TAMILNADU AGRICULURAL UNIVERSITY

COMMUNITY SCIENCE COLLEGE AND RESEARCH

INSTITUTE
MADURAI - 625 104

SMS LAB SERVICES PRIVATE LIMITED, PUTHUCHATRAM,

CHENNAI

AN INTERNSHIP PROGRAMME REPORT

DURATION - (01/10/2019 TO 15/11/2019)

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SUBMITTED BY

S.ABINAYA 2016008001
BHARATHI.V 2016008007
DIVYA.R 2016008011
NIDHARSHANA.S 2016008020
PAVITHRA.T 2016008021
RAMYA.R 2016008025
VIDHYA SHRI.P 2016008039
C.VIJAYA 2016008040

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 ACKNOWLEDGEMENT
We express our sincere gratitude to the management of SMS lab
services private limited (SMSLA) for having given us an opportunity to work
with them and make the best out of our internship. Our heartfelt gratitude also
goes out to the staff and employees at SMSLA for having co-operated
throughout 45 days of internship period.

We have gained both the analytical skills and managerial skills

throughout the internship period. We are forever grateful for the cooperation
during the period of our internship.

TABLE OF CONTENTS

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 S.N CONTENTS PAGE
O NO

1 Introduction 5

2 About the lab 5

3 Login 5

4 LIMS 7

5 Food 8

6 Water 25

7 Microbiology 34

8 Instrumentation department 46

9 Extraction 46

10 HPLC 53

11 ICPMS/AAS 58

12 Mechanical 61

13 Quality Control 61

14 Inference 64

INTERNSHIP REPORT

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Introduction:

We the students of 3rd B.Sc.,(Food Nutrition and Dietetics) of Community Science

College and Research Institute, TamilNadu Agricultural University, Madurai has been
undergoing an Internship Programme for our course FSN402 in SMS Lab Pvt Ltd, Chennai for
45 days from Oct 1 to Nov 15, 2019.

There we had been placed in different departments like Login, Quality Control,
Proximate, Microbiology, Water, Instrumentation (Extraction, HPLC, and ICPMS/AAS)

About the lab:

It is one of the commercial labs situated in Chennai, started in 2012 with a mission of
holistic quality services in Testing, Inspection, Training and advisory services in food, water, air
and materials. It also carries out Environmental analysis, method development, R&D, nutritional
profile and shelf life studies. The lab is NABL accredited and recognized by FSSAI, AGMARK,
MOEF, TEA BOARD, BIS, EIC, ISO17025.

The ISO 17025 has the clauses regarding the resource requirements, structural
requirements, terms and definitions, process requirements, management system requirements and
other general requirements for the competence, impartiality and consistent operations of
laboratories.

It tests the following samples:

 Food
 Air
 Water
 Metals

LOGIN:

In Login department, it has various underlying procedures before sending the sample for
analysis. It consists of

 Sample receiving
 Storing of sample before booking
 Checking
 Giving Quotation
 Booking
 Decoding
 Sending for analysis

Sample receiving:

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 Samples are received either by courier or the sampling persons are allowed to bring sample
samples from concern places. TRF (Test Request Form) is filled by the client or by the sampling
person. It contains all the information about the sample including the sample name, quantity of
the sample, then it is placed in the login area. As soon as the sample is received it must be
decoded (removing all the information and details of the sample present in the packaging
material). Various type of samples are received for analysis .They would segregate the samples
as

 APEDA
 FSSAI
o EIC
o BIS
o Water and Environment
o Food
o Container

Checking:

All the information about the sample which are present in the client letter and the TRF-
Test request Form is checked by the booking person. Then the sample is checked for its
condition whether it is fit for analysis, its expiry date and the quantity required for sample
analysis was checked.

Quotation:

All the information must be checked again. Based on the parameters that are quoted by
the client the quotation is made for the sample and it is sent to accounts department.

