A PROJECT ON

INDUSTRIEAL BIO TECHNOLOGY

KBT354 Mini Project or Internship Assessment
Submitted
by
KM ANSHIKA VERMA
Roll No-1903610540038
In
Department of Biotechnology
(2020-21)
Under the supervision
Of
Institute of Transgene life Sciences

Submitted
to
Dheerendra Kumar
Head,
Department of Biotechnology

R. R. INSTITUTE OF MODERN TECHNOLOGY

NH-24, Bakshi Ka Talab, Sitapur Road Lucknow
(U.P.) India
Website : www.rrimt.ac.in.
Email: [email protected], [email protected]

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 ACKNOWLEDGEMENT

This mini project is a culmination of task undertaken by us during the course at Institute of
Transgene life Sciences Lucknow. Acknowledgement is not a mere formality or ritual but a
genuine opportunity to express the indebitness to all those without whose active support and
encouragement this project wouldn’t have been possible. One of the most pleasing aspects in
collecting the necessary information, it is the opportunity to thank those who have actively
contributed to it.

I am grateful To Mr. Dheerendra Kumar & other faculty members, Department of

Biotechnology, RRIMT, Lucknow whose valuable guidance and support constant
encouragement at every stage of work.

I would like to express my thanks to Mr. Dheerendra Kumar for his guidance and
cooperation rendered for allowing me to undergo training under his guidance. Diction is not
enough to express my gratitude to my Parents who have molded my career through enormous
sacrifice throughout the years.

Name: Km Anshika Verma

Branch: Biotechnology (2nd year)

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 DECLARATION

I Km Anshika Verma, hereby declare that the mini Project Report on "Industrial Bio
technology” is done by me under the guidance of, Training institute supervisor name-Kavita
Yadav from 07 July 2020 to 06 Aug. 2020 at Institute of Transgene life Sciences,
Lucknow is submitted in partial fulfillment of the requirements for the award of the degree in
B.Tech in Biotechnology.

DATE:

PLACE: Lucknow

Signature of the candidate

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 CONTENT
1. Lab Rules and Regulations
2. Bio-instrumentaion
3. Solutions Preparation
4. Food Safety Management System
5. Quality Management
6. Food Product Analysis
a. Cereal and Cereal Product (wheat, bread, gram flour)
1. Determination of foreign matter
2. Determination of moisture content of wheat flour
3. Determination of alcoholic acidity wheat flour
4. Determination of gluten content wheat flour
5. Determination of moisture content of bread
6. Determination of alcoholic acidity of bread
7. Detection of kesari dall(besan) in gram flour
b. Milk Analysis
1. Detection of adulterants present in milk
2. Detection of preservatives present in milk
3. Turbidity test for milk
4. Sensory evaluation of milk
c. Water Analysis
1. Determination of alkalinity of water
2. Determination of acidity of water
3. Detection of Ethylene di-amine tetraacetion acid (EDTA) in water
4. Determination of total water hardness
5. Determination of disinfectant present in water
d. Microbial Analysis
1. Isolation of microorganism from (soil, water and milk)
a. Media preparation
b. Sample collection
c. Serial dilution
d. Spread plate and pour plate
2. To obtain pure culture of bacteria by steak plate method
3. Staining and biochemical activities of purified culture
a. Gram staining
b. Catalase test
c. Amylase test
d. Citrate test
7. Result and Discussion
8. Reference

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 CHAPTER 1

LAB RULES REGULATIONS

1. To wear an apron before you enter a laboratory for protecting the clothes that you wear.

2. Before starting work, clean the working table with disinfectants like 95% alcohol or dilute
detergents like savlon or lyzol.

3. The table should have a notebook and only glass-wares and equipment that are needed.

4. Do not smoke, drink or eat in the laboratory.

5. In case of injury, either apply an anti-bacterial cream or inform your instructor.

6. When culture of an organism is spilt cover the area, treat it with ethyl alcohol (after putting
off the Bunsen flame) or any other disinfectant for some time and then only clean the area.

7. Don’t work with open hair (ladies).

8. Turn off the Bunsen flame when it is not in use.

9. All microbial cultures should be handled with care.

10. Keep used liquid cultures, supernatants and glass-wares in autoclavable containers.

11. Discard contaminated plates and plastic containers in autoclavable bags.

12. Discard organic solvents (phenol and chloroform) in waste containers.

13. Do not pipette out broth cultures, concentrated acids and alkalis by mouth.

14. All culture tubes must be kept in up-right position in baskets or stands.

15. Labeling of petriplates, tubes, flasks etc. should be done before starting an experiment.

16. Materials like chemicals, stains, reagent bottles, unused glass-wares etc. must be replaced
in their original place.

17. Tools like scalpel , forceps, inoculation needles etc. that come in contact with cultures or
agar medium (sterile) should be sterilized by making the portion that goes into tube or petri-
plate, red hot on Bunsen flame, cool in 95% alcohol and flame heat it by passing over the
flame to burn off alcohol.

18. Clean microscope lens before and after use.

19. Keep the doors and windows closed while inoculating or during transfer of sterile
cultures.

20. Toxic chemicals should be handled with precaution and while discarding used ones they
should be discarded in labeled containers.

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21. Broken blades, sharp instruments and broken glass pieces should be disposed in separate
containers.

22. First aid kits must be placed in each laboratory.

23. Portable fire extinguishers and a fire blanket should be kept ready.

24. Use specific toxic and mutagenic chemicals under fume hood.

25. Do not expose yourself to UV rays.

26. Wear safety glass, (UV) gloves, masks, hot gloves and rubber aprons while handling
concentrated acids and bases.

27. Clean the table and inoculation chamber, wash your hands and then only leave the
laboratory.

28. Prepare a flowchart prior to each experiment.

The Lab Notebook & Lab Report

 Documentation in a lab notebook is an essential skill for any biotechnician.

 Documentation is done for one or all of the following reasons.
- to record what an individual has done and observed.
- to establish ownership for patent purposes and other legal use.
- to establish criteria used to evaluate a finished product or the process to make it.
- to prove that a procedure was done correctly .
- to adhere to, evaluate and develop standard operating procedures (SOP).

Most common hazardous chemicals biotechnology lab

 Carcinogens – formaldehyde
 Mutagens – ethidium bromide
 Neurotoxins – acrylamide
 Teratogens – formamide
 Nephrotoxins – acetonitrile
 Hepatotoxins – chloroform
 Corrosives – phenol, strong acids & bases

Safe Handling of Chemicals

Flammables :- Do not heat these reagents unnecessarily, and never in the presence of a flame
or source of a spark. In general, only open containers in fume hoods.

Corrosives :- Wear personal protective equipment (PPE) such as lab coats, goggles and
gloves, and always add strong acids or bases to water when making solution. Neutralize
slowly to avoid rapid generation of heat and gases.

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Reactive chemicals :- Wear PPE such as lab coats, goggles and gloves, and know the
reactive properties of the chemical. Always store oxidizing chemicals away from flammable
materials.

