EXPERIMENT NO.

11

GLUCOSE DETERMINATION

ENZYMATIC- COLORIMETRIC GLUCOSE OXIDASE-PAP METHOD (TRINDER REACTION)

 SERUM SAMPLE Preparation (ONE PER GROUP)

• Materials: Red Top, Adapter, Tourniquet, Wet & Dry Cotton, 12 x 75mm, parafilm.
1. Obtain blood sample via ETS (Red top)
2. Centrifuge 2500 RPM (#7) (10 Mins)
3. Transfer serum to 12 x 75 mm.
4. Label serum tube: Group #
5. Place a parafilm after use. (To be used for next experiment)

 TEST PROPER:
1. Label 13 x 100 mm test tubes: blank, standard, control, test (patient).
2. Pipette 1.0 ml working glucose reagent to all tubes and place in a 37 C heating bath (@ Spectro) for at least 5 minutes.
3. Add 0.01 mL (10 uL) of sample to respective tubes, mix and incubate at 37 C (@ Spectro) for exactly 10 minutes.
4. After incubation, zero the spectrophotometer with the reagent blank at 500 nm.
5. *Read and record the absorbance of all tubes. USE SI UNIT (mmol/L) TO REPORT FINAL RESULTS.

Set-up:

1 ml glucose
reagent
10 uL Distilled Water
10 uL Standard
Test (patient)

10 uL Control
Standard

Control
Blank

10 uL Serum
Serum Sample

Test tube rack

*How to use spectrophotometer:

1. Clear (2x)
2. Select Filter: (Press Key # according to nearest Wavelength needed)
1. 340nm
2. 405nm
3. 450nm
4. 505nm
*520 nm: Clear (2x) -> 1 -> 5 Enter -> 4 Enter
5. 545nm
6. 600nm
3. Enter
4. No
5. Enter
6. Insert BLANK -> STD -> SAMPLE
Note: Always wipe the bottom of the tube every time it is inserted in the well of the spectrophotometer.
 EXPERIMENT NO. 13
HDL- CHOLESTEROL DETERMINATION
Procedure:
A. HDL- cholesterol separation:

Sample
Control

Sample supernatant
Control supernatant
1. Label 13 x 100 mm test tubes: Control, Sample 1, etc.
2. Dispense 0.2 mL (200uL) sample to each labeled tube.
3. Add 0.2 mL (200uL) HDL-cholesterol reagent.
4. Mix well and then allow to stand at ambient temperature (room temp) for 10 minutes.
5. Centrifuge at 1000 x g for 10 minutes.
6. Separate the supernatant into separate test tubes accordingly labeled.

200 uL HDL-Chole reagent

200 uL Control
200 uL Serum
B. HDL-Cholesterol assay:
1. Label test tubes accordingly as Reagent blank, Standard, Control, sample 1, etc.
2. Dispense 1.0 mL Cholesterol (enzyme) reagent.
3. Prewarm for 5 minutes at 37 C.
4. Place 0.1 mL (100uL) of supernatant from each respective sample into each labeled tube.
5. Incubate all tubes at 37 C for 10 minutes.
6. Zero the spectrophotometer at 520 nm using reagent blank.
7. Read and record absorbance readings of the standard (use the HDL-Cholesterol standard), control, sample1, etc.

Set-up:

1 ml Chole reagent
100 uL Distilled Water
10 uL HDL-Chol Standard
10 uL Control Supernatant
10 uL Sample Supernatant
Standard

Control

Sample
Blank

Test tube rack

 EXPERIMENT NO. 12
TOTAL CHOLESTEROL DETERMINATION
ENZYMATIC COLORIMETRIC – CHOLESTEROL OXIDASE-PAP METHOD (TRINDER REACTION)

Procedure:
1. Label 13 x 100 mm test tubes: “Blank”, “Standard”, “Control”, “Test”, etc.
2. Pipette 1.0 mL Cholesterol reagent into each tube and prewarm at 37 C for at least 5 minutes.
3. Add 0.01 mL (10uL) of sample to respective tubes.
4. Cover each tube with nescofilm/ parafilm (not necessary). Mix by gentle swirling.
5. Incubate the tubes to the 37 C water bath (spectro) for 5 minutes.
6. Zero the spectrophotometer with Blank at 500 nm
7. Read and record absorbance of all tubes.
 EXPERIMENT NO. 14
TRIGLYCERIDES DETERMINATION
Procedure:
1. Label test tubes as “reagent blank”, “standard/ calibrator”, “control”, “patient sample 1”, etc.
2. Pipette 1.0 mL of reagent into appropriate tubes and prewarm at 37 C for 5 min.
3. Add 0.010 mL (10uL) of the appropriate sample to their respective tubes. Swirl gently to mix.
4. Incubate all tubes for 5 minutes at 37 C.
5. Zero the spectrophotometer with reagent blank, at 500 nm. (500- 520 nm is acceptable).
6. Read and record absorbance of all tubes. The final color is stable for 60 minutes.
 EXPERIMENT NO. 15
BLOOD UREA NITROGEN (BUN) DETERMINATION
(KINETIC: UREASE- GLUTAMATE DEHYDROGENASE REACTION)

