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Abstract This experiment addressed the following central question Is it possible to distinguish different blood cells from other blood components using different staining procedures? Identical blood smears were stained separately using Wright-Giemsa and Periodic Acid-Schiff Staining methods illustrated in Dr. Jessen-Marshall's lab handout. An experimental slide underwent an alternative staining method. By following explicit instructions, smears produced dark-staining blood components for which they were explicitly intended. The results of this lab answered the central question affirmatively as blood components were easily identified under a microscope.
Introduction In this lab, some simple staining procedures will be learned along with the viewing of the stained cells. Also, the stains will be observed and identification techniques will be used to recognize different blood cell types. Is it possible for different types of blood cells to be distinguished from each other using different staining procedures?
If the blood cell smear were left in diastase for 15 minutes as opposed to 0 during the Periodic Acid-Schiff stain, then the cells would not stain as well, and therefore, be less visible under the microscope. The blood smear will not be as visible in this instance because the carbohydrates would not absorb as much of the stain.
Procedure First, the field of view of each magnification was measured and drawn to use for later reference. A stage micrometer was used to measure the 4x, 10x and the 40x objectives. These measurements will be used to estimate sizes of blood cells later in the lab.
Blood was drawn from Ben Liston's finger and drops were placed on slides. The drops were smeared thinly across the slides using the procedure illustrated in Dr. Jessen-Marshall's lab handout (Page 6, Lab Handout, Jessen-Marshall, 00). Smears were allowed time to dry before the staining processes began.
One slide was stained using the Wright-Giemsa method in order to identify blood cell. First the slide was dipped in the Wright-Giemsa for 10 seconds, a phosphate buffer for 0 seconds, rinsed in distilled water and permitted to dry.
Two slides were stained according to Periodic Acid-Schiff Staining method. Before the staining could occur the slides were fixed for ten minutes with absolute ethanol-formulin to improve stain quality. These slides were then rinsed under running tapwater for 10 minutes and then doused with distilled water. One slide was placed aside for examination later. Two blood smears were then placed in a 1% solution of diastase; one was placed in the jar for 0 minutes according to process outlined in the lab manual. The second slide, the experimental, was only placed in the solution for 15 minutes to observe the results if it had not been properly treated with diastase.
After the slides were placed in diastase, they were moved to the periodic acid solution for ten minutes. The slides were rinsed with distilled water, transported to Schiff's reagent and incubated for ten minutes in the fume hood. Slides were washing under running water and were counterstained with Harris Hematoxylin for three minutes. Afterwards the slides were washed in running water, dipped in ammonia water, rinsed with distilled water and allowed to dry.
Results
Staining the slides was successful because specific cell types were visible under a microscope when the correct staining procedure was followed. Under the 400x objective, of the Wright-Giemsa stain, Neutrophils and Basophils were identified as they were stained a darker hue (Figure A). The blood smear that underwent the PAS staining with the diastase treatment was found to contain Monocytes, which were also darkly stained (Figure B). The blood smear that underwent PAS staining without diastase treatment contained Platelets, which appeared black (Figure C).
The experimental blood smear slide that was left in the diastase treatment for only 15 minutes proved to be ineffectively stained for any visible blood component. This result supports the hypothesis that leaving a slide in diastase for an inadequate amount of time would result a failure to stain.
Discussion The central question was "Is it possible for different types of blood cells to be distinguished from each other using different staining procedures?" The hypothesis that was formulated was that if one altered the staining times by decreasing them, the stains would not take to the blood smears. The conclusion that was drawn from the experiments and careful observation was the blood smears, when left in diastase for 15 minutes less than the correct amount of time, failed to stain for any blood components. The data collected from the experimental slide supported this fact because there was no specific staining that made any blood component stand out. This blood smear appeared all pink colored with no cell definition.
In this experiment, multiple slides should be put through the procedures to rule out any random error. Also, more accurate timekeeping devices, such as a handheld stopwatch, could have been used as opposed to a wall clock with a minute hand. The results, other than the experimental, were pretty conclusive in that cell components took the stain and were easily identified. This procedure, when used in a laboratory setting, could be used to determine if an individual has a high count of any of the stainable blood components. In the future, this experiment could be repeated using a different experimental design in order to better understand the complex staining procedures.
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