Toxic chemicals :- Wear PPE such as lab coats, goggles and gloves, and know the toxic
properties of the chemical. When working with a dry powder, wear a mask to avoid breathing
the dust.

Waste Disposal

 Most districts have specific policies for waste disposal.

 Most hazardous waste must be collected and disposed of by professionals.
 Need biohazard bags for biological hazards = plates (no sharp items),fill only ½ full.
 Autoclave bio-contaminated items 15-20 min at 15-20 psi before trash.
 Bio-contaminated loops and tubes can be soaked in 10% bleach for 30 min before
regular trash.
 Many chemicals may not go down drain, i.e. ,CuSO4, silver nitrate, EtBr, etc.
 Label waste with type/concentration/date.

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 CHAPTER 2

BIO-INSTRUMENTATION

Micro Centrifuge :- A laboratory centrifuge is a piece of laboratory equipment, driven by

motor, which spins liquid samples at high speed increasing the effective gravitational force
will more rapidly and completely cause the precipitate to gather on the tube as a “pellet”. The
remaining solution is called the “supernatant”. The rate of centrifugation is specified by the
acceleration applied to the sample, typically measured in revolutions per minute(rpm) or
relative centrifuge force (RCF). The particle settling velocity in centrifugation is a function
of their size and shape, centrifugal acceleration, the volume fraction of solid present the
density difference between the particles and liquid and viscosity. The use of centrifuge is
known as centrifugation.

Analytical Balance :- An analytical balance is used to measure mass to a very high degree of
precision and accuracy. The measuring pan pan of high precision(0.1mg or better) analytical
balance are inside a transparent enclosure with doors so that does not collect and so any air
currents in the room do not affect the balance operation. The use of vented balance safety
enclosure, which has uniquely designed acrylic airfoils, allow a smooth turbulence free air
flow that prevents balance fluctuation and measure of mass down to 1μl without fluctuations
or loss of product.

Gel Electrophoresis :- Gel electrophoresis is a technique used for the separation of DNA,
RNA or protein molecules using an electric field applied to a gel matrix. DNA gel
electrophoresis is usually performed for analytical purposes, often after amplification of DNA
via PCR, but may be used as a preparative technique prior to use of other methods such as
mass spectrometry, RELP, PCR, cloning, DNA sequencing or southern blotting for further
characterization. The term gel in this instance refers to the matrix used to contain, and then
separate the target molecules. In the most cases, gel is a polymer whose composition and
porosity is chosen based on the specific weight and comption of the target to be analyzed
when separating proteins or small nucleic (DNA, RNA or oligonucleotides). The gel is
usually composed of different concentrations of acrylamide and a cross linker producing

8
different sized mess networks of polyacrylamide. When separating larger nucleic acids, the
preferred matrix is purified agarose. In both cases, the gel froms solid, yet porous matrix.
Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using
appropriate safety precautions to avoid poisoning. Agarose is composed of long un branched
chains of unchanged carbohydrate without cross link resulting in a gel with large pores
allowing for the separation of macromolecules and macromolecular complexes.

pH Meter :- A pH meter is an electronic instrument used to measure the pH of a liquid. A

typical pH meter is consist of a special measuring probe connected to an electronic meter that
measures and displays the pH reading. For very precise work the pH meter should be
calibrate before each measurement. For the normal use calibration should be performed at the
beginning of each day. The reason for is that the glass electrode does not give a reproducible
e.m.f. over longer periods of time. The calibration should be performed with at least two
standard buffer solutions that span the range of pH values to be measured. For general
purpose buffer at pH 4 and pH 10 are acceptable. The pH meter has one control to set the
meter reading equal to the value of the first standard buffer and second control which is used
to adjust the meter reading to the value of second buffer.

Hot Air Oven :- Hot air oven are electrical devices used in sterilization. The oven used dry
heat to sterilize article. Generally they can be operated from 50 to 300°C. There is a
thermostat controlling the temperature. These are digitally controlled to maintain the
temperature. Their double walled insulation keeps the heat in and conserves energy the inner
layer being a poor conductor and outer layer being metallic.

Vortex Mixer :- A vortex mixer is a simple device used commonly in lab to mix small vial
of liquid. It consists of electric motor with the drive shaft oriented vertically and attached to a

9
cupped rubber piece mounted slightly off center. As the motor runs the rubber piece oscillates
rapidly in a circular motion.

Water Bath :- A laboratory water bath is a tool used to maintain a very stable temperature
much like an incubator. Water baths can hold often temperatures with in tenth of degree
celsius, the water is often circulated. Sometimes beads are used as a waterless option.
Scientistific water baths are also used in commercial kitchens to cook.

UV Trans-Illuminator :- Trans illuminator is a technique of sample illumination by

transmission of light through the sample. Trans illumination is used in a variety of method of
imaging. This indtrument uses U.V. light to identify agarose and polysaccharide gels.

Laminar Air Flow :- A laminar air flow cabinet or laminar flow closet or tissue culture hood
is a carefully enclosed bench designed to prevent contamination of semiconductor wafers,
biological samples or any particle sensitive device. Air is drawn through a HEEPA filter and
blown in a very smooth, laminar air flow towards the user. The cabinet is usually made of
stainless steel with no gape or joints where spores might collect.

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Bod Incubator :- A bod incubator is an incubator designed to maintain necessary temperture
to perform a test called biochemical oxygen demand(BOD). It involves incubating samples
saturated with oxygen at necessary temperture for five days. Such an incubator has a
compressor to depress the temperature below ambient and a heater to bring it back. These
work against each other and produce very precise temperature control, often as close as +/-
0.1°C.

Autoclave :- An autoclave is a device to sterilized equipment and supplies by subjecting to

high pressure saturated steam at 121°C or more, typically for 15-20 min depending on the
size of the load and contents. It was invented by Charles Chamber land in 1879, although a
precursor known as the steam digester was created by Denis Papin in 1679.

11
 CHAPTER 3

SOLUTION PREPARATION

A solution is a homogeneous mixture created by dissolving one or more solutes in a

solvent. The chemical present in a smaller amount, the solute, is soluble in the solvent (the
chemical present in a larger amount). Solution with accurately known concentration can be
referred to as standard (stock) solution. These solutions are bought directly from the
manufacturer or formed by dissolving the desired amount of solute into a volumetric flask of
a specific volume. Stock solutions are frequently diluted to solution of lesser concentration
for experimental use in the laboratory.

(1 molar solution :- molecular weight of solute dissolve in 1litre of water solution is called 1
molar. “1 litre = 1000ml, 1ml = 1000 μl”)

CALCULATION OF RELATED PREPARATION

(1) Molar Solution

(2) % Solution

(3) X Solution

(1) Molar Solution :- The molarity of a solution is defined as the number of moles of solute
per one liter of solution

𝑀𝑜𝑙 = MW X no. of moles X given quantity

1000 (or ltr.)

