laboratory medical

1. ALLAHUMMA RABBI ZIDNI ILMAA
WELCOME
to
BIOCHEMISTRY LABORATORY

2. Under discussion
• Self introduction
• Lab-introduction,(GENERAL SAFETY RULES)
• Pipetting
• Calorimetry/spectrophotometry

3. A. General Safety Rules
1. Listen to or read instructions carefully
before attempting to do anything.
2. Wear safety goggles to protect your eyes
from chemicals, heated materials, or
things that might be able to shatter.
3. Notify your teacher if any spills or
accidents occur.

4. A. General Safety Rules
4. After handling chemicals, always wash
your hands with soap and water.
5. During lab work, keep your hands away
from your face.
6. Tie back long hair.

5. A. General Safety Rules
7. Roll up loose sleeves.
8. Know the location of the fire
extinguisher, fire blanket, eyewash
station, and first aid kit.
9. Keep your work area uncluttered. Take to
the lab station only what is necessary.

6. A. General Safety Rules
10. It is suggested that you wear
glasses rather than contact lenses.
11. Never put anything into your
mouth during a lab experiment.
12. Clean up your lab area at the
conclusion of the laboratory period.
13. Never “horse around” or play
practical jokes in the laboratory.

7. B. Glassware Safety
1. Chipped or cracked glassware should
not be used. Show it to the teacher.
2. Broken glassware should not be
disposed of in a classroom trashcan.
There is a special glass disposal container
for it.
3. When pouring liquids into glassware,
make sure the container you are pouring
into is resting on a table at least a
handsbreadth from the edge.

8. B. Glassware Safety
4. Pour down a glass stirring rod to prevent
liquids from spilling.
5. If a piece of glassware gets broken, do
not try to clean it up by yourself. Notify
the teacher.
6.When inserting glass tubing into a rubber
stopper, apply a lubricant to the glass
and use a twisting motion.

9. C. Chemical Safety
1.Wear protective goggles and a lab apron
whenever heating or pouring hazardous
chemicals.
2. Never mix chemicals together unless you
are told to do so (and then only in the
manner specified).
3. Never taste any chemicals (you should
never taste anything in the lab).

10. C. Chemical Safety
6. Follow the instructions of your teacher
when disposing of all chemicals.
7. Wash your hands after handling
hazardous chemicals.

11. D. Electrical Safety
1. Lay electrical cords where no one
can trip on them or get caught in
them.
2. Be sure your hands and your lab
area are dry before using electrical
equipment.
3. Never poke anything into electrical
outlets.

12. D. Electrical Safety
4. Unplug cords by pulling the plug and not
the cord.
5. Unplug all electrical equipment at the end
of the lab period.

13. E. Heating Safety
4. The only type of glassware that may safely
be heated is either Kimax or Pyrex.
5. Always point the top ends of test tubes
that are being heated away from people.
6. When heating a test tube, move it around
slowly over the flame to distribute the heat
evenly.

14. E. Heating Safety
7. Only glassware that is thoroughly dry
should be heated.
8. Heat glassware by placing it on a wire
gauze platform on a ringstand. Do not hold
it in your hand.

15. E. Heating Safety
9. When lighting a burner, wait until a match
is struck or the striker is in place before
you turn on the gas.
10. The amount of air can be adjusted by the
air supply valve below the tube of the
burner. This regulates the flame
temperature and color.
11. Never leave a burner or hotplate
unattended.

16. First Aid
Injury: Burns
To do: Immediately flush with cold water
until burning sensation is lessened.

17. First Aid
Injury: Cuts, bruises
To do: Do not touch an open wound without
safety gloves. Pressing directly on minor
cuts will stop bleeding in a few minutes.
Apply cold compress to bruises to reduce
swelling.

18. First Aid
Injury: The eyes
To do: Flush eyes immediately with plenty
of water for several minutes. If a foreign
object is lodged in the eye, do not allow
the eye to be rubbed.

19. First Aid
Injury: Poisoning
To do: Find out what substance was
responsible for the poisoning and alert the
teacher immediately.

20. First Aid
Injury: Electrical shock
To do: Shut off the current at the source.
Remove wire with rubber gloves. Alert the
teacher immediately.

21. Micropipetting

22. What is a Micropipette
• A pipette designed for the measurement of very
small volumes.
• The unit we are using is Microliters. μL
• A Microliter is 1/1000 of a Mililiter
• Laboratory science often involves working with
very small volumes of liquid; frequently millionths
of liters are used. One millionth of a liter is equal
to one Microliter, abbreviated 1 µL.

