1. Identification of Spermatozoa From Challenged Samples Chelsea Ellis and Reena Roy, Ph.D ABSTRACT This research involved the detection of human spermatozoa from challenged samples using fluorescent staining with Sperm HY-LITER TM. The fluorescently stained human male cells were detected using fluorescence microscopy without the aid of computer software. Extracted spermatozoa were stained with reagents contained in SPERM HY- LITER™ kit developed by Independent Forensics. The spermatozoa deposited on various substrates, subjected to various conditions were identified using a Leica DM2500 P fitted with a Prior Lumen 200 Fluorescence Illumination System. MATERIALS AND METHODS RESULTS CONCLUSION Negative controls showed no transfer of spermatozoa in this experiment (controls in the same bag) Fabrics washed with hot water and detergent yielded less spermatozoa than fabrics washed with cold water Spermatozoa binds more tightly to cotton fabric than black leather This method can accurately and efficiently detect spermatozoa without a fluorescent microscope or an expensive computer software The results obtained from this research indicates that a manual method of detection of fluorescently stained spermatozoa can benefit forensic biology laboratories. A Leica DM2500 P microscope was used for the detection of the samples on the slides. The fluorescently labeled spermatozoa were identified with a Prior Lumen 200 Fluorescence Illumination System attached to the Leica DM2500 P. The human spermatozoa were searched for manually without the aid of any computer software or microscope stage. Normal human semen was diluted with water and deposited on fabrics such as leather, denim, fleece, polyester, and polyester-cotton blend. Together, the fabrics deposited with semen and fabrics without semen (negative control) were subjected to two different types of wash cycles (hot or cold) and with and without Tide ® detergent. The fabrics were dried at room temperature overnight and then extracted in 20 ul of water for 30 minutes. The extracted semen was deposited on a 25x75 mm, 1.0 mm thick, pre-cleaned VWR Scientific Micro slides and stained with SPERM HY-LITER™ reagents. SubstrateBody FluidTime Left at RTWash TimeExtraction TimeExtracted InResults Denim1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterMany Observed Denim1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterMany Observed Denim1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterMany Observed Denim1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterMany Observed White Polyester1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterMany Observed White Polyester1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterMany Observed White Polyester1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterMany Observed White Polyester1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterMany Observed Black Leather1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterFew Observed Black Leather1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterFew Observed Black Leather1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterFew Observed Black Leather1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterSeveral Observed Black Fleece1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterSeveral Observed Black Fleece1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterSeveral Observed Black Fleece1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterSeveral Observed Black Fleece1µL Human SemenOvernightCold Wash Cycle30 Minutes20µL WaterSeveral Observed SubstrateBody FluidTime Left at RTWash TimeExtraction TimeExtracted InResults Denim5µL Human SemenOvernightHot Wash Cycle30 Minutes20µL WaterA few Observed Denim5µL Human SemenOvernight Hot Wash Cycle+ Detergent 30 Minutes20µL WaterA few Observed Denim10µL Human Semen5 daysHot Wash Cycle30 Minutes50µL WaterMany Observed Denim10µL Human Semen5 days Hot Wash Cycle+ Detergent 30 Minutes50µL WaterA few Observed Cold Water Without Detergent Hot Water With and Without Detergent Sperm HY-LITER TM Black Leather Sperm HY-LITER TM Cotton Sperm HY- LITER TM Black Fleece Sperm HY-LITER TM White Polyester Sperm HY-LITER TM Cotton KPIC Human Spermatozoa KPIC Bull Spermatozoa KPIC Human Spermatozoa