But, There are some reagents that cannot be weighed (as they are in liquid form) therefor
there molar solution is prepared by another method in which first we have to find out its
molarity.

Calculation :- Molarity = purity% X specific gravity X 1000

Molecular weight

Eg:- To make of HNO3 in 200ml given purity 70%, specific gravity 1.4, molecular weight
69.

Molarity = purity% x specific gravity x 1000 = 70 x 1.4 x 1000 = 14.20

Molecular weight 69 x 100

(2) % solution :- A percentage solution is an amount or volume of chemical or compound

per 100ml of a solution. It is a relative expression of solute to solvent : x amount/100ml = x
% . There are two types of % solutions commonly used in histology:

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 1. % weight by volume (w/v) 2. % volume by volume (v/v)

Calculation :- Molarity = % solution x 10

Molecular weight

Eg :- Convert 6.5% solution in molarity when molecular weight 325.6

= % solution x 10 = 6.5 x 10 = 0.199

Molecular weight 325.6

% weight by volume (w/v) :- Given gram weight in 100ml of solution or distilled water.

% volume by volume (v/v) :- Given volume in ml dissolve in 100ml of solution.

(3) X solution :- Many buffer solutions are made as concentrated solution such as 5x, 10x
which means 5 time concentration of that particular solute that is dissolved in solution.

Calculation :- M1V1 = M2V2 or C1V1 = C2V2

Where : M1/C1 = Stock solution concentration

V1 = Volume needed from making working solution

M2/C2 = Working solution concentration

V2 = Given amount of working solution to be made

Eg :- How many ml of 5 molar K2Cr2O7 solution must be diluted to prepare to 250ml of 0.1
ml solution.

M1V1 = M2V2, 5 x V1 = 0.1 x 250, V1 = (0.1 x 250)/5, V1 = 5

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 CHAPTER 4

FOOD SAFETY MANAGEMENT SYSTEM

The Codex Alimentarius Commission brought out an International Standards on Food Safety
Management System (HACCP) in 1997. The food industries keenly awaiting for such an
international standards implemented it in their operations and got certification though
implementation in their operations.

The international Organization for standardization stepped in and brought out ISO
22000:2005 Food safety management systems - Requirements for any organization in the
food chain.

The aim of this International Standard is to harmonize on a global level the requirements for
food safety management for businesses within the food chain.

ISO 22000: 2005

Since ISO 22000 is a generic food safety management standard, it can be used by any
organization directly or indirectly involved in the food chain. It applies to all organizations in
the food chain. It doesn't matter how complex the organization is or what size it is, ISO
22000 can help ensure the safety of its food products. 

ISO 22000 combines generally recognized key elements to ensure food safety along the food
chain
 Interactive Communication
 Systems approach to Food Safety Management
 Prerequisite Programme
 HACCP Principles

The food chain consists of the entire sequence of stages and operations involved in the
creation and consumption of food products. This includes every step from initial production
to final consumption. More precisely, it includes the production, processing, distribution,
storage, and handling of all food and food ingredients. 

The food chain also includes organizations that do not directly handle food. These include
organizations that produce feed for animals. It also includes organizations that produce
materials that will eventually come into contact with food or food ingredients.

Why to use ISO 22000: 2005

• ISO 22000 will help you to achieve the following objectives: 

• To establish a food safety management system (FSMS).
• To ensure that products do not cause adverse health effects.
• To demonstrate compliance with external safety requirements.
• To evaluate customers' food safety requirements.
• To provide safe products and enhance customer satisfaction.

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 • To export food products and penetrate international markets.
• To communicate safety issues throughout the food chain.
• To ensure compliance with company’s food safety policy

ISO 22000 Food Safety Management System

FSMS is a set of interrelated or interacting elements (system) to establish policy and

objectives and to achieve those objectives used to direct and control an organization with
regard to food safety.
An ideal food safety management system in an enterprise should be one that:
meets the food safety policy (overall intentions and direction of an enterprise related to food
safety as formally expressed by top management), and achieve the measurable objectives
related to the policy 
meets performance of “effectiveness” (extent to which planned activities are realized and
planned results achieved) and “efficiency” (relationship between the results achieved and the
resources needed).
applies proven management principles (comprehensive and fundamental rule or belief) for
leading and operating an enterprise, aimed at continually improving performance over the
long term by focusing on customers while addressing the needs of all other stakeholders

15
 CHAPTER 5.
QUALITY MANAGEMENT
Quality Management

Definition

 The Process that Include all the activities of the performing organization that
determines Quality policies , objectives and responsibilities so that the project will
satisfied the needs for which it was undertaken.
 Quality refers to the sum of the attributes or properties that describe a product.
 These are generally expressed in terms of specific product characteristics such as
length ,width and color etc.

Evolution Of Quality Management

 1924 – Statistical Process Control Charts.

 1930 – Tables for acceptance sampling.
 1940 – Statistical sampling techniques.
 1950 – Quality Assurance/TQC.
 1960 – Zero Defects.
 1970 – Quality Assurance in Services.

Principles Of Quality Management

 Customer Focused Organization.

 Leadership.
 Involvement of People.
 Process Approach.
 System Approach to Management.
 Continual Improvement.
 Factual approach to Decision making and
 Mutually Beneficial Supplier Relationship.

Critical Projects Of Quality Management

(1) Quality Planning

(2)Quality Control

(3)Quality Assurance

Quality Planning :- It is defined as which quality standards are relevant to the project and
determining how to satisfy them.

Quality Control :-It is that part of GMP Concerned with sampling , specification and
testing , documentation and release procedure which ensure that the necessary and relevant
tests performed and the product is released for its use only after ascertaining its Quality.

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Quality Assurance :- It is a wide ranging concept covering all matters that individually or
collectively influence the quality of a product. It is the totality of the arrangements made with
the object of ensuring that products are of quality required for their intended use.

Are QA and QC Same terms ?

 Big NO , These both the terms are effectively different. Most of the time we used
both terms randomly , hence to study and understand the difference between them is
important.

Difference B/W QA & QC

• Lets differentiate on the basis of following Points –

Definition, Focus on, Goal, How to Achieve, Example, Responsibility.

Quality Control Focuses on

 Quality Control aims to Identify and Correct defect in the finished products.
 It is a reactive process.

Quality Assurance Focuses on

 Quality Assurance aims to prevent defects with a focus on the process used to make
the product.
 It is a proactive quality process.
 It identifies weaknesses in process to improve them.

Goal of Quality Control

 The Goal of Quality Control is to Identify defects after a product is developed and
before its released.

Goal of Quality Assurance

 The Goal of Quality Assurance is to improve development and test processes so that
defects do not arise and when the product is being developed.

How to Achieve ? Goal of Quality Control

 Finding and Eliminating sources of Quality problems through tools and equipment
so that customer requirements are continually met.
 The activities and techniques used to achieve and maintain product quality , process
and services.

How to Achieve ? Goal of Quality Assurance

 Established a good Quality Management System and the assessment of its adequacy.
 Periodic conformance audits of the operation of the system.

