Glucose test
Ms. Ibtisam alaswad
taim
Ms. Nour A.
Introduction
Blood glucose :_
• The main sugar that the body makes from the food
in the diet. Glucose is carried through the
bloodstream to provide energy to all cells in the
body. Cells cannot use glucose without the help of
insulin.
• Glucose is a simple sugar (a monosaccharide). The
body produces it from protein, fat and, in largest
part, carbohydrate. Ingested glucose is absorbed
directly into the blood from the intestine and
results in a rapid increase in blood glucose.
Glucose is also known as dextrose.
The Objectives to be achieved
• To understand the principle to spectrophotometer
A spectrophotometer is used to measure the amount
of light a sample (be it liquid or solid) absorbs.
Light is passed through the sample and a detector
measures the intensity of that light that passes
through the sample and hits that detector.
• To determine the blood glucose
concentration as an application of
spectrophotometer use
• To know how to use some devices in the
lab
The principle of Glucose test
Glucose present in the plasma is oxidized by the enzyme
glucose oxidase( GOD) to gluconic acid with the liberation
of hydrogen peroxide, which is converted to water and
oxygen by the enzyme peroxidase( POD.)
4_aminophenazone ,an oxygen acceptor, takes up the
oxygen and together with phenol forms a pink coloured
chromogen which can be measured at 515nm.
Specimen type, collection and
storage
• Plasma is the specimen of choice for glucose
estimation. Plasma glucose levels have been
checked to be quite stable for 6 hours at room
temperature (25 -350C) in the author’s laboratory. It
is important that plasma should be separated from
the cells soon after collection, preferably within 1
hour.
• About 2 ml of the patient’s blood should be
collected by venipuncture into a tube containing a
mixture of potassium ethylene diaminetetraacetate
(EDTA) sodium fluoride at a ratio 1:2 (W/W). Five mg
of the mixture is adequate for 2 ml of blood. The
tube should be gently but thoroughly shaken for
complete mixing.
Procedure
Blank
Standard
Standard
__
Sample
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__
Control
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Reagen
1.0 ml
10µl
1.0 ml
Sample
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10µl
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1.0 ml
Control
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1.0µl
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Procedure
• Mix. Incubate 10 min. at 37ºc. or 30 min at
room temperature. Measure the
ABSORBANCE at 505nm (490-550)
against the blank.
• The color is stable 30min
Discussion
• Calculation
Glucose (mg/dl) = (Abs. sample/Abs. standard)* standard
conc.
• Linearity
This method is linear up to 500mg/dl.
If the glucose concentration is greater than 500mg/dl. , dilute
the sample 1:2 with saline solution and repeat the
determination and multiply the result by 2
• Reference value
Serum: 55-110mg/dl. Plasma glucose: Fasting: 70 –110 mg/dl
How to Use a Micropipettor
• The micropipettor is used to transfer small amounts (< 1
ml) of liquids. The scales on micropipettors are in
microliters (1000μl = 1 ml).
How to Use a Micropipettor
1.
Never exceed the upper or lower limits of these
pipettors.
2. Set the desired clockwise to increase volume by turning the
centrally located rings volume or counterclockwise to
decrease volume.
3. Place a tip on the discharge end of the pipettor. NOTE: If
sterile conditions are necessary do
not allow the pipet tip to touch any object (including your
hands).
How to Use a Micropipettor
4. The plunger will stop at two different positions when it is
depressed each point indicate the volume to be taken.
5. Depress the plunger until you feel the initial resistance and
insert tip into the solution, just barely below the surface of
the liquid and not as deep as possible.
How to Use a Micropipettor
6 .Carefully and slowly release plunger. NOTE: If the
solution you are pipetting is viscous, allow the pipet tip to
fill to final volume before removing it from solution to
avoid the presence of bubbles in the plastic tip which will
result in an inaccurate volume.
7. Discharge the solution into the appropriate container by
depressing plunger. This time, depress the plunger to the
point of initial resistance, wait one second, and then
continue pressing the plunger as far as it will go in order to
discharge the entire volume of solution.
8.Remove tip by pressing down on the tip discarder.
NOTES
􀂾 Never point a pipettor up. This may cause liquid to run
down into the pipettor destroying it.
􀂾 When withdrawing liquids with the pipettor, always release
the plunger slowly. This prevents liquid from rushing into
the end of the pipette and clogging it up. This is especially
important with large volume pipettors (200-1000 μl(.
􀂾 Be sure you use the proper size tip for each pipettor.
􀂾 Always use a new tip for each different liquid.
􀂾 Use the correct pipettor for the volume that is to be
dispensed. Never use the 200-1000 μl pipette to dispense
volumes below 200 μl. going below or above the range of
the micropipettor may damage the instrument.