EXPERIMENT No.

AIM: To estimate the blood glucose level by Orthotoluidine method

Principle: Glucose reacts with o-toluidine in glacial acetic acid on heating to yield a blue-green
N-glycosylamine derivative. The intensity of this colour is proportional to the concentration of
glucose present.

Procedure: Take three test tube mark it T (Test), S (Standard) , B(Blank).

Reagents Blank Standard Test

Sample - - 0.1ml

Standard - 0.1ml -

Distilled water 0.1ml - -

O- Toluidine reagent 5ml 5ml 5ml

Mix the contents of each test tube and keep in a boiling water bath for 10 minutes.

Cool and take the OD on colorimeter reading at 625 nm.

OBSERVATION:

 OD of blank -
 OD of standard -

 OD of test –

Calculation:

Concentration of glucose in mg/100 ml blood.

= ODT - ODB / ODS - OD B * 100mg%

Result: The given sample contains …….mg/dl of blood sugar.

Interpretation:

In normal healthy persons

Fasting plasma glucose is 70-110 mg/dl

Post parandial glucose is less than 140 mg/dl

When blood glucose concentration is within the normal range, it is referred to as
normoglycemia.

When values are higher than the normal range, it is known as hyperglycemia.

When the values are lower than the normal limits, it is called as hypoglycemia.

FBS > 140 mg/dl Indicative of diabetes mellitus

PPBS > 200mg/dl

Hyperglycemia is observed in

 Diabetes mellitus
 Hyper function of the pituitary and adrenal glands (acromegaly, Cushing syndrome,
pheochromocytoma, etc)
 Emotional states like fear, anger and anxiety lead to elevation in blood sugar levels
because of secretion of epinephrine which has a hyperglycemic action
 Meningitis, encephalitis, shock
 Severe hemorrhage
 Nephrotic syndrome, brain tumour and sepsis

Hypoglycemia is seen in

 Insulin overdose in patients of diabetes

 Pancreatic tumours affecting beta cells
 Severe liver disease
 Von Gierke’s disease
 Functional or reactive hypoglycemia
VARIOUS METHODS OF ESTIMATION OF BLOOD GLUCOSE

NON ENZYMATIC METHOD ENZYMATIC METHOD

1. Nelson – Somogyi method Hexokinase method

2. Folin Wu method Glucose oxidase – peroxidase

method

3. Asatoor king method Glucose dehydrogenase method

4. O – toluidine method

5. Benedict’s/ Fehlings test