forensic seminal analysis.pptx.forensics

seminal stain analysis

1. “DETECTION OF SEMEN STAIN & SEMINAL FLUID”

2. Table of content

3. INTRODUCTION :

4. STRUCTURE OF HUMNA SPERM A mature sperm (also called spermatozoa) consists of the following four parts – Head Neck Middle piece Tail

5. collection Use a clean and sterile plastic container to transfer liquid semen in that container. Label the container with the date, time, location, and name of the collector. Keep the specimens refrigerated and submit to the laboratory as soon as possible. I collected my human semen sample in the clean and sterile plastic container from KGMU, Lucknow and label the container with the date, time, location and name of collector. Keep the samples refrigerated as soon as possible.

6. methods During my work, I have used three methods for detection of seminal stains- Physical method Chemical method Microscopic method

7. Physical Method Include visual examination to naked eye seminal stains generally appear translucent or opaque spots at the times with yellowish tint and darker border depending on the color and thickness of substrata .

8. Chemical method Fluorescence Test - Equipment, Materials- U.V. Torch Stained cloth piece Procedure- U.V. light (250-365nm wavelength) is generally useful for locating seminal stains on larger material such as cloth carpet and bed covers. I used U.V. torch for detecting seminal stain on the cloth piece. Throw the U.V. light on stained cloth piece. Result- Seminal stain gives a pale bluish fluorescent.

9. Acid Phosphatase Test (Walker Test) Procedure- Place a small cloth piece of seminal stain on What man filter paper. Add 1-2 drop of Step 1 Reagent( Buffer, Sodium Alpha- Naphthyl Phosphate) and allow to react for 30 sec.(No color should develop at this stage). Add 1 drop of Step 2 Reagent( Buffer, Brantamine Fast Blue-B salt) .Record the result after 10 sec. Results – A positive reaction is recorded upon rapid development of a purple color, which indicative of semen. This is not a confirmatory test for semen.  

10. Florence crystal test for choline Procedure- Measure the potassium iodide, iodine and put into the porcelain crucibles. Mix the potassium iodide, iodine and distilled water into the beaker. Prepare an extract of seminal stain in distilled water. Extracted drop is placed on a glass slide and allowed dry at the room temperature. Place a cover slip over it and allow a drop of Florence solution to run under cover slip. Observe microscopically at 100X magnification. Results – If semen is present dark brown crystals of choline iodide appear immediately

11. Microscopic Examination Christmas Tree Test - Procedure- Cut the stained cloth into small pieces. Dip the cloth pieces overnight into water. Scrap the stain and transfer the material on the slide. Dry the smear slide on the room temp. Add few drop of alum sol. on the slide and leave for 45min. Wash the slide with tap water. Add few drop of eosin sol. and leave overnight. Wash the slide with tap water. Add few drop of methylene blue and wash the slide immediately. Dry the slide on the room temp. Add 1 drop of cedar wood oil on the slide. Keep the slide in microscope for microscopic view.(100*0.25 magnification). Results – Purple or pink colour spermatozoa show in microscopic view.This is a confirmatory test for semen detection.    

12. The Effect of Fabric Type and Laundering on the Detection of Semen Stains The effect of fabric type and different laundering conditions on the ability to detect semen stains on washed fabrics. Three potential factors affecting semen identification on laundered clothing: fabric type, water temperature during washing, and whether the stain was dry at the time of washing. Following laundering, semen stains on four fabric types (cotton, polyester, denim, and wool) were examined and tested with three common methods used to detect semen; screening with an alternate light source, acid phosphatase press test, and histological staining of spermatozoa. It was determined that semen was difficult to detect if it was still wet when the semen-stained article was washed.

13. Sample Preparation – 150μL of semen was deposited onto 1 of 4 different fabric types; Cotton Polyester Denim Wool. This was replicated to create 4 groups based on the washing conditions; Hot water with dried stains Hot water with wet stain Cold water with dried stains Cold water with wet stains.

14. Cotton Polyester Denim Wool Hot/Dry n=1 n=1 n=1 n=1 Hot/Wet n=1 n=1 n=1 n=1 Cold/Dry n=1 n=1 n=1 n=1 Cold/Wet n=1 n=1 n=1 n=1 Unwashed n=1 n=1 n=1 n=1 All samples were washed in the hot standard cycle (~60°C) or cold standard cycle (~30°C). Wet stains were washed within 30 minutes of the semen being deposited. No detergent was used during any of the washing cycles, and samples were air-dried after washing.