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  Prevention of quality problem through planned and systematic activities including
documentation.

Quality Control Example

 A QC Review, Performing Inspections, Performing testing.

Quality Assurance Example

 A QA Audit, Process Documentation, Establishing Standards, Developing

Checklists, Conducting Internal Audits.

Responsible for Quality Control

 Quality control is usually the responsibility of a specific team that test the product for
defects.

Responsible for Quality Assurance

 Everyone on the team involved in developing the product is responsible for Quality
Assurance.

Working together QA & QC

QC detected a recurrent problem with the quality of the products. QC provides feed
back to QA person that there is a problem in the process or system that is causing product
quality problems. QA determines the root cause of the problem and then bring changes to
the process to ensure that there are quality related issues in the future.

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 CHAPTER 6

FOOD PRODUCT ANALYSIS

Standards for cereals, pulse sand their products are laid do wnin section 2.4 of food safety
and standards(food product standard sand food additives) regulations, 2011. These in clude
standards for food grains, their milled products and processed cereal products. In addition
standards formalted foods and solvent extracted edibleoil seed flours are also included under
this items.

Definition of Refractions

Refractions mean all components of food grains, which differ from normal grains such
as foreign matter, other food grains, damaged grains , we evilled grains, broken, shrivelled
grains etc. The definition of various refractions is given under “explanation” in 2.4.6.15 for
items 2.4.6(2-14) in food safety and standards (food product standards and food additives)
regulations,2011. Additional definitions are –

(1) Karnal Bunt :- Grains of wheat having a done appearance and blacks in colour, the
blackness spreading a long the long to done furrow on the ventral size given the karnals a
bunt like appearance. The grains are affected by a field fungus “neovossia indica”.

(2) Ergot :- Grains of wheat showing as lightly curved body in the ear in place of karnal.
Ergot is produced by fungus claviceps pupurea.Ergot produces ergo toxin and occurs in rye,
millets and wheat.

(3) Filth :- Any objectionable matter contributed by animal contamination of the product
such as rodent, in sector bird matter, or any other objectionable matter contributed by in
sanitary conditions.

(a) Heavy filth – Heavier filth material separated from product by sedimentation based
on different densities of filth, food particle sand immersion liquids such as CHCl3 etc.
Examples of such filth are in sectand rodent excreta pellets and pellet fragments, sand and
soil.

(b) Light filth – Lighter filth particles that are oleophilicand are separated from product
by floating them in an oil – aqueous liquid mixture. Examples are in sect fragments, wholein
sects , roden thair sand fea the rbarbules.

(c) Sieved filth – Filth particals of specific size ranges separated quantitatively from
product by use of selecteds ievemesh sizes.

Procedure

Examine the test sample for its general condition, such as appearance freedom from
moulds, insect in festation of fodour, poisonous and deleterious matter.

a. Cereal and Cereal Product (wheat, bread, gram flour)

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1. Determination of foreign matter
Determine foreign matter by transfer ring entire 500gm of the sample over the set of the
largest perforation comes at the top and those with smaller perforationoli dpanat the bottom.
A gitate the sample thoroughly to strain out the foreign matter at various levels. As a result
of this training, other food grains and foreign matter like bold pieces of clay, chaffetcsh all
remain on the first three sieves according to their sizes. The top most sieve would contain
bold grains, big piecevs of clay and other big sized foreign matter, while the lower sieves
would contain smaller, shriveled and badly insect damaged grains and smaller foreign matter.
Separate the sieves after straining and pick up all foreign matter and addit to the foreign
matter collected on the bottom pan. Weigh the total foreign matter of the bottom pan and
calculate the percentage in the case of rice, millets and smaller sized the quantity of sample
for test should be 250gm. For the purpose of reducing the quantity of the test sample, spread
the entire sample in at ray, divide it in to four equal portions, collect the two opposite
portions and repeat this process till the required quantity of sample is collected.

2. Derermination of Moisture content of wheat flour

Preparation of sample

Invert and roll container several times to ensure homogeneous mixture. Avoid extreme
temperatures and humidities while opening the containers for analysis. Keep sample in a
colsed container.

Procedure

Weight accurately about 5gm of sample in a previously dried and tared dish and place the
dish with its lid underneath in the oven maintained at 130°-135°C. for 2 hours. The time
should be reckoned from the moment the oven attains 130°C. after the dishes have been
placed. Remove the dish after 2 hours, cool in the dessicator and weight.

Calculation

Moisture % = (W1 – W2) x100 = (37.060 – 36.550) x 100 = 10.2%

W1 –W 37.060 – 32.060

Where,

W1 = Weight in gm of the dish with the material before drying =37.060gm

W2 = Weight in gm of the dish with the material after drying = 36.550gm

W = Weight in gm of the empty dish = 32.060gm

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 Moisture is an indicator of grain storability. Wheat flour with high moisture content
(14.5%) attractd molts bacteria and insects all of which because deterioration during storage.

3. Determination of alcoholic acidity of wheat flour

Reagents

 Neutral Ethyl alcohol – 90% (V/V)

 Standard Sodium Hydroxide solution – approx. 0.05 Normalty
 Phenolphthalein indicator – Dissolve 0.1gm in 100ml of 60% Ethyl alcohol.

Procedure

Weight 5gm sample in a stoppered conical flask and add 50ml of neutral ethylalcohol.
Stopper, swirl gently and allow to stand for 24hours with occasional swirling. Filter the
alcoholic extract through a dry filter paper. Titrate 10ml of the alcoholic extract with standard
sodium hydroxide solution to a pink end point using phenolphthalein as indicaror

 Whatman filter paper No. 1 is to be used for filtration process

Calculation

Alcoholic acidity with 90% alcohol calculated as H2SO4 on dry bases

= Volume of titre x .00245 x 50 x 100 x 100 = 40 x 0.05 x100 = 0.2

gm
10 x exact wt of sample x (100–moisture content) 1000

Requirment :- NaOH =0.05 N ,

Note : Titration reading = 1.2ml

4. Determination of gluten content of wheat flour

Requirement : Wheat flour, A beaker.
Procedure
Weight 25gm of wheat flour in transfer at into 100ml of beaker. Add 15ml of water into
the water and needed it slowly and make it dough. Add sufficient amount of water to deep
the dough into it for 15min. Take out the dough from water and wash in slowly under the tap
water. Remove access of water by squeezing the dough in the palm. Take the weight of
wheat gluten. Dry it in the oven for 5min (at105°C). Take the weight for the dry gluten.
Calculation

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 Gluten of dry weight = (Wt. of dry gluten x 100 x 100
exact wt of sample x (100–moisture content)

= 3.95 x 100 x100 = 17.51gm.

25 x (100 – 9.8)

5. Determination of Moisture content of bread testing

Preparation of sample

Cut the sample into small pieces and mix together so as to from a composite sample and
transfer to a clean dry air tight glass container.