23. Glass pipettes are not highly
accurate for volumes less than 1
milliliter (1 ml), but the
automatic pipettes are both
accurate and precise

24. VOLUME ADJUSTMENT
KNOB
PIPETTE 3 VOLUMES

25. Step 1: (Continued) Read the Volume
How to Read the Volume Indicator:
(a): P-20 Model
6.86 m l = 0.00686
or 6.86 x 10-3 ml
(c): P-1000 Model
262 m l= 0.262
or 2.62 x 10-1 ml
(b): P-200 Model
132.4 m l = 0.1324
or 1.324 x 10-1 ml

26. Example of tip sizes:
Attaching the
disposable tip
Operating the Micropipette
Step 2: Attach the Disposable Tip

27. Operating the Micropipette
Step 3: Depress the
Plunger to the First
Stop
Step 5: Draw up the
sample
Step 6: Pause
To aspirate the sample into the tip,
allow the pushbutton to return slowly
and smoothly to the fully extended UP
POSITION. NEVER LET THE
PLUNGER SNAP UP! This draws the
exact calibrated volume into the tip if
the tip remains below the liquid
surface during withdrawal.
Wait a few seconds to ensure that the full volume of sample is
drawn into the plastic tip. WAIT LONGER FOR LARGER
VOLUMES. WAIT LONGER FOR MORE VISCOUS ("SYRUP-
LIKE") SUBSTANCES.
Step 4: Immerse Tip in Sample

28. Operating the Micropipette
Step 7: Withdraw the Tip
Remove the tip from the sample liquid. No liquid should remain on the OUTSIDE of the tip.
Wipe away any droplets on the outside of the tip with a lint-free tissue, such as KIMWIPES,
but only wipe droplets from the side of the tip. NEVER TOUCH THE TIP OPENING or you
may absorb part of your sample.
Proper Droplet Removal WRONG Droplet Removal

29. Operating the Micropipette
Step 8: Dispense the Sample
To dispense the sample from the pipette:
a) Touch the tip end to the side wall of the receiving vessel and
b) Depress the plunger to the FIRST STOP.
c) Pause for at least one second-- 1-2 seconds for P-1000, 2-3 seconds for P-5000,
or longer for viscous liquids.
d) Press the plunger to the SECOND STOP (the second point, of greater resistance,
at the bottom of the stroke) to expel any residual liquid in the tip (like "blowing
out" a glass pipette).
(a) Start
Dispensing
(b) 1st Stop =
Dispense
(c) 2nd Stop =
Expel

30. Operating the Micropipette
Step 9: Release the Plunger
Gently allow the plunger to return to the UP position.
DO NOT allow it to SPRING BACK!
Step 10: Discard the Tip
Discard the tip by depressing the tip
ejector button, as shown below. A fresh tip
should be used for each sample to prevent
sample carryover.
Press ejector button to discard tip.

31. WATER BATH/CONSTANT
TEMPERATURE BATH
• Used to carry out various chemical
reactions at specific temperature.
• It is controlled by thermostatic arrangement.
• Components---- 1.nickleplated tank(20-30lt)
. 2.strip heater
3.thermostat ,controlled
knob.
4.thermometer

32. applications
USE
• Dterm of serum enzymes
• Enzymatic determination of
glucose, urea , cholesterol,
TAG.
• Serological determinations
• Saponification
TEMPERATURE
37°
37°
56°
60°---70°

33. SPECTROPHOTOMETRY
Definition : The measurement of the intensity
of light at selected wave lengths
Light  [Solution]  Transmission
Light Lens Slit Monochromator
Sample Detector Quantitative Analysis
Slits

34. Applications
• Protein
• Amino Acids (aromatic)
• Pantothenic Acid
• Glucose Determination
• Enzyme Activity (Hexokinase)

35. Visible Spectrometer Application
• Niacin
• Pyridoxine
• Vitamin B12
• Metal Determination (Fe)
• Fat-quality Determination (TBA)
• Enzyme Activity (glucose oxidase)

36. spectrophotometry
Lamp Monochromator
Cuvette
Detector

37. Beer’s Law
Optical density of solution is directly
proportional to the concentration of the
solution
A = abc = kc
A = absorbance
a = absorptivity
b = light path length
c = concentration
k = constant (k-value)

38. Lambert’s Law
• Optical density of a colored solution is
directly proportional to the path of light
(diameter of the cuvette)

39. Components
• Light source: tungsten
• Monochromator: diffraction grating
• Cuvette
• Photodetector: PMT, photocell
• Readout

40. LIGHT SOURCES
• UV Spectrophotometer
• 1. Hydrogen Gas Lamp
• 2. Mercury Lamp
• Visible Spectrophotometer
• 1. Tungsten Lamp
• IR Spectrophotometer
• 1. Carborundum (SIC)

41. CELLS/ CUVETTES
• UV Spectrophotometer
• Quartz (crystalline silica)
• Visible Spectrophotometer
• Glass
• IR Spectrophotometer
• NaCl

42. Absorption Measurement
• Colorimeter with filter
• Spectrophotometer
• Prism
• Diffraction grating

43. Color Wavelength (nm)
Ultraviolet 400 and under
Violet 400 - 450
Blue 450 - 500
Green 500 - 570
Yellow 570 - 590
Orange 590 - 620
Red 620 - 650
Infrared 750 & over
VISIBLE REGION WAVELENGTH

44. Spectrum of Radiation

46. Spectrophotometry
1. Turn instrument on
2. Select correct wavelength 
3. Block light, set Zero (no light = infinity
absorption = 0% T)
4. Choose and clean cuvette
5. Open light, insert Blank (maximum light =
no absorption = 100% T)
6. Measure absorption of Standards, Controls
and Patient samples to 3rd decimal place

47. SPECTROPHOTOMETR
READINGS
SPECTROPHOTOMETR READINGS

48. Standards
• Precisely prepared = known concentration
• Usually pure solution of single compound
• Plot absorbance vs concentration: standard
curve

49. NEXT LAB
ON
24th SEPTEMBER
ESTIMATION OF SERUM
TOTAL PR0TIENS

50. THANK YOU