Booking:

After the quotation is made, the received samples are booked in LIMS software. In booking,
all the information about the samples are uploaded like

 Client information
 Sample name
 Sample description
 Quantity of the sample
 Sample batch no
 Sample identification
 Parameter (chemical, biological, physical, antibiotics, pesticides etc.)
 Testing method
 Price for analysis

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 All the information are filled in concerned places in the software. Parameters are either
decided by the standard method or based on the client requirement. Based on the sample and the
parameters, quotation is prepared by the lab.

Decoding:

Decoding- Removal of all the information about the sample present in the sample
containing material.

Allocation of code:

Based on the sample, the codes are assigned as FD- food, EN-environment, etc.. After
booking, the persons in the login inform about the sample to concern departments like

 Water and environment

 Container
 Microbiology
 Food department
 HPLC
 GCMS
 LCMS
 ICPMS

LIMS:

LIMS stands for Laboratory Information Management System. It is also called as Q

LIMS. In this, the booked sample details are uploaded which would be helpful for further
reference. In this all the details about the analysis are updated here.

1. In parameter column, there are

 Parameter name
 Description
 Test method
 Appropriate Lab
 Class of the parameter
 Quantity
 Lab unit
 No of days required for analysis
 Price for the detection of the parameter

2. In product definition column, the products are segregated and also code was given. For
eg: Edible oils and fat –EOF. Parameters are added to the concerned product.

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 3. In parameter worksheet, Details about the permissible limit, LOQ, Instrument that is used
to analyze the sample are given.

4. In TEST method, IS method, IS method, APHA method, ASTM method etc which are
followed to analyze the sample are given.

NABL scope:

NABL stands for National Accreditation board for testing and calibration Laboratories.
NABL certification number for this lab is TC-6118. Every accredited lab has a scope of
accreditation. Scope has the information about the NABL accredited parameters that are
analyzed in the lab with its method and the list of products for which the parameters are
analyzed.

FOOD DEPARTMENT:

MATRIX ANALYSED:

All the samples from farm to fork are analyzed here.

 Milk & milk products

 Oils and fats
 Fruits and vegetables
 Cereals and cereal products
 Herbs, spices and condiments
 Meat and meat products and marine products

Standard methods based on which analysis is done;

 Indian standards
 Food safety and standard authority of India (FSSAI)
 Association of official analytical chemists
 Internal SOP

FRUITS AND VEGETABLES: (FSSAI MANUAL-3)

 pH
 Acidity
 Total soluble solids
 Drained weight
 salt
 Fruit content
 Filled capacity

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FILL OF CONTAINER:

Principle: This method determines the percent total volume of a container occupied by
the contained food. It is designed primarily for cans but can be used for wide mouth glass
containers also.

DRAINED WEIGHT:

Principle: The sample is drained on a standard mesh sieve. The weight of the material
remaining on the sieve is expressed as percentage of the can contents.

Capacity of Sieve
Sieve size can(ml) diameter(mm)

Canned fruits & veg <than 850 20

>850 30

Canned tomatoes <850 20

>850 30

Other products - 20

DETERMINATION OF SOLUBLE SOLIDS:

 Principle: Measurement of the refractive index of the test solution at 200C, using a
refractometer, and use of tables correlating refractive index with soluble solids content
(expressed as Sucrose), or direct reading of the soluble solids content on the refractometer.

 Preparation of test solution:

a) Clear liquid products- mix thoroughly & use it

b) Semi thick products: mix thoroughly & pres part of sample , leave the first drop of liquid
& use remainder of the liquid.

c) Thick products(jam, jellies): weigh 40g of sample- add 100-150ml of distilled water-heat
the contents-cool-after 20 min-filter it & use the filtrate.

DETERMINATION OF SODIUM CHLORIDE (SALT CONTENT) IN BRINE:

 Principle:

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 Direct titration of sodium chloride in brine with standard silver nitrate solution based on the
Mohr’s method is adequate for routine analysis.