15. Examination of Samples Alternate Light Source Examinations   All of the samples were examined with U.V. Torch at a 455 nm wavelength setting. The fluorescence of the stains was recorded as strong, moderate, weak, or undetected.   Acid Phosphatase Testing   All of the samples were tested using the acid phosphatase test method as mentioned earlier. A positive reaction was recorded if a purple color reaction occurred within two minutes, and the time of the initial color change was noted. If no color reaction occurred within two minutes, the sample was deemed negative. Christmas Tree Test-   All of the samples were tested using the Christmas Tree Test as mentioned earlier.The slides were observed under and scored based on the number of spermatozoa present

16. Sperm Density Score No Sperm Visible Negative Sperm Hard to Find 1+ Some Sperm in Some Fields, Easy to Find 2+ Many or Some Sperm in Most Fields 3+ Many Sperm in Every Field 4+

17. RESULTS Alternate Light Source Examination- Cotton Polyester Denim Wool Hot/Dry Weak Negative Negative Negative Hot/Wet Negative Negative Negative Negative Cold/Dry Weak Negative Negative Weak Cold/Wet Negative Negative Negative Negative Unwashed Strong Negative Weak Moderate

18. Acid Phosphatase Test- Results of AP test. “+” denotes positive, “-“denotes negative. Cotton Polyester Denim Wool Hot/Dry + + + + Hot/Wet + _ _ _ Cold/Dry + + + + Cold/Wet + _ _ _ Unwashed + + + +

19. Unwashed acid phosphatase test Cold water dry acid phosphatase test. Cold water wet acid phosphatase test Hot water dry acid phosphatase test

20. RESULT AND DISCUSSION: IAA production P solubilization Drought stress tolerance bacteria The strain C6.3 has shown the maximum production of indole acetic acid followed by C5.2 The strain C6.3 has shown the maximum P solubilization activity followed by R6.1

21. Temperature stress tolerance bacteria IAA Production Siderophore production: The only drought stress tolerance bacterial strain which able to siderophore production is C8.2. The bacterial strain which has shown the maximum production of IAA is PL6 followed by PL3

22. Siderophore production P solubilization The bacterial strain which has shown the maximum P solubilization is PL16 The bacterial strain which has shown the maximum siderophore production is PL10

23. Colony PCR The temperature stress tolerance bacterial strains showing the bands with ladder L PL9 PL3 PL11 PL10 PL27 PL25 PL20 PL14

24. CONCLUSION: Plant growth promoting rhizobacteria (PGPR) are soil beneficial bacteria with a wide range of activity in the soil affecting plant growth and the environment. The production of secondary metabolites, i.e. plant growth substances , changes root morphology resulting in greater root surface area for the uptake of nutrients, siderophores production , antagonism to soil-borne root pathogens, phosphate solubilization , and di-nitrogen fixation . Drought stress affects the growth, dry mater and harvestable yield in a number of plant species, but the tolerance of any species to this menace varies remarkably. The drought stress and temperature stress tolerance bacteria both are able to produce IAA, P solubilization and siderophore production at a different level of manner.

25. REFERENCES: 1. Anandham , R., Gandhi, P.I., Madhaiyan, M. and Sa, T., 2008. Potential plant growth promoting traits and bioacidulation of rock phosphate by thiosulfate oxidizing bacteria isolated from crop plants.  Journal of basic microbiology ,  48 (6), pp.439-447 2. Conway Morris, A., Gadsby, N., McKenna, J.P., Hellyer, T.P., Dark, P., Singh, S., Walsh, T.S., McAuley, D.F., Templeton, K. and Simpson, A.J., 2016. 16S pan-bacterial PCR can accurately identify patients with ventilator-associated pneumonia.  Thorax . 3. Datta , C. and Basu, P.S., 2000. Indole acetic acid production by a Rhizobium species from root nodules of a leguminous shrub, Cajanus cajan.  Microbiological research ,  155 (2), pp.123-127. 4. Gill , S.S. and Tuteja, N., 2010. Polyamines and abiotic stress tolerance in plants.  Plant signaling & behavior ,  5 (1), pp.26-33 . 5. Khan , M.S., Zaidi, A., Ahemad, M., Oves, M. and Wani, P.A., 2010. Plant growth promotion by phosphate solubilizing fungi–current perspective.  Archives of Agronomy and Soil Science ,  56 (1), pp.73-98 . 6. Lynch, J.M. and Bragg, E., 1985. Microorganisms and soil aggregate stability. In  Advances in soil science  (pp. 133-171). Springer, New York, NY.