BCM 221 Practical Biochemistry

1. Pharos University in Alexandria Faculty of Dentistry BCM 221 Practical Biochemistry

2. About the course … Course Grades Grading Schedule Laboratory Procedures Biosafety Measures Material Downloading Colorimetry

3. About the course … BiochemistryII is an extension of the last year Biochemistry course, The practical part of the course is clinically oriented towards the laboratory diagnosis of pathological conditions mostly encountered in patient healthcare.

4. Course Grades The course is allotted 2 Credit hrs (200 marks). Midterm Exam = 40 marks Practical =60 End term =100

5. Practical Grades Total mark “60” divided as follows: 20marks:practical work 10marks:(presentation) 10 marks:Theoretical background 20marks:finalpracticalexam

6. Laboratory Policies

7. Attendance Attendance in the lab is obligatory. You are allowed to enter the teaching room within the first 10 min of the lab session. If you miss a lab session, you will NOT be allowed to repeat the missed lab experiment under any circumstances.

8. Downloads you are required to download the week’s experiment and bring the hard copy to the lab, and no excuses will be accepted if you come to the lab without your own copy. The download procedure is similar to that of the earlier semesters.

10. Downloads (cont.) After downloading experimental protocols, you are required to study the lab exercise before coming to the lab to avoid any confusion. At the end of the lab session, a report with completed lab work will be submitted to the lab instructor.

11. Material Downloading Experimental protocols will be available on the website regularly. You are required to study the lab exercise before coming to the lab to avoid any confusion. At the end of the lab session, a report with completed lab work will be submitted to the lab instructor.

12. Laboratory Procedures For determination of glucose, uric acid, cholesterol, triglycerides, urea, creatinine and bilirubin, laboratory kits depending on colorimetric techniques will be used.

13. Laboratory Procedures For fluid sampling, automatic pipettes with disposable tips will be used. Automatic pipettes are precision instruments that are used to deliver small volumes accurately. They must be handled with respect and extra care.

14. Laboratory Procedures A colorimeter will be finally used for measurement of absorption values of the colored reaction products.

15. Biosafety Measures In the biomedical lab, there are many hazards. Most accidents can be prevented by following the rules, being prepared for the lab, and by thinking about what you are doing. Always behave with consideration for others’ safety. Always remember you are handling biological fluids. Be very careful concerning the glass ware because there was a great loss last term and if any student broke anything it will be deducted from his money and if it was repeated there will be )investigation )تحقيق اداري)

16. Cleanup You should cleanup your work area after the lab experiment and return it back the way you found it. Both detergent & disinfectant solutions are available in the lab in spray bottles, and every student should bring his own soft tissue paper. The lab instructor will check your work area before you leave the lab.

17. Waste disposal There are 3 types of containers in the lab: • Green (for general waste) • Violet (for sharp objects) • Red (for biohazard material)

18. Safety measures & rules Always wear your lab coat before entering the lab. Use goggles & gloves if necessary as directed by your lab instructor.

19. Safety measures & rules Behave in a respectable and responsible way allover the lab. Only take your notebook and pen to the lab area; bags, coats, …etc should be kept in your locker. Mobile phones are allowed in condition to be kept on a silent mode.

20. Safety measures & rules Tie up long hair to keep it away from flame.

21. Safety measures & rules Do not touch anything from the lab equipment unless told to do so by the lab instructor.  Assume all chemicals in the lab are dangerous. Do not taste any solution or chemical.

22. Safety measures & rules Do not pipette any solution by mouth. Do not carry reagents around the lab. No food or drink is allowed in the lab.

23. Safety measures & rules Note the position of the safety showers in the lab. Wash your hands thoroughly before leaving the lab.

24. Biosafety Measures Disposable tips, test tubes and gloves are disposed of in the biohazard waste. Clean up your lab bench after you terminate working and wash your hands with detergent and disinfectant before you leave the lab.

25. Colorimetry

26. Coloremetric Estimation of Body Fluids Principle: Many important biological solutions are estimated as coloured solutions, in which the intense of the colour is proportional to the concentration of the biological substance. When light passes in a coloured solution, it has specific wave length, therefore, the intense of the colour is measured as its optical density or absorbance of its wave length.

27. Coloremetric Estimation of Body Fluids Colorimeter measures the absorbance or optical activity of the coloured solutions, and the intense of colours are read on its scale. The zero point of the colorimeter should be first adjusted with a solution known as the blank. A water blank or a reagent blank may be used. Sometimes the zero point is adjusted against air, this depends upon the procedure used.

28. Coloremetric Estimation of Body Fluids The sample unknown solution is compared with a known standard to obtain the exact concentration of the test unknown sample. The following formula is usually used: A sample Conc.sample _________ * Conc.Standard=……mg/dl A standard

29. Coloremetric Estimation of Body Fluids A sample: Absorbance (optical density, reading) of the sample. A standard: Absorbance (optical density, reading) of the standard. Conc.sample: Concentration of the test unknown sample. Conc. Standard: Concentration of the known standard solution.

30. Colorimeter A colorimeter is a device to measure the intensity of light after it has passed through an absorbing medium. In its simplest form it consists of:

31. Components of Colorimeter • Light Source: • A low level of illumination (usually from 6-12 V lamp) is used. • 2. Condenser lenses: They give parallel rays. • 3. Filter: • A filter consists of a material that selectively transmits the desired wave length and absorbs all other wavelengths. Its colour is complement to that of coloured solution to allow maximum absorption and transmission.

32. Components of Colorimeter 4. Cuvette: Cuvettes are optically transparent cells used to contain the “coloured” solution under study. In general, the length of light path across the cuvette is usually kept constant at 1 cm. Scratched or contaminated cuvettes result in inaccurate work.

33. Components of Colorimeter 5. Photocell: A photocell measures the intensity of the light transmitted through the solution after absorption. It converts quanta of radiation to electricalenergy which may be amplified, detected and recorded. Thus, the photocell has the function of generating an electric current proportional to the light falling on it from the coloured solution according to the intense of its colour.

34. Components of Colorimeter 6. Galvanometer: It contains the scale of reading. The output (electrical current) of the photo cell can now be read by a sensitive meter system. The reading is proportional to the intensity of the electric current emitted from the photocell and consequently to the intensity of colour of the solution of the unknown test sample under investigation.

35. Application • Colorimetry helps in measuring the concentration of any unknown substance. If the substance by itself is coloureless, it can be chemically converted to a coloured substance. • The colorimeter can also be used to measure the turbidity of solutions (e.g. bacterial suspension), but the nepheometer is a better measure of turbidity.

36. Thank You