Efficacy of slide fixative methods for examining sperm
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The identification of semen is very important in forensic cases, especially in sexual assault cases. Microscopic examination for spermatozoa is the only confirmatory test for the presence of semen since sperm is unique to semen and because other testing methods for components of semen are still not specific enough to truly be confirmatory. To prepare slides for microscopic examination, the sample is centrifuged to pull the sperm to the bottom of the sample tube to make a pellet. The supernatant is removed and some of the pellet is placed onto a microscopic slide. The slide is then fixed and commonly stained with Kernechtrot Picroindigocarmine Stain (KPIC) prior to examination. The first stain used in the KPIC method is nuclear fast red which is a metallic dye that stains nuclear material, including sperm heads, red. After the nuclear fast red is rinsed off using distilled water, a second stain, picroindigocarmine, is used. Picroindigocarmine is a saturated picric acid and indigocarmine solution that will stain sperm tails and the cytoplasm of epithelial cells a light green color. This second stain is then rinsed off using ethanol. The objective of this research was to determine how much sperm is lost, if any, during the slide preparation process and if one fixative method was better than others at preventing the loss of sperm during the staining process. Three traditional heat fixative methods (hot plate, flame from a Bunsen burner, and oven) were evaluated and five chemical fixatives (100% ethanol, 100% methanol, Carnoy’s solution, 2.5% glutaraldehyde, and 4% paraformaldehyde) as well as without the use of any fixative were also evaluated. This was done by collecting both the distilled water and ethanol washes from the staining process in separate centrifuge tubes and preparing a slide from those to identify any sperm that may have been dislodged from the slide during the staining process. Since there was no sperm loss was detected in any of the fixative methods, all are suitable for use in identifying semen. Due to cost, time, and ease of use, heat fixatives may be preferable. Since the different temperatures of the heat fixatives did not appear to have an effect on sperm loss, the hot plate would be the easiest and quickest method to use. Additionally, hot plates don’t require a gas line into the lab. However, the results also demonstrated how difficult it is to count the same number of sperm on multiple slides made from the same semen dilution. For all slides, 5 µL of a 1:2500 dilution of semen was used, however, the number of sperm observed on the slides varied widely for all fixative methods. Multiple samples from three different tubes of the same dilution of semen were quantified to see if this variability was also observed in DNA quantification. The results showed that even though there was some variation in quantities between samples, the results were not statistically significant. One avenue of future research would be to evaluate other steps of slide preparation that may decrease sperm yield.