Apparatus

 Moisture dish – made of porcelain, silica, aluminium, stainless steel (7.5 x 2.5cm)
 Oven, electrical maintained at 105 ± 2°C
 Dessicator

Procedure

Weight accurately about 5gm of the prepared sample in a moisture dish previously dried
in the oven at 105°C. Place the dish in the oven maintained at 105 ± 2°C for 4 hours. Cool in a
dessicator and weigh. Repeat this process of heating, cooling, weighing till the difference
between two consecutive weighing is less than 1mg.

Calculation

Moisture % = (W1 – W2) x 100 = (54.33 – 52.60) x 100 = 34.6%

W1 – W 54.33 – 49.33
Where,
W1 = Weight in gm of the dish with the material before drying = 54.33

W2 = Weight in gm of the dish with the material after drying to constant weight =
52.60

W = Weight in gm of the empty dish = 49.33

6. Determination of Alcoholic acidity of bread testing

Reagents

 Neutral Alcohol 90% (V/V)

 Standard NaOH solution – 0.05 Normalty
 Phenolphthalein solution – 1% solution in ethyl alcohol

22
Procedure

Weight about 5.0gm of dried sample into a stoppered conical flask and add 50ml of 90%
neutral alcohol previously neutralized againts phenolphthalein. Stopper shake and allow to
stand for 24 hours with occasional shaking. Filer the alcoholic extract through a dry filter
paper. Titrate the combined alcoholic extract against 0.05 normalty standard sodium
hydroxide solution using phenolphthalein as an indicator.

Calculation

No. of ml of 1 normal NaOH required for neutralization of 100gm of sample

= Titer x Normality of NaOH x 100 = 1.4 x 0.05 x 100 = 0.7ml

Wt. of the sample taken 10

Requirment :- NaOH = 0.05 , Sample taken = 10ml

Note : Titer reading = 1.4

7. Determination of Kesari dal (besan) powder (lathyrussativus) of gram flour

Principle

The presence of kesari dal powder is detected on the basis of the presence of an unusual
amino acid namely beta-N-oxaly I L amino alanine which is not present in the seed of other
legumes

Apparatus

 Steam bath/water bath

 Air oven
 Chromatographic paper whatman No.1

Regents

 Ethyl alcohol 70%

 Isoproponol solution – 10%
 Liquified distilled phenol – water solution – (4:1)
 Ninhydrin solution – 0.1% in acetone or ethanol
 Buffer – Pyridine : Aceticacid : Wter(0.5 : 5 : 95) pH 3.60

Procedure

Weight approximately 5gm powdered sample and extract it with 100ml mlethyl
alcohol(70%) by keeping over night whith occasional shaking. Filter the extract and
evaporate to dryness on a steam / waterbath. Extract the residue with 10ml of iso – proponol
solution, filter and use this solution for chromatography. Spot 20μl of the extract using a
haemoglobin pipette or capillary tube at a distance of 1cm from the bottom of the

23
chromatographic filter paper. Keep in a solvent chamber saturated with phenol – water
solution over night. Remove from the chamber, dry filter paper in a current of air at room
temperature for 4-5 hours or in an oven at 80°C for 1 hour and spray with ninhydrin solution.
Dry the chromatogram in the oven for 15minutes. The appearance of bluish – purple spot at
about Rf values 0.1 shows presence of BOAA which is present only in lathrussativus. Other
proteins extracted simultaneously also give similar colour but at different Rf values. Always
run known sample of kesari dal powder simultaneously and compare the spot of control with
sample under test.

24
 B. MILK ANALYSIS

5.b.1. Methods for detection of adulterants present in milk

Preparation of sample

Sample are received after few days of drawl and contain preservation (0.4% for malin).
Warm the sample to 37°C by transferring it to the beaker and keeping it in a water bath
maintained at 40 – 45°c stir slowly for proper homogenisation. Mix sample thoroughly by
pouring back into the bottle mixing to dislodge any residual fat sticking to the sides and pour
it back in the beaker. During mixing do not shake the bottle vigorously. Allow the sample to
come to room temperature (26 – 28°C) and withdraw immediately for analysis. If small clots
or lumps are observed in the sample which cannot be dispersed a few drops of liquor
ammonia may be used during homogenisation. If even after homogenisation the sample
shows lumps or clots or droplets of oil are visible suggestive of curdling /splitting of milk the
sample should be deemed unfit for analysis and rejected.

Detection of Cane sugar in milk

Sucrose is absent in milk and its presence in milk indicate adulteration. Presence of
sucrose in milk can ce determined by the following method. Fructose in cane sugar (sucrose)
reacts with resorcinol in HCl to give red colour.

Reagent

Resorcinol solution (0.5%) :- weight 0.5gm of resorcinol in about 40ml of distilled water.
Add 35ml of concentrated HCl (12 normality) to it and make up the volume to 100ml using
distilled water.

Note : The resorcinol flakes should be white in colour.

Procedure

Take 1ml of milk in a test tube. Add 1ml of Resorcinol solution and mix. Place the tube
in boiling water bath for 5min. Withdraw the tube and observe the colour. Appearance of
deep red colour indicates presence of sucrose or a ketose sugar. In pure milk samples no such
red colour is developed and sample remains white in nature.

25
Detection and Quantification of Starch in milk

Reagent

Iodine Solution :- Dissolve 2.6gm of iodine and 3gm of potassium iodide in a sufficient
quantity of water and make upto 200ml.

Procedure

Take about 5ml of milk in a test tube. Bring to boilong condition and allow the test tube to
cool to room temperature. Add 1 to 2 drops of iodine solution to the test tube. Development
of blue colour indicates presence of starch which disappears when sample is boiled and
reappears on cooling.

Detection of Added Urea in milk

Urea is a natural constituent of milk and it from a major part of the nonprotein nitrogen
of milk. Urea concentration in milk is variable within herd. Urea content in natural milk
varies from 20mg/100ml to 70mg/100ml. However, urea content above 70mg/100ml in milk
indicates milk cotaining “ added urea”. The addition of urea to milk can be detected by using
para dimethylaminobenzaldehyde (DMAB). This method is based on the principle that urea
forms a yellow complex with DMAB in a low acidic solution at room temperature.

Reagent

DMAB reagent(1.6% w/v) :- Dissolve 1.6gm DMAB in 100ml ethylalcohol and add 10ml
concentrate HCl

Procedure

Mix 1ml of milk 1ml of 1.6% DMAB reagent . Distinct yellow colour is abserved in milk
containing added urea. The control (normal milk) shows a slight yellow colour due to
presence of natural urea.

26
Detection of Ammonium compounds in milk

Reagents

Nesslers reagent : Dissolve the following chemicals separately.

a. 8.0gm of mercuric chloride in 150ml distilled water.

b. 60.0gm of sodium hydroxide in 150ml distilled water.
c. 16.0gm of potassium iodide in 150ml distilled water.

Add reagent “a” to reagent “b” and mix well. To this mixture, add reagent “c” mix and dilute
the contents to 500ml. Leave this solution undisturbed and decant the clear upper layer of the
solution and store in a stoppered glass bottls.

