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Aim: Estimation Of Reducing Sugars By Cole's Method

INTRODUCTION:
Sugars having free aldehyde group or ketone groups are known as simple or
reducing sugars. They are highly reactive in reducing oxidizing system like-Cuso4
(copper sulfate) is converted into Cu2O (Cuprous oxide) during such a reaction, the
sugar itself gets oxidized. Examples of reducing sugar are glucose, fructose, maltose,
lactose etc.
Sugars not having free aldehyde or ketone groups are known as non-reducing
sugars. They fail to participate in oxidizing reducing systems. Examples of non-reducing
sugar are sucrose, starch, glycogen, dextran. However, if such sugars are hydrolysed,
(when heated under acidic condition) the product behaves as a reducing sugars e.g. When
sucrose is hydrolysed, breaks down into glucose and fructose, so, after hydrolysis, the
previously strongly dextrorotatory solution becomes levorotatory. This is due to fact that
the fructose molecules are more strongly levorotatory than the glucose molecule id
dextrorotatory. This phenomenon is called "inversion" and the mixture of glucose and
fructose obtained is called "invert sugar".
Sugars present in natural samples (like sugarcane juice and its product jaggery)
are glucose, fructose, sucrose, xylose, mannitol, starch, pectin and cellulose. Among
these sugars sucrose is largely present. Fructose is the sweetest, being excelled only by
glucose and sucrose.

Relative sweetness of some of the sugars, considering sucrose as 100

SUGAR RELATIVE SWEETNESS

Galactose 32
Maltose 32
Glucose 74
Sucrose 100
Invert sugar 130
Fructose 173
Saccharin 45,000

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Total sugar present in sugarcane juice is 110-120 mg/ml out of which sucrose is 95-
105mg/ml and reducing sugar is 8-10mg/ml. In jaggery, sugar concentration is variable.
It depends on quality of sugarcane used for jaggery production and concentration of
sugarcane syrup.

METHODS FOR ESTIMATION OF SUGAR

Sugar can be measured by three types of methods: oxidation-reduction, condensation,
and enzymatic measurement. The chemistry of these methods is briefly described as
follows:
1. Oxidation - reduction Methods: Enediol form of a sugar is highly reactive & easily
oxidized. Upon oxidation, carbon chain is ruptured with the formation of acids of
shorter chain length. Folin-Wu, Nelson-Somogyi, Shaffer-Hartmann-Somogyi,
Neocuproinecolor reaction, alkaline ferrocyanide method are based on the reducing
property of glucose.
2. Condensation Methods: Glucose (and other aldoses) can undergo condensation
with variety o aromatic compounds (phenols and primary aromatic amines) in hot
acid solution to yield colored products. Hydroxymethylfurfural is formed from
glucose in hot strongly acidic solutions. The aldehyde group of this product
condenses with phenols (resorcinol, thymol, anthrone, 𝛼-napthol, m-aminophenol,
phloroglucinol) and aromatic aines (o-toludine, m-aminophenol) to yield colored
compound that can be quantitated spectrophotometrically.
3. Enzymatic Methods: Enzymes lie glucose oxidae, hexokinase, acylphosphate:D-
glucose-6-phosphotransferase, and glucose dehydrogenase are used for estimation of
glucose. The hexokinase and glucose oxidase enzymatic methods have been used for
routine determination of glucose in the body fluids.

COLE’S METHOD (Oxidation-Reduction method)

Principle:
Sugar having free –CHO or –CO groups, when heated in alaline solution, their aldehyde
or ketone group is converted to form enediol. This has more reducing power and reduces
K3Fe(CN6)-3 to K4Fe(CN6)-4 ad sugars are oxidized to complex mixture of acids.

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The amount of ferricyanide reduced depends upon the concentration of sugar in the
given sample. Ferricyanide ions (yellow solution) are reduced to ferrocyanide ions
(colorless solution).
Potassium fericyanide solution is treated with alkali and boiled. The mixture is titrated
with the sugar solution. When the yellow color has nearly disappeared a drop of
methyleneblue is added. The titration is continued until the color is discharged. Both
methyelene blue and ferricyanide are reduced by sugar in hot alkaline solution, but the
indicstor is not affected until the whole of the ferricynidehs been reduced. The end point
is reached when the solution is decolorized.