 Calculation:

NaCl % = Titre value x Normality of AgNO3 x 58.45 x 100 /Weight of the sample x 1000

DETERMINATION OF PH VALUE:

pH is the measurement of H+ ion activity; It measures active acidity. pH may be

determined by measuring the electrode potential between glass and reference electrodes; pH
meter is standardized using standard pH buffers. Use homogenized sample for the determination
of pH.

DETERMINATION OF ACIDITY (APPLICABLE TO JAMS, JELLIES ALSO):

 Titrable acidity can be expressed conveniently in gm acid per 100 gm or per 100 mL as
appropriate, by using the factor appropriate to the acid as follows:

1 mL of 0.1 N NaOH equals

 Malic acid - 0.0067 gm

 Oxalic acid - 0.0045 gm
 Citric acid monohydrate - 0.0070 gm
 Citric acid anhydrous - 0.0064 gm
 Tartaric acid - 0.0075 gm
 Lactic acid – 0.0090 gm
 Acetic acid – 0.0060 gm
 Oleic acid – 0.00282 gm

 Calculation:

Acidity= eq.wt of acid* normality of NaoH *100/sample wt /vol.of alcohol*1000

Report acidity as, ml 0.1N NaoH per 100 gram or 100ml as required.

DETERMINATION OF FRUIT CONTENT:

 PRINCIPLE: By the addition of Formaldehyde one H ion is liberated per molecule of

amino acid. It is titrated with alkali. The secondary amino group of histidine does not react,
those of proline and hydroxy proline react to about 75 %. Tertiary nitrogen and guanidine –
groups undergo no reaction.

 APPARATUS: pH meter

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 Calculation:

The amount of alkali used in titration expressed as ml 0.1N alkali & referred to 100ml fruit
juice or 100g concentrate is equal to formal no.of sample.

For orange juice % fruit content is 1.05F/1.4.

EDIBLE OILS AND FATS: (FSSAI MAUAL-2)

Oils and fats are a rich source of dietary energy and contain more than twice the caloric value
of equivalent amount of sugar. Their functional and textural characteristics contribute to the
flavour and palatability of natural and prepared foods. They contain certain fatty acids which
play an important role in nutrition and are also carriers of fat soluble vitamins. Important
parameters are;

 Acid value
 Free fatty acids
 Peroxide value
 Iodine value
 Saponifiable matter
 Unsaponifiable matter

DETERMINATION OF SAPONIFIABLE MATTER:

 DEFINITION: The saponification value is the number of mg of potassium hydroxide

required to saponify 1 gram of oil/fat.

 PRINCIPLE: The oil sample is saponified by refluxing with a known excess of alcoholic
potassium hydroxide solution. The alkali required for saponification is determined by
titrating the excess potassium hydroxide with standard hydrochloric acid

 ANALYTICAL IMPORTANCE: The saponification value is an index of mean molecular

weight of the fatty acids of glycerides comprising a fat. Lower the saponification value,
larger the molecular weight of fatty acids in the glycerides and vice-versa.

 CALCULATION:

Saponification value=56.1(B-S) N/ W

B-vol in ml std Hcl req for blank, S- vol in ml std Hcl req for sample

N-normality of std Hcl, w-sample weight

DETERMINATION OF UNSAPONIFIABLE MATTER

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 Principle: Unsaponifiable matter is defined as the substances soluble in the oil, which after
saponification are insoluble in water but soluble in the solvent used for the determination.

 Calculation: Weight in g of the free fatty acids in the extract as oleic acid = 0.282 V N

Where, V = Volume in mL of standard sodium hydroxide solution N = Normality of standard

sodium hydroxide solution

Unsaponifiable matter = 100 (A-B)/ W

Where, A = Weight of the residue in gm B = Weight of free fatty acids in the extract in gm
W = Weight of the sample in gm

DETERMINATION OF ACID VALUE:

 DEFINITION: The acid value is defined as the number of milligrams of Potassium

hydroxide required to neutralize the free fatty acids present in one gram of fat. It is a relative
measure of rancidity as free fatty acids are normally formed during decomposition of
triglycerides. The value is also expressed as per cent of free fatty acids calculated as oleic
acid, lauric, ricinoleic and palmitic acids.