Procedure

Take 5ml of milk sample into a test tube. Add 1ml of nesslers reagent. Mix the contents
of the tube thoroughly. Observe and note the colour. Appearance of yellowish or grey colour
confirms the presence of added ammonium salts in milk.

Tests for Presence of Sulphates in milk

Presence of sulphate salts, which may be added to milk to raise its SNF level in milk,
can be detected by using barium chloride.

Reagents

 Barium chloride (BaCl .2HO) 5% (w/v) aqueous solution : Dissolve 5.0gm barium
chloride in distilled water and make the final volume to 100ml.

27
  Trichloroacetic acid (TCA), 24% (w/v) aqueous solution : Dissolve the 24gm of TCA
into distilled water and make the final volume to 100ml obtain 24% TCA.

Procedure

Take 10ml of milk in a 50ml stoppered test tube. Add 10ml of TCA solution. Filter the
coagulated milk through whatman filter paper grade 42. Take 5ml of clear filtrate. Add few
drops of barium chloride solution. Observe for any visible precipitatesin the tube. Formation
of milky white precipitates indicates the presence of added sulfated like ammonium sulphate,
sodium sulphate, zince sulphate and magnesium sulphate etc to milk.

Addition of skim milk powder

Take 50ml of milk in each of two centrifuge tubes and balance properly in the centrifuge.

 Centrifuge at 3000 rpm for 30 min.

 Decant the supernatant liquid carefully.
 Dissolve the residue in 2.5ml of concentrated nitric acid.
 Dilute the solution with 5ml of water.
 Add 2.5ml of liquid ammonia and observe for colour development.

Skim milk powder being highly proteinacious in nature gives orange colour with nitric acid
while unadulterated milk being low in protein content gives only a yellow colour.

28
5.b.2. Detection of preservatives present in milk

Addition of chemicals is illegal there for common methods to detect their addition is
important.

Boric acid /Borax in milk

Reagents

Turmeric paper : Weight 2gm of turmeric powder and mix in it with 100ml of 80% of
ethenol. Filter the solution and calect the filtret. Deep the filter paper into the filtret dry it and
cut it to except size in storege.

Procedure

Take 5ml of milk in a test tube and add 1ml of concentrated HCl and mix well. Deep
streat of turmeric paper and dry it. If turmeric paper terns red colour its show the presence of
boric acid/borax.

Formaline detection (Hehnes test) in milk

Procedure

Take 10ml of milk in a test tube and 0.5ml of 1% phericchloride solution. Add slowly
5ml of concentrated H2SO4 through the sides of the test tube. Formation of purple ring will be
seeing at the junction. If formaline is present.

29
Anionic detergen test in milk

Procedure

Take 0.5ml of milk in a test tube and add 0.5ml methylindie. Add 1ml of chloroform and
vortex the mixture the 15sec. Centrifugsed the tube and 10000rpm for 3min. Observed the
tube colour distribution dark blue colour in the lower tube confirme this test.

Benzoic detection in milk

Procedure

About 20ml of milk of strits of equail valume of consentrated HCl until the cut is
dissolved. It is now allowed to cool about 25ml of mixture of either or petrolium ether is
added to the mixture of milk and hydrochloric acid and its scken. The ether layer separation
and gives a precipeted with the drop of amonium hydroxide in the presence of benzoic acid.

30
β napthol test in milk
Procedure

Milk is extract with chloroform and heated with potassium hydroxide for few min. If a
deep blue colour apears it indicate the presence of β napthol.

Benzoic acid Salicylic acid in milk

Procedure

5ml of milk taken a test tube and few drop of consentrated H2SO4 is added. Milk ppt will
be form add 0.5% phericchloride solution drop by drop. If buffs colour will apear that shows
the benzoic acid is present. And if voilet colour will be apear shows the presence of salicylic
acid.

Soap test in milk

Procedure

Take 4ml of milk in a test tube and 10ml hot water and 1 – 2 drops of phenolpthline
indicators. If pink colour will be obtain its shows the presence of soap.

31
Detection of Ammonium compound in milk

Reagent

Nessler’s reagent : Dissolve the following chemicals separately.

a. 8.0gm of mercuric chloride in 150ml distilled water.

b. 60.0gm of sodium hydroxide in 150ml distilled water.

c. 16.0gm of potassium iodide in 150ml distilled water.

Add reagent “a” to reagent “b” and mix well. To this mixture, add reagent “c” mix and
dilute the contents to 500ml. Leave this solution undisturbed and decant the clear upper layer
of the solution and store in a stoppered glass bottles.

Procedure

Take 5ml of milk in a test tube and add 1ml of nesslers reagent. Observed the colour
devloped. If amonia compound is present yellowish or gray colour is apear.

Determination of qualiti of a milk sample by MBRT test.

Principle

32
 The reduction test is based on the oxidation reduction activites of the bacteria present the
faster is the reduction.

Procedure

Prepare methylin blue solution by dissolving 1ml methylin blue in 250ml sterlized distil
water.Transfer 10ml of milk into the test tube and add 1ml of methylin blue in it. Mix the
content of test tube by inverting it 2 – 3 times. Incubater the test tube at 37°C for 6 hsd ours.

Observation

Observe the incubated milk tube for every 30min for 3 to 6 hours for the reduction of
methylin bluu, i.e. change in the colour of the sapmple from blue to white. Record the time
required form de-colourlisation.

5.b.3. Turbidity test for milk

Procedure

Take 20ml of milk in a flask and add 4gm of amonium sulphate keep it for 5min and filter
its. Place in the test tube in boiling water bath. Transfer the test tube into the another beaker
and cooling its.

Inter preation : The milk is consider sterlize when the filteret show no turbidity.

Result : The given sample was showing slidy turbidity hence the sample is consider not
properly sterlized

33
5.b.4. Sensory evaluation of milk

Sensary evaluation may be define as a scientific method to evoke, measure, analysis and
interperent result of those characture stick of foods persivid through 5 sensory of sight, smell,
test,taugh and hearing.

Where sensary envaluation is apply in dairy

 Inspection of row material.

 New product devlopment or improvement of exisiting product.
 Cost reduction.
 Quality conterol.
 Salection of packeing material.
 Self-life study.

The basic test

A basic test is one of which specific test birds have been identified which is responsible
for that particular test sensetion.

There are fives basic test : Sweet, Sour, Soltin, Bitter, Umami (umami is also good
flevers in the glutamate)

Sequence of observing sensory characteristics of milk and milk products

When a dairy product is offered for sensory evaluation its appearance aspect is first
noticed. It is perceived quickly and non-evasively. These four factors combine to determine
the colour of any food product. Appearance is the first attribute perceived by the consumer
and often become deciding factor in purchasing decisions. After appearance, the ortho-nasal
perception of odour or aroma of the food takes place. Upon taking the food in the mouth the
retro-nasal (in nose) perception of aroma continues. Perception of foods consistency, texture
and possibly sound especially for crispy and crunchy foods are also perceived inside the
mouth.

