this property, cells with a thinner peptidoglycan layer can be stained a different color than gram positive cells within a culture. The primary color is applied to the culture and imparts a purple color to all bacteria in your sample. Adding Gram’s iodine, a mordant used by the method’s namesake, makes the violet color more intense, and creates a bond with the primary stain that makes it less soluble. The third step is to add a decolorizing agent, Ethyl Alcohol.
In gram negative cells the alcohol makes the outer cells porous, which allows the primary stain to be washed away. The gram positive cells have pores that are actually dried out by the alcohol, which makes the primary stain fix into the membrane, and not get washed away. The final stain is the counter stain that lends a different, pinkish color to the gram negative cells that have been washed of the primary stain. Methods The method used in this experiment was derived from Cappucino and Sherman (2011) as detailed in experiment 10 with substitutions for the unknown bacteria.
Aseptic lab technique was followed. One slide of the #7 unknown bacteria and one slide which included both unknown #7 and unknown #2 were prepared using the following stain technique. The initial smears were made from broth cultures using a loop, air dried, and then heat fixed. The smears were covered with crystal violet stain and allowed to absorb for one minute, before gently rinsing with a controlled stream of water from a squeeze bottle. Next, Gram’s iodine was applied to the smears with a dropper and left to sit for one minute before rinsing with water.
Next, the 95% Ethyl Alcohol mixture was dropped slowly onto each smear and allowed to run off until the alcohol appeared clear and then gently rinsed with water. Lastly in the staining procedure, Saffranin was dropped onto the smear until it was covered, for 45 seconds. The smears were rinsed with water final time, then placed between sheets of bibulous paper to dry. Each smear was viewed under the light microscope using oil immersion at 1000x magnification. These smears were compared to smears prepared in the same fashion, of the known bacteria E. coli, B. cereus, S. ureus, and a mixed smear containing both E. coli and S. aureus. Table 1 shows how the three different cells were observed after staining. The only pink and gram negative bacteria were the E. coli which were rod shaped and collected in a strepto arrangement. Bacillus cereus was purple in color, appeared rod shaped characteristic of bacilli and was gram positive, appearing purple as expected. The other gram positive and purple bacteria, Staphylococcus aureus were spherical in shape, and clustered into large groups with some lone cocci visualized.
The mixture in Table 1 showed a good contrast between the pink rods of E. coli and the purple circles of S. aureus. * Table 2 shows Unknown # 7 as a rod shaped, chain forming, purple bacteria. Unknown #7 was visualized as a clustering purple, spherical organism. Both appeared gram +. The mixture of the two showed the same shapes, but in this case the rods and spheres tended to cluster and were more difficult to observe as they were the same color. But rods and spheres could be clearly discerned upon careful examination. Table 1 served as a control for table 2, as each the unknowns in Table 2 were going to be one of the Table 1 organisms, barring contamination. If a bacteria was visualized in Table 2’s data that was not present in Table 1, there was an obvious error which could be corrected by redoing the slides. The information for Table 2 came from observations on Thursday following the original day for the unknowns on Tuesday, as we observed contamination on the slides from gram negative bacteria that should not have been present with gram positive stains.
Discussion B. cereus and S. aureus appeared purple upon observation through the microscope. This is consistent with knowledge about the two bacteria that indicate they are gram positive bacteria. The purple coloration means that their outer layers are of the more highly cross linked type with more peptidoglycan, which gives their cell membrane shape and firmer structure. This also makes the membranes open to dehydration without weakening.
When the decolorizing agent was applied in this experiment, the cell walls of the gram positive organisms shrank up, not allowing the primary stain to leave. They remained purple even after treatment with Saffranin. E. coli’s outer layer also has peptidoglycan but it is much thinner, and surrounded by lipids. When the decolorizing agent was applied to the slide containing this bacteria, it’s membrane let the stain wash easily out of the thin membrane. The saffranin as the second stain was then picked up easily, giving the E. oli a pink shade. The results of the study show that unknown # 7substance was B. cereus. The purple color and gram positive reaction ruled out E. coli as the identity of the unknown. Rods arranged in strips were observed in the unknown # 7, so B. cereus became the leading contender. When unknown #2 and unknown # 7 were mixed, a contrast between the rods and spheres could be observed, which made it clear that unknown #7 showed a clear bacilli structure. Contamination on Tuesday of the experiment led to misleading results.
Because unknown #2 had been visualized as purple cocci, and unknown #7 had been visualized as purple bacilli, when unknown #2 and unknown # 7 were mixed, it was unclear as to what the pink gram negative bacteria appearing in the slide were. The pink bacteria clung to the purple bacteria in the slide. It was decided that the unknown #2 had been mixed up with another unknown during testing, or the aseptic technique had been compromised. The unknowns were heat fixed and stained a second time, visualized individually and in a mix . The final results were unambiguous.
It is unclear whether the gram-negative bacteria were E. coli or another gram negative bacterium as the pink structures were behind the purple structures in the slide and were very translucent. It also possible the pink bacteria were a result of decolorizing the bacteria for too long with ethyl alcohol during the staining process, or the pink bodies were contaminant material and not cells at all which happened to take on the Saffranin stain by chance.