 PRINCIPLE: The acid value is determined by directly titrating the oil/fat in an alcoholic
medium against standard potassium hydroxide/sodium hydroxide solution.

 ANALYTICAL IMPORTANCE: The value is a measure of the amount of fatty acids,

which have been liberated by hydrolysis from the glycerides due to the action of moisture,
temperature and/or lipolytic enzyme lipase.

 CALCULATION: Acid value = 56.1 V N /W

Where, V = Volume in mL of standard potassium hydroxide or sodium hydroxide used

N = Normality of the potassium hydroxide solution or Sodium hydroxide solution; and

W = Weight in gm of the sample

Expected acid value Mass of test portion

<1 20

1-4 10

4-15 2.5

15-75 0.5

>75 0.1

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FREE FATTY ACIDS (FFA):

The acidity is frequently expressed as the percentage of FFA in the sample. The
percentage of FFA in most oils and fats is calculated on the basis of oleic acid; although in
coconut oil and palm kernel oil it is often calculated as lauric acid, in castor oil in terms of
ricinoleic acid and in palm oil in terms of palmitic acid.

FFA as oleic acid % by weight 28.2V *N/W

FFA as lauric acid % by weight 20V*N/W

FFA as risinoleic acid %by

29.8V*N/W
weight

FFA sa palmitic acid % by

25.6V*N/W
weight

IODINE VALUE:

The iodine value of an oil/fat is the number of grams of iodine absorbed by 100gm of the
oil/fat, when determined by using Wijs solution.

 PRINCIPLE: The oil/fat sample taken in carbon tetrachloride is treated with a known
excess of iodine monochloride solution in glacial acetic (Wijs solution). The excess of iodine
monochloride is treated with potassium iodide and the liberated iodine estimated by titration
with sodium thiosulfate solution.

 ANALYTICAL IMPORTANCE: The iodine value is a measure of the amount of

unsaturation (number of double bonds) in a fat.

 CALCULATION: Iodine value = 12.69 (B – S) N /W

Where, B = volume in mL of standard sodium thiosulphate solution required for the blank.

S = volume in mL of standard sodium thiosulphate solution required for the sample.

N = normality of the standard sodium thiosulphate solution.

W = weight in gm of the sample.

CEREALS AND CEREALS PRODUCTS: (FSSAI MANUAL-4)

Cereals are a staple food in most countries and are considered important source of
nutrients. They contain carbohydrate, protein and fiber, as well as micronutrients such as vitamin

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E, some of B-vitamins, magnesium and zinc. Cereals are raw materials that are used to make
cereal products.the important parameters are,

 Refractions
 Moisture
 Total ash
 Ash insoluble in dilute HCl
 Gluten
 Crude fibre
 Alcoholic acidity
 Total carbohydrates

REFRACTIONS:

Refraction means all components of food grains, which differ from normal grains such as
foreign matter, other food grains, damaged grains, weevilled grains, broken, shriveled grains etc.

QUANTITY OF SAMPLE TO BE TAKEN:

Bolded grains such as wheat/maize/barley/whole pulses- 50g

Smaller grains such as rice/split pulses/ millets- 20g.

MOISTURE: 5g sample, temp- 130-1330C, time-2hrs

TOTAL ASH: 5g sample, temp- 550-6000C, time-3hrs

ASH INSOLUBLE IN DIL.HCL:

The sample is ashed and added with dil.HCl and kept in water bath for 10mins. Then it is
cooled and filtered till it is acid free. It is then kept in oven for 3hrs, then kept in muffle furnace
at 550-6000C until grey ash is obtained.

GLUTEN:

The sample is mixed with water and made into dough. It is then soaked in water for 1hr
and washed in running water. The insoluble gluten is then dried and gluten content is calculated.