34
 5.c. WATER ANALYSIS

5.c.1. Determination of Alkalinity test of water

Procedure

Pippet 10ml of test solution into a conical flask add a few drops of phenophthline indicator
the solution is turns pink. Titret with 0.1molar HCl untill the pink colour just disappear.
Measure the number of ml of acid used.

Calculation

Active alkalinity = Titer x Molarity HCl x 4 = 2.1 X 0.1 X 4 = 0.084

Volume of sample taken 10
Reading :- 2.1ml

5.c.2. Determination of Acidity test of water

Procedure

35
 Pippet 10ml of test solution into a conical flask and add few drop of phenophthyline
indicater. Titer with 0.1molar NaOH until the first permanent pink colour appear.
Calculation
Acid volume = Titer x Molarity of NaOH x 56.1 = 3.5 X 0.1 X 56.1 = 1.96
Volume of sample 10
Reading :- 3.5ml

5.c.3. Ethylene di-amine tetraacetion acid (EDTA) in water

Procedure
Pipette 25ml of test solution into a conical flask and adjust the pH to neutral by adding
hydrochloric acid. Add 50ml of ammonia buffer and 3 – 4 drops of solution black indicator. In
the presence of tree EDTA the solution is to blue. Titrate with 0.01molar zinc chloride until
the solution just change to violet/red.

Calculation

EDTA = Titer x Molarity of zinc x 29.2 x 10-4 = 7.5 X 0.01 X 29.2 X 10-4 = .0876 X
10-4
Volume of sample taken 25
Reading :- 7.5

5.c.4. Determination of total water hardnes

36
Procedure
Pipette 50ml of water to be tests into a conical flask and add 10ml of ammonia buffer and 5 – 6
drops of solochrome black indicator. Titrate with 0.01molar EDTA until all traces of red
colour disappear when the end point of blue gray colour appears load the titer of EDTA.
Calculation
Total hardness = Titer x Molarity of EDTA x 105 = 4.2 X 0.01 X 105 = 84 ml
Volume of sample taken 50
Reading :- 4.2ml
Note : water hardness = calcium ion + magnesium ion.

5.c.5. Determination of disinfectant present in water

Available chloride
Procedure
100ml of sample was taken and was mix with 10ml of potassium iodide.10ml of H2SO4 is also
added and titrated with 0.1molar sodium thyosulphate until a colourless solution is obtain.
As brown colour fade drop a starch solution to the solution, it will become dark blue titrate
till it become colourless.
Calculation
Avlable chloride = Titer x Molarity of Na2S2O3 x 35500
Volume of sample taken

To detect the presence of iodine

Procedure

37
 100ml of sample is taken and was 50ml of deiodeze water. Titret with it 0.01molar
Na2S2O3 until brown yellow colour disappear.

Calculation

Avlable iodine = Titer x Molarity of Na2S2O3 x 105x 1.27

Volume of sample taken

Alkalinity of H2O

A high pH (>13) and a high titrable active alkalinity whould normaly indicate the
presence of sodiumhydroxide in industrial detergents. However there may be other inorganic
compaund such as silicate, corbonate and phosphate that make a significant contribution to
alkalinity.

Silicate

Procedure

2ml of weight sample in a test tube and 3ml of concentrated HCl added. Boil for 5min
cool and add on axise of amoniahydraoxide solution. A white ppt that sloculates on boiling in
this present of silicate.

Phosphate

Procedure

38
 Add 2ml of weight solution in a test tube and add 3ml of concentrated nitric acid and 5ml
of solution amonia molibadate solution. Boil the solution for 5min the appearance of yellow
ppt indicates the presence of phosphate.

Carbonate

Procedure

Add about 2ml of solution to a test tube and 1ml of concentrated sulphuric acid.
Effervescence indicater the presence of carbonate.

Acid detection

Sulphuric acid

Procedure

Add about 2ml of solution in a test tube and 2ml cancentrated HCl and add exces of
beriumchloride solution. A white ppt indicates the presence of sulphuric acid.

Nitric acid

Procedure

Add 3ml of solution in a test tube and 3ml of pheric sulphate solution and shake it to mix
properly. Add 3 – 5 drops of concentrated sulphuric acid solution slowlly down the side of
the tube so that the acid from a lear be need the equecous mixture. It possetive a brown ring
will be formed between were the to liquid meets.

39
Reducing reagent

Procedure

Add about 2ml solution in a test tube 2ml of concentrated sulphuric acid add 2ml of
potaccium permagnate solution. Heat to over 28°C for few min decolori sation of per magnet
indicates the presence of reducing reagent.

Oxidizing reagent( chlorine, peroxide)

Procedure

10ml of sample 2ml of concentrated sulphuric acid and 2ml of potaccium iodid add in a
test tube. A brown colour indicate chlorine or peroxide.

5.D. MICROBIAL ANALYSIS

5.d.1. Isolation of microorganism from (soil, water and milk)

5.d.1.a. Media preparation

Made 200ml of nutrient agar taking 0.5% peptone, 0.3% yeast extract, 0.5% NaCl,
1.5% agar. Dissolved 1.5% agar in another 100ml of water. Boiled both dissolved solution
and quickly mixed into a 250ml of conical flask. Closed the mouth of the flask tightly with
cotton pulg and wrapped it with alumunium foil. Autoclaved the media for 20min with at 15
psi pressure.

Procedure

Made four agar plates by pouring autoclaved media to 1/3 rd of the petri plate and marked
them by different codes for each microorganism. Kept all four petri plates aside to let the
media

Solidify in each plate and slightly covered all. Took 100 microliter of bacillus subtilis
suspension in the center of the plate and uniformly spread throughout the media plate with
the help of spreader. Placed different discs to the microbe spred region with the help of a fine
forceps and marked the region over the cover. Kept all the petri plates in the incubator
inverted at 34°C temperature. Observed the plates at different time intervals i.e. after 16
hours, 24 hours, 40 hours, 48 hours and 64 hours.

40
5.d.1.b. Sample collection

For the isolation of bacteria, soil sample was collected from NBFGAR garden, milk
sample was collected from greencity chauraha, water sapmple was collected from tedipuliya
in lucknow.

5.d.1.c. Serial dilution

Microorganisms are isolated from soil by using serial dilution so that the number of
bacteria present in soil decreases by serially diluting the crude sample obtained. About 6 test
tubes are taken each containing 9ml of saline solution of .85% NaOH. First test tube is called
crude.

5.d.1.d Spread plate and poure plate

Spread plate:- 100 micro liters of inoculums is taken by micro pipette poured on petri
plates and the colonies do not overlap.

Pour plate:- In contrast to spread plate method, culture is either added to molted
media, mixed and poured into plates or media is first poured on plate and them molten media
is added ( done for anaerobes).