Procedure:
1. Reconstitute reagent with 12 mL of triple distilled water.
2. Zero the spectrophotometer with water and set at 340 nm wavelength.
3. Pipette 1.0 mL of BUN reagent and preheat at 37 C for 3 min.
4. To one cuvette at a time add 0.01 mL (10 uL) of sample / standard.
5. After 30 seconds measure and record the absorbance (A1).
6. After an additional 60 seconds take a second absorbance (A2).
7. Determine the delta change in absorbance between two reading (A1-A2).
 EXPERIMENT NO. 16
CREATININE DETERMINATION
(Colorimetric Method- JAFFE Reaction)
Working Reagent Preparation:
Mix well 1-part Picric acid reagent to 1 part base reagent. The working reagent is stable for 8 hours at room
temperature.
Procedure:
1. Dispense 1.5 mL of working reagent into tubes labeled reagent blank, Calibrator, Control, Patient’s Sample,
2. Add 0.05 mL sample into each appropriate tube and mix well. Use distilled water for reagent blank.
3. Incubate at 37 C for 15 minutes.
4. Zero instrument at 510 nm using the Reagent blank.
5. Read and record absorbance readings.
 EXPERIMENT NO. 17 URIC ACID DETERMINATION
(COLORIMETRIC: URICASE – TRINDER REACTION)
Procedure:
1. Label test tubes, “Reagent blank”, “Standard”, “Controls”, “Unknown”, etc.
2. Pipette 1.0 mL of working reagent into all tubes.
3. Prewarm all tubes at 37 C for 3 minutes.
4. Add 0.025 mL (25 uL) of sample to respective tubes and mix.
5. Incubate all tubes at 37 C for 10 minutes. 99
6. Read the samples against Reagent blank at 520 nm. (Wavelength range 500- 520 nm).
7. Record the readings.
 EXPERIMENT NO. 18
TOTAL PROTEIN DETERMINATION
(COLORIMETRIC METHOD- BIURET REACTION)
 EXPERIMENT NO. 19
SERUM ALBUMIN DETERMINATION
(COLORIMETRIC METHOD – BROMCRESOL GREEN DYE BINDING REACTION)
LABORATORY PRACTICES:
#1 READ YOUR PROCEDURES BEFORE COMING TO CLASS.
#1 READ YOUR PROCEDURES BEFORE COMING TO CLASS.
#1 READ YOUR PROCEDURES BEFORE COMING TO CLASS.
#1 READ YOUR PROCEDURES BEFORE COMING TO CLASS.
#1 READ YOUR PROCEDURES BEFORE COMING TO CLASS.
#1 READ YOUR PROCEDURES BEFORE COMING TO CLASS.

ASSIGN MEMBERS: PER EXPERIMENT

• TWO PERFORMERS
• TWO for OUTPUT (TO CALCULATE)
• OTHERS- WASH AND CLEAN MATERIALS.

THERE SHOULD BE AN OUPUT! EVERY EXPERIMENT A GROUP WITHOUT VALUES READ IS NOT
GRADED FOR THE EXPERIMENT. (GOAL: PERFORMER SHOULD BE FAST AND ACCURATE)

ONE SIGNED OUTPUT PAPER SHOULD ONLY BE THE ONE SUBMITTED EVERY AFTER EXPERIMENT
AVOID ANY ERASURES.

ALL LAB. MATERIALS ARE STATIONED AND SHOULD NOT BE MOVED.

IF NOT, A PERFORMER PLEASE STAY SEATED AT YOUR AREA.

ALL BAGS SHOULD NOT BE PLACE ON YOUR COUNTER TOPS.
STAY SEATED! ANYONE OUT OF POST WOULD HAVE A GROUP DEDUCTION
STAY QUIET! ANY UNNECESSARY QUESTIONS AND NOISE CALLS FOR A QUIZ.