Gluten content on dry basis = weight of dry gluten*100*100/ exact weight of sample*(100-
moisture content)

ALCOHOLIC ACIDITY:

Sample is mixed with neutral ethyl alcohol and allowed to stand for 24hrs. Then it is
filtered and the filtrate is titrated against Std NaOH solution with phenolphthalein as an
indicator.
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TOTAL CARBOHYDRATES:

It is calculated after determining the percent of moisture, total protein, fat and total ash. It
includes sucrose, dextrose, dextrin, maltose or lactose percent by mass

= 100-(A+B+C+D)

ENERGY: Energy value (kilocal/g)= (4*C)+(9*F)+(4*P)

SPICES AND CONDIMENTS: (FSSAI MANUAL-7)

Spices and condiments are added to foods in small amounts but they make important
contribution to the sensory qualities due to presence of volatile and fixed oils. The important
parameters are;

 Moisture
 Total ash
 Ash insoluble in dil.Hcl
 Non-volatile ether extract
 Volatile oil
 Cold water soluble extract
 Alcohol soluble extract

MOISTURE:

DEAN AND STARK TOLUENE DISTILLATION METHOD:

PRINCIPLE: The amount of water is determined by distilling the material with an

organic liquid immiscible with water & collecting the distillate in a graduated tube.

VOLATILE OIL:

PRINCIPLE: The determination of volatile oil in spice is made by distilling the spice
with water, collecting the distillate in a graduated tube in which aqueous portion of the distillate
is automatically separated & returned to distilling flask, & measuring the volume of the oil. The
content of volatile oil is expressed as percentage v/w.

NON- VOLATILE ETHER EXTRACT:

Extract 2g of ground sample in a continuous extraction apparatus with diethyl ether for 8
hrs. Remove the ether by distillation followed by blowing with stream of air with flask on
boiling water bath. Dry in oven at 1108c and weighed. Shake the residue with 2-3ml diethyl
ether at RT. Allow to settle & decant the ether. Repeat it until no more residue dissolves. Dry it
& weigh again.

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COLD WATER SOLUBLE EXTRACT:

Weigh 2g sample in 100ml vol.flask & make up with distilled water. Shake vigorously &
filter the extract through a dry filter paper, evaporate 50ml aliquot portion. Heat in oven at
130*c. Cool and weigh.

ALCOHOL SOLUBLE EXTRACT:

Weigh 2g sample in 100ml volumetric flask and makeup with ethanol. Then allow to
stand for 4 hrs. Filter the extract & evaporate 50ml aliquot portion. Heat in oven at 103 0c. Cool
and weigh.

MEAT AND MEAT PRODUCTS, FISH &FISH PRODUCTS: (FSSAI MANUAL-10)

Meat, fish and their products are important components of diet of a large majority of
people. Their nutritive value and palatability are widely appreciated. The important parameters;

 Moisture
 Total phosphorous
 Total volatile basic nitrogen
 Extract release volume
 Sodium chloride
 Acidity of brine

MOISTURE: 5g sample, temp-100*c, time-5hrs

TOTAL PHOSPHOROUS:

PRINCIPLE: Mineralization of test portion with H2SO4 & HNO3, precipitation of

phosphorous as quinoline phosphomolybdate & drying & weighing of the precipitate.
Alternatively the sample can be ashed and ash taken up in 15ml conc.nitric acid in a conical
flask, adding water to make up 75ml, heating on a boiling water bath for 30 min, cooling &
makeup into known volume.

DETERMINATION OF NITRITE:

Principle: The present method describes a spectrophotometric method for the

determination of nitrite based on the reaction of nitrite with sulfanilamide to form a diazonium
salt, then coupling the diazotized sulfanilamide with N- (1 Napthyl) ethylenediamine
dihydrochloride to form an intensely purple coloured azo-dye which is measured
spectrophotometrically.