5.d.2. To obtain pure culture of bacteria by streak plate method

Principle

Colony of bacterial (whose pure culture is to be obtained) is taken by a inoculation loop

and streaked on fresh meadia (in a petri – dish). There are 2 types of streaking –

(a) Zig Zag streak (b) Quadrant streak

Requirements

41
 Bacterial culture (to be streaked) nutriant agar media Of 100ml (NAM) :- NaCl – 0.5gm,
Beef extract – 0.3gm, Pepton – 0.5gm, Agar – 1.8gm, Distilled water – 100ml.

Procedure

Prepared the NAM media autoclaved it and poured in petri – dish. After media got
solidified transferred the culture to be streaked onto the petri – dish. Kept the petri – dish in
an incubator at 37°C for 24 hours.To preserve the purified culture.

5.d.3. Staining and biochemical activities of purified culture

5.d.3.a. Gram staining

Principle

Gram staining is performed to check the purity of culture whether it is contaminated by

any other bacterial culture or not. This technique was developed by Hans Christian Gram to
classify the bacteria’s into 2 groups –

(a) Gram positive (b) Gram negative

Gram positive :- Bacteria’s have more % peptidoglycan and a thin layer of lipids.
Peptidoglycan takes the stain crystal violet and does not washed with 95% alcohol and appear
blue or purple.

Gram negative :- Bacteria’s have thick layer of lipids in their cell wall composition i.e.
about 80% lipid where as 20% peptidoglycan. Therefore on washing with 95% alcohol the
crystal violet stains washes away as alcohol dissolves the lipids and causes pore formation on
the cell wall due to which stain washed off. On treating with counter stain safranin cells
appear pink in color under microscope. Iodine act as mordent and it helps better binding of
crystal violet and red to bacterial cell wall.

Materials required :- Crystal violet, Iodine, 95% alcohol, Safranin, Microscope, Distilled
water.

5.d.3.b. Catalase test

42
Principle

During aerobic respiration in the presence of oxygen microorganisms produce hydrogen

peroxide which is lethal to the cell. The enzyme catalase present in the organism breaks down
hydrogen performed by adding H2O2 to water and oxygen and helps them in their survival.
Catalase test is performed by adding H2O2 to the culture taken on glass slide directly. Release
of free oxygen gas is a positive test.

Requirements :- Hydrogen peroxide (3%)

Procedure

2 to 3 drops of hydrogen peroxide is taken on three glass slide. One loop full of the
culture was kept on each slide of milk water and soil sample. Slide was observed after 10
minutes.

Observation

If bubbles are observedon the slide them they are catalase and if bubble is not observed
them it catalase negative.

Result :- There was no bubble observed so all the sample gave catalase negative test.

5.d.3.c. Amylase test

Principle

Amylase is an exoenzyme that hydrolyses starch a polysaccharide into maltose a

dissachride and some monosacchride and such as glucose. These dissachrides and
monossachrides enter into the cytoplasm of the bacterial cell through the semipermeable
membrane and thereby used the endoenzyme. Strach is a complex carbohydrate composed of
two constituents-amylase a straight chain polymer of 200 – 300 glucose units amylopectin a
larger branched polymer with phosphate group. The ability to degrade strach is used a
criterion for the determination of amylase production by a microbe. Strach is the presence of
iodine produces a dark blue coloration of the medium and a clear zone around the colony
indicates amylolytic activity. The production of amylase is supported by minimal agar media
containing a particular substrate.

43
Requirements

Minimal agar medium, Nutrient agar slant cultures, Gram’s iodine solution, Petri-dishes,
Inoculation loop, Bunsen burner

Procedure

(1) Preparation of minimal agar media (composition for 100ml) :- Potassium dihydrogen
phosphate – 0.3gm, Disodium hydrogen phosphate – 0.6gm, NaCl – 0.5gm, Magnesium
sulphate – 0.02gm, Ammonium chloride – 0.2gm, Starch – 0.8gm, Agar – 2gm.

(2) A singal streak inoculation was made into the centre of the approximately labeled
petri dishes containing minimal agar media.

(3) The inoculated petri dishes was inoculated for 48 hours at 37°C in inverted position.

(4) The surface of the plate was flooded with gram’s iodine solution with a dropper and
was left for 30minutes. The excess iodine is poured off.

Observation

The petri plates were examined for starch hydrolysis around the colony that the colour
change of the medium.

Result

After adding gram’s iodine colour did not changed to blue so it gave negative test.
Showing no colour change after the addition of gram’s iodine so bacterial sample does not
possesses amylase activity.

5.d.3.d. Citrate test

Principle

44
 Cirate test is used to differentiate among enteric bacteria no the basis of their ability to
utilize or ferment citrate as sole carbon source. The utilization of citrate depends on the
Presence of enzyme citrate produced by the organism that breaks down the citrate to
oxaloacetic acid and carbondioxide. The citrate test is performed by inoculating the
microorganisms into organic synthetic medium simmon’s citrate agar. When citric acid is
metabolized the carbon dioxide generated combines with sodium and water to from sodium
carbonate alkaline product that changes the colour of the nutrient broth from green to blue
and this constitutes a posetive test.

Requirements

Nutrient broth culture of bacteria, Simmon’s citrate agar slants, Bunsen burner,
Inoculation loop.

Procedure

(1) Preparation of simmon’s citrate agar slants (composition for 1000ml) :- Ammonium
dihydrogen phosphate – 1.0gm, Sodium chloride – 5.0gm, Sodium citrate – 2.0gm,
Magnesium sulphate – 0.2gm, Agar – 15.0gm. Dipotassium phosphate – 1.0gm, Bromotymol
blue indicator, Distilled water – 1000.0ml.

(2) All the constituents except phosphates which are to be dissolved separately in 100ml
of water were dissolved and the volume was made up to 1 litre. The pH was set to 7.3 and the
medium was poured in culture tubes and sterilized by autoclaving at 15 lb pressure for 15
minutes.

(3) The simmon’s citrate agar slants were inoculated with nutrient broth by means of an
inoculation loop.

(4) The test tubes were inoculated at 37°C for 48 hours to observe the colour change.

Observation

The colour changed from greenish to blue of the broth due to the citrate utilization

Result

The broth colour observed was red in due to the break down of citrate in simmon’s citrate
media.

45
 REFERENCES

 Biochemistry and molecular biology- Keith Wilson and John Walker

 Google
 Fssai Manual_Water_Analysis by FOOD SAFETY AND STANDARDS
AUTHORITY OF INDIA, MINISTRY OF HEALTH AND FAMILY WELFARE,
GOVERNMENT OF INDIA
 2016 Manual of methods of ananlysis of food related to milk and its products by
FOOD SAFETY AND STANDARDS AUTHORITY OF INDIA, MINISTRY OF
HEALTH AND FAMILY WELFARE, GOVERNMENT OF INDIA

 2012 Manual of methods of ananlysis of food related to cereals and its products by
FOOD SAFETY AND STANDARDS AUTHORITY OF INDIA, MINISTRY OF
HEALTH AND FAMILY WELFARE, GOVERNMENT OF INDIA

46