SERUM

Principle : Molar solution contain gm molecular wt of a substance in 1L of solution.
Amount of Solution : 72ml of 25% glucose solution.Requirement :
a. chemical : Glucose, Distilled water.
b. Apparatus : Digital balance, Beaker, Stirrer, Funnel, Volumetric.

Procedure :
1.72ml of 25% glucose solution is measured by measuring cylinder and poured into the volumetric flask. With the help of funnel. Then the solution diluted with distilled water up to the marked of 100ml.
2.Then the volumetric flask is closed with cork & greatly shaking. Thus 100ml of 1(M) glucose solution is prepared by supplied solution.
Precaution :
1. weight should be taken properly.
2.Lower end of the meniscus of water should be taken into count.
3. Distilled water should be used

Principle :
Cholesterol is hydrolyzed to free cholesterol and fatty acid by cholesterol esterase. The cholesterol is oxidized in to cholesterol -3-one and H202 in the reaction catalyzed by cholesterol oxidase. The peroxidase catalyzes the reaction between H202 and 4-aminophenaz one to produce red quinoneimine whose intensity of color is proportional to the total cholesterol present in the sample.
Cholesterol ester cholesterol esterase => cholesterol + Fatty acid
Cholesterol cholesterol oxidize => cholesterol + H202
H202 + phenol + 4-amino pherazone peroxidase quinoneimine + H20

Requirements :
   a. Equipment’s :
1. Test tube
   2. Cuvette
3. Centrifuge machine
4. Pipette
5. Micro pipette
6. Photoelectric colorimeter with filler
7. Inculcator
8. Test tube rack
b. Enzyme regent :
1. Phosphate butter
2. Cholesterol esterase
3. Cholesterol oxidase
4. Phenol
5. Peroxidase
6. 4-aminophenezon
7. Mutarotase
   c. Standard regent :
   cholesterol solution (200 mg/clt)
Specimen :serum
Procedure : arranged three cuvettes and marked them as regent blank (RB), Standard (sto), Sample (S), Pipetting sheme.
Pipette into cuvette Sample Standard Regent blank
Sample 10 ml - -
Standard 10 ml    -
Regent blank 1000 ml 1000 ml
  These are mixed and incubated for 10 minutes at 20-25’C temperature and then measure the absorbance of the sample against regent blank with one hour.
Assay :
1. Wave length : 530 mm (500-546 mm)
2. Optical pathway : 1 cm
3. Temperature : 20-25’C4.
Measurement : Against reagent blank
Result :
Name of the subject : sajid anwar
Age : 28
Sex : Male
Serum cholesterol level : 120 mg/dl or 3.125 mmol/L
Normal serum cholesterol level : 150 to 220 mg/L or 3.9-5.72 mmol/L
Comment : The serum cholesterol level of the subject is.

Principle :albumin bound bilirubin is released by a detergent. The total bilirubin reacts with diazotized 2.4-diclour alinin to form red azody.
Regent Preparation : Mixed DCA with NIT in a rato of 1:1 before use and alow it to stand for at list 15 miniuts at room temparature protected from light. Blank is ready for use.
Specimen : No Hemolytic EDTA or heperinized plasma stored in dark until use.

Apparatus :Test tube, cuvette,autopipette, Centrifage machine, Photoelectric colorimeter with filter,Incubator, test tube rack,
Pi petting Scheme :
                                          Normal assay    Paediatric
   sample s.blk sample s.blk

Specimen   100 ml 100 ml 20 ml 20 ml
Working regent 1000 ml - 1000 ml -
Blank - 1000 ml - 1000 ml
Mixed and allowed to stand for all least 10 minutes at 20 to 25'C projected from light. measure absorbency of sample against the sample blank with in 60 minutes.

preparation of regent blank : Mixed 100 ml distilled water with 1000 ml working regent.
Assay :
Wave length : 530
mm optical pathway : 1 cm
Temperature : 20-25'C
Measurement       : Against regent blank.
Normal assay :
Bilirubin concentration,
C=A absorption of sample x 12.5 mg/dl
C=A absorption of sample x 214 limol/L
Calculation :
A absorbance of sample = Absorbance of sample
=absorbance of blank
= 0.07-0.05
= 0.02Bilirubin concentration c = 0.02 x 12.5 mg'dl
= 0.25 mg/dl
Paediatric assay :
C= A absorpance of sample x 58 mg/dl
C= A absorpance of sample x 992 mg/L
Result :
Name of the subject    : Sajid
age       : 19
sex    : Male
Serum bilirubin level : 0.25 mg/dl
Normal serum bilirubin level : 2-1 mg/dl
Comment : The serum bilirubin concentration level of the subject is with in normal physiological limit.
Precation :
  a. Blood should be dream with aseptic precation.
b. powring of blood from syringe to cuvette,middle must be avoided.
c. incubation time must be maintained.

Principle : In alkaline solution creatinine form an areange & red color complex with picric acid.
The absorbance of teir complex is proportional to the creatinine concentration it in the sample.
creation's + picric acid = creatinine picrate complex.
apparatus recurred :
    1. test tube
    2. Pipette
    3. Micropipette
    4. photo electric colorimeter with filter.
    5 Test tube rack.
Regent Recquired :
    1. Picric acid
    2. NAOH
    3. Standard (2mg/dl)
    4. Deproteinization solution

Speciemen : Serum , distilled water.
working regent : Mixed picric acid with NAOH in the ratio of 1:1
Calculation :
conc of creatinine    = A absorbonce of sample      x standerd
                                 A absorbonce of sample
                              = 0.03 x 2
           0.07 x
        = .45 mg/dl
Regent preparation :
popette incuvate    Regent Blank    Standard    Sample
serum       -   -          1ml
standerd       -      1ml      -
diatilled water       1ml          -          -
deproteinzation       -      -                -
solution      1ml         1ml      1ml
Mixed carefully & centrifaged the sample at high speed for 5-10 minutes.

pipette in cuvate   Regent        standerd    sample
centrifaged sample   -          -          1ml
Dilute standerd       -                 1ml        -
dilute ragent blank    1ml          -       -
working reagent        1ml       1ml       1ml
Mixed & in cubet for exactly 20 minutes at 25'C temparature than heat & absorbance of standard & sample regent blank.
Assay :
web length    - 546 nm
optic pathway    - 1 cm
Temparature        - 25'C
Mewsurement against in regent
Result :
Name of the subject    : Flora
Age              : 19
Sex          : Female
serum creatinine level   : .85 mg/dl
Normal serum creatinine level : 17 to 19 ml mol/L or 1.1 mg/dl
Comment :the blood creatinine level of the subject is with in normal level.
Precaution :
1. Blood should be drawn with aseptic preacaution.
2. Centrifugation should be completed property.
3. during incubation appropriate temparature should be maintain.
4. appropriate filter should be used.

PREPARATION OF 100ml 1(M) SOLUTOIN OF GLUCOSE FROM 25% GLUCOSE SOLUTION.

ESTIMATION OF SERUM CHOLESTEROL BY ENZYMETIC COLORIMETRIC METHOD.

ESTIMATION OF SERUM BILIRUBIN BY PHOTOELECTRIC COLORIMETER.

ESTIMATION OF SERUM CREATININE BY JAFFE REACTION.