TESTS FOR PRESENCE OF POLYPHOSPHATES:

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 Principle: Extraction of meat or meat product with trichloro acetic acid, cleaning of the
serum obtained with ethanol / diethyl ether mixture, separation of the phosphates by thin layer
chromatography and detection of polyphosphates by spraying with reagents for colour
development.

DETERMINATION OF PH:

The pH is a measure of the acidity or alkalinity in solutions or water containing

substances. pH values lower than 7 are considered acidic, while pH values higher than 7 are
considered alkaline. A pH of 7 indicates neutrality. pH values are related to the concentration of
hydrogen ions (H+) in the substance.

Product pH value

Meat mixes in jelly+ vinegar 4.5-5.2

added

Raw fermented sausage 4.8-6.0

Beef 5.4-6.0

Pork 5.5-6.2

Canned meats 5.8-6.2

Blood 7.3-7.6

Curing brines 6.2-6.4

Blood sausages 6.5-6.8

DETERMINATION OF EXTRACT RELEASE VOLUME (ERV):

Principle: The technique is based on the volume of aqueous extract released by

homogenate of meat when allowed to pass through the filter paper for a given period of time, by
this meat of good organoleptic and microbial quality release large volume of extract, whereas
meat of poor quality releases smaller volume or none.

ERV (ml) Meat quality: > 25 mL Good quality, > 20 mL incipient spoilage, < 20 mL spoiled
meat.

DETERMINATION OF TOTAL VOLATILE BASIC NITROGEN (TVBN):

Principle: Meat extract is treated with relatively weak alkali and the volatile base is
distilled or diffused over into standard acid or boric acid.

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DETERMINATION OF SODIUM CHLORIDE:

Take 1-2 gm of the dried material (obtained after determination of moisture) in a 250 mL
beaker and add 50 mL of distilled water free from chloride and heat on a water bath till all the
Sod. Chloride goes into solution. Filter in a 250 mL conical flask and wash with distilled water
till the washings are free from chloride. Add 20 mL of dilute nitric acid and a known volume of
standard silver nitrate sufficient to precipitate all the chloride. Add 1 mL of ferric alum indicator
and titrate with standard Potassium thiocyanate solution until a permanent light brown colour
appears.

DETERMINATION OF ACIDITY OF BRINE:

Take 25- 40 mL of the brine solution, (previously filtered to remove suspended matter if
any) in a 200 mL flask, add 25- 50 mL water if desired and titrate against standard Sodium
hydroxide solution using phenolphthalein as indicator till a faint pink colour persists for 15
seconds.

MILK AND MILK PRODUCTS: (FSSAI MANUAL -1)

Dairy product, milk and any of the foods made from milk, including butter, cheese, ice
cream, yogurt and condensed and dried milk. The important parameters are;

 Moisture
 Total solids
 Total ash
 Acid insoluble ash
 Titrable acidity as lactic acid

MOISTURE: 1g sample, temp-102*c, time-2hours

DETERMINATION OF TOTAL SOLIDS (GRAVIMETRIC METHOD):

In this procedure, a known quantity of milk is dried on a boiling water bath. Subsequently
sample is dried in hot air oven at 102 ±2°C and from the weight of the residue, the total solids
content in milk is determined.

Total Solid Content =M2 − M0/ M1 − M0 x 100

DETERMINATION OF FAT IN MILK:

 METHOD 1: GERBER METHOD: The milk is mixed with sulphuric acid and iso-amyl
alcohol in a special Gerber tube, permitting dissolution of the protein and release of fat. The
tubes are centrifuged and the fat rising into the calibrated part of the tube is measured as a
percentage of the fat content of the milk sample. The method is suitable as a routine or
screening test.
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 METHOD 2: ROSE-GOTTLIEB METHOD: The milk sample is treated with ammonia
and ethyl alcohol; the former to dissolve the protein and the latter to help precipitate the
proteins. Fat is extracted with diethyl ether and petroleum ether. Mixed ethers are evaporated
and the residue weighed.

