Abstract – The Purpose of this exercise is to gain experience in bacterial morphologies in prepared wet-mounted slides and interpreting the findings of bacteria through direct and indirect staining technique.
Hypothesis – The experiment will allow for further insight into stained organisms, allowing extended contrast, differentiating shape and structure through utilization of microscope.
Procedure – Instructions followed as per procedure, specimens viewed through v-scope, using 10x, 20x, and 40x stained wet-mount. Observations compared amongst one another from cd-rom.
Analysis – Using the virtual microscope, the images seemed to be more clear and concise. Using the light microscope again for another experiment has increased my confidence in its utilization. Although the procedure was different, the experiment itself was easy to follow. The only challenging aspect of this lab was getting the supplies in order to do the experiment. Fortunately, there were no wait times necessary to view the images, and observation became readily accessible.
Conclusions – The experiment was interesting as the first portion consisted of a virtual microscope, it allowed for a different magnification view and I was able to view these as often as necessary. The other three exercise were not difficult to do, however it was getting the supplies in order for the experiments that I found much difficulty in.
A. List three reasons why you might choose to stain a particular slide rather than view it as a wet-mount. Gram stains provide a more intricate view of shape
Gram stains provide better views of structure.
Gram stains show changes in bacteria.
B. Define the following terms:
Chromophore: It is part of the molecule that makes color when light is passed through certain wavelengths Acidic dye: They are soluble in water possessing an affinity for amphoteric fibers, lacking cellulose fibers. Basic dye: A cationic stain and reacts with material that is negatively charged, which is synthetic and soluble in water.
C. What is the difference between direct and indirect staining? The difference between staining is the dye that is used, direct staining uses basic dye and indirect staining uses acidic dye
D. What is heat fixing?
When you take the slide with the bacterial smear and place it over a fire to help it adhere to the slide.
E. Why is it necessary to ensure that your specimens are completely air dried prior to heat fixing? If you do not make sure that the specimen is dry before heat fixation, it will essentially boil the specimen instead of increasing its adherence to the slide.
F. Describe what you observed in your plaque smear wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences? With the wet mount the bacteria was in a more aqueous state looked more transparent. With the specimen being directly stained, I was able to make out borders of the bacteria and the shapes were more easily identifiable. With indirect staining, I was able to make out the inside of the bacteria more clearly being that it seemed clearer, due to the contrast.
G. Describe what you observed in your cheek smear wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences? With the cheek smear on a wet mount, it looked more aqueous than in its original state. With the different magnifications, I was able to make out the structure and the shape. With direct and indirect staining, I did see a difference with the cheek smear, they looked almost like opposites, with the background changes. The cells borders were more clearly defined with direct stain and with increased magnification.
H. Describe what you observed in your yeast wet-mount, direct stained slide, and indirectly stained slide. What were the similarities? What were the differences? In the yeast, the shape and structure were easy to identify in all the different techniques. I found many differences in the procedure. I was able to clearly see the bacteria and their cell wall with increased magnification.
I. Were the cell types the same in all three specimen sets: yeast, plaque, and cheek? How were they similar? How were they different? The cell types had some similarities but they differed in size shape and structure. The aqueous state of the specimens is something that I must say that stood out the most for my experiments. According to microbehunter.com specimens that are used in a wet mount should be blotted with a piece of paper towel if the specimen seems to aqueous. Something I did not think to try because of disrupting the specimen in its authentic state, in future experiments I will incorporate this technique with any other wet mount procedures.