Glucose + Fe(CN6)-3OH- (boiling) Fe(CN6)-4 + Sugar acids

(yellow) (colorless)

Requirements:
1. 1% w/v aq. Potassium ferricyanide solution (to be stored in dark bottles).
2. 2.5 N NaOH solution, 1% Methylene blue (aqueous).
3. 1 ml & 10 ml graduated pipettes, 100 ml Erlenmeyer flask, glass beads/small
porcelains pieces.

Procedure:
A. Approximate Titration (pilot Reading)
1. Measure 20 ml of ferricyanide solution and 5ml of sodium hydroxide solution in
100 ml flask.
2. Add 4-5 glass beads or small porcelain piece in the flask and place it on wire gauze
over a flame. (Glass beads provide nuclei for the bubbles of the vapour, and thus
ensure sturdy, gentle boiling. If glass beads are omitted, the liquid may become
superheated and then suddenly boil with great violence and bumping).
3. Heating should be arranged in such a way that the mixture begins to boil within 2
minutes. As soon as the mixture boils, the flame can be lowered to avoid excessive
concentration. Active boiling should be maintained during the whole of the
titration.
4. Fill the 1 ml graduated pipette with the sugar solution, and add few drops of sugar
solution in the boiling mixture at intervals till the color of the ferricyanide changes

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from dark yellow to 7disperse and continue the titration until the entire ferricyanide
solution becomes colorless.
5. Note the amount of sugar solution consumed for titration. This is known as pilot
reading.
6. It is advisable to have 2-3 pilot reading as the accuracy of estimation depends on it.

B. Dilution of Sample
7. If less than 9 ml of sugar is required, it is advisable to dilute a small amount with
water, so that the final volume required is between 9 and 11 ml (i.e., dilution is
done up to 10 ml).
8. Prepare sufficient dilution enough for at least 3 readings.

C. Second Titration
9. Start afresh, and titrate the alkaline ferricyanide solution using the diluted sugar
solution in exactly the similar way as to that the approximate estimation.
10. Note the amount of sugar solution consumed for titration. This is known as final
reading.
11. Final readings should be between 9 and 11 ml. Take the average of final readings
and calculate according to following formula.

CALCULATION OF RESULTS
Formulas used for calculation of various sugars are as follows:
Glucose: 20.12 + (0.035x X) mg/X ml
Lactose: 23.60 + (0.1xX) mg/X ml
Maltose: 26.80 + (0.06xX) mg/X ml
Sucrose: 19.20 + (0.065xX) mg/Xml (after inversion with boiling acids and
neutralization).
Where X = average amount of sugar solution consumed during the final reading.

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Aim: To perform Vital Staining of Saccharomyces Cerevisiae.

INTRODUCTION:
Vitality refers to the activity or the metabolism of the cell. In case of yeast, vitality has been
linked to its fermentative performance. Numerous staining techniques are available for
visualization, differentiation and separation of organisms in terms of morphological
characteristics and cellular structures. Organisms may frequently be stained without first
killing them, this type of staining is known as intravital staining.

Principle:
Methylene blue readily permeates yeast cells, but it is enzymatically reduced to a
colorless compound in living cells. Dead cells will appear dark blue when stained with
methylene blue while live cells will be colorless.

Requirements:
1. GYE broth
2. Methylene blue solution: prepare 1% methylene blue solution and dilute it50
times.
3. Sterile Suspension Vials
4. Sterile Distilled Water
5. Clean Grease free glass slide and cover slip.

Procedure:
1. Inoculate 100 ml sterile GYE broth with Baker’s yeast aseptically and incubate
the flask overnight at 37⁰ C.
2. Centrifuge the content at 10,000 rpm for 10 minutes and resuspend the pellet in
sterile distill water.
3. Take equal amount of cell suspension and methylene blue in a suspension vial
and mix properly. Take one drop of suspension from this vial on grease free glass
slide and place clean cover slip on it carefully.
4. Observe under 40X lens to observe the vitality of given culture.

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