Fat % (w/w) = Weight of Extracted Fat/ Weight of milk X100

 METHOD 3: ACID DIGESTION METHOD (WERNER SCHMIDT METHOD): In

this method, milk proteins are digested with concentrated hydrochloric acid. Liberated fat is
extracted with alcohol, ethyl ether and petroleum ether. Ethers are evaporated and residue left
behind is weighed to calculate the fat content.

CALCULATION: Fat, % (w/w) =100 (W1 − W2) /W3

TITRABLE ACIDITY:

The method is based on the titration of the sample with sodium hydroxide to
phenolphthalein end point and by comparing the colour with colour obtained by mixing
rosaniline acetate or cobalt sulphate to a known volume of milk sample.

=Volume of 0.1 NaOH x 100 x 100 x 10 /Weight of MSNF x weight of sample

TOTAL ASH:

Weigh accurately about 3 g of the dried milk sample in the crucible, previously dried in a
hot air oven and weighed. Heat the crucible gently on a burner or hot plate at first and then
strongly in a muffle furnace at 550  20°C till grey ash is obtained. Cool the crucible in a
desiccator and weigh it. Heat the crucible again at 550  20°C for 30 min. Cool the crucible in a
desiccator and weigh.

CALCULATION: Total ash (on dry basis), % by mass = M2 − M /100 − M0 x ( M1− M) 𝑥 100

BAKERY PRODUCTS: (IS 12711):

Bakery products which include bread, rolls, cookies, pies, pastries and muffins are
usually from flour or meal derived from some form of grain. The important parameters are;

 Moisture
 Total ash
 Total solid content
 Fat
 Fruits in fruit bread/ cake
 Non-fat milk solids in milk bread
 Acidity of extracted fat

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MOISTURE: 5g sample temp-105*c time- 4hours

TOTAL ASH: 5g sample, temp-500+10*c

TOTAL SOLID CONTEN T: =100- moisture (%)

FAT: Dried sample (10-30g)-dry (2hrs, 100+2*c)-soxhlet extraction (16hrs)-dry 100*c for 1
hour

ACIDITY OF EXTRACTED FAT:

Same procedure as fat, then add 50ml mixed benzee alcohol-phenolpthalein reagent.
Then titrate against KOH solution. End point (pnik colour).

CALCULATION: Acidity as extracted fat= 1.41*V/M1-M, M1- flask with sample, M-empty
flask weight

FRUITS IN FRUIT BREAD /CAKE:

Both dry fruits and preserved fruits are picked out and weighed.

COMMON SALT: (IS 253)

Salt has long been used for flavouring and for preserving food. It commonly features as
the table or in the kitchen as free-flowing table salt, rock salt, sea salt, or kosher salt. The
important parameters are

 Moisture
 Matter insoluble in water
 Total chlorides
 Sulphate
 Matter soluble in water other than NaCl
 Alkalinity

MOISTURE: 20g sample, temp-140-150*c, time- 4 hours

MATTER INSOLUBLE IN WATER:

Take 20g sample and dissolve in water and then boil& cool. filter and wash the residue
until free from soluble salts. Collect the filtrate &preserve it. Dry the crucible containing
insoluble residue.

CALCULATION: =100*M1/M2, M1- residue weight(g), M2- dried sample wt.

TOTAL CHLORIDES:

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 10 ml of preserved solution & add 1ml of potassium chromate indicator solution. Titrate
against with silver nitrate. End point: reddish brown tinge.

CALCULATION: Total chloride(cl) % by mass= 354.6*V*N/M

MATTER SOLUBLE IN WATER OTHER THAN NACL:

=100-(NaCl + matter insoluble in water)

SULPHATE:

Dissolve 10g of dried common salt in 400ml water. Filter & wash residue free from soluble salts.
Collect the filtrate & washings, add 1 drop of methyl orange, 10ml of dil.Hcl and then boil.
During boiling, add 10-12ml barium chloride solution, continue boiling-until granular precipitate
is obtained. Filter through GC, wash the precipitate free from chlorides, then dry it in oven at
105-1100c.

 CALCULATION:

Sulphate(as SO4) % by mass= 41.13*M1/M2, M1-wt of barium sulphate,M2-dried sample wt.

IS STANDARDS:

1.ANIMAL FOOD AND

FEED

IS 7874 PART-1 ANIMAL FEEDS & FEEDING

STUFFS(GENERAL METHODS)

IS 7874 PART-2 ANIMAL FEEDS & FEEDING STUFFS-

MINERALS & TRACE ELEMENTS

IS 13433 PART-1 ANIMAL FEEDS & FEEDING STUFFS-

CALCIUM

IS 7219 ANIMAL FEEDS & FEEDING STUFFS-

PROTEIN

IS 1374 POULTRY FEEDS

2.COSMETICS &
ESSENTIAL OILS

IS 11142 SPECIFICATION FOR HENNA POWDER

IS 7159 METHODS OF TEST FOR HENNA

POWDER

21
IS 326 PART-3 NATURAL& SYNTHETIC PERFUMERY
MATERIALS-DETERMINATION OF
RELATIVE DENSITY

IS 326 PART-10 ATURALS & SYNTHETIC PERFUMERY

MATERIALS-DETERMINATION OF
RESIDUE ON EVAPORATION

IS 5383 SPECIFICATION FOR TOOTH POWDER

IS 286 METHODS OF SAMPLING & TEST FOR

SOAPS

IS 3959 SPECIFICATION FOR SKIN POWDER

IS 7679 SPECIFICATION FOR HAIR CREAM

IS 9740 SPECIFICATION FOR SHAVING CREAM

3.FOOD AND
AGRICULTURAL
PRODUCTS

IS 12711 BAKERY PRODUCTS

IS 1011 BISCUITS

IS 3752 ALCOHOLIC DRINKS

IS 3506 TABLES FOR ALCOHOLOMETRY

IS 4079 SPECIFICATION FOR CANNED

RASAGOLLA

IS 4333 PART-1 FOOD GRAINS-REFRACTIONS

IS 4333 PART-2 FOOD GRAINS-MOISTURE CONTENT

IS 4333 PART-5 FOOD GRAINS-URIC ACID

IS 1155 SPECIFICATION FOR WHEAT ATTA

IS 1009 SPECIFICATION FOR MAIDA

IS 1435 MACARONI,SPAGHETTI, VERMICELLI

& EGG NOODLES-SPECIFICATION

IS 9487 SPECIFICATION FOR RTE-RICH

EXTRUDED FOODS

22
IS 1158 SPECIFICATION FOR CORN FLAKES

IS 2639 SPECIFICATION FOR PAPAD

COFFEE & COCOA

PRODUCTS

IS 3077 SPECIFICATION FOR ROASTED &

GROUND COFFEE

IS 2791 SOLUBLE COFFEE POWDER-

SPECIFICATION

IS 3309 SOLUBLE COFFEE-CHICORY POWDER-

SPEC

IS 1164 COCOA POWDER-SPEC

IS 8833 COCOA BEANS- MOISTURE CONTENT

IS 11923 COCOA MASS-SPEC

EDIBLE OILS & FATS

IS 548 PART-1 METHODS OF SAMPLING & TEST FOR

OILS & FATS-PHYSICAL & CHEMICAL
TEST

IS 548 PART-2 METHODS OF SAMPLING & TEST FOR

OILS & FATS- PURITY TEST

IS 9037 SPECIFICATION FOR PEANUT BUTTER

IS 7224 SPEC- IODIZED, VAPOUR

EVAPORATED IODIZED, REFINED
IODIZED SALT

IS 253 SPECIFICATION FOR COMMON SALT

FOOD ADDITIVES AND

PRESERVATIVES

IS 4467 SPECIFICATION FOR CARAMEL

IS 1320 SPECIFICATION FOR BAKER’S YEAST

IS 6031 SPEC-CALCIUM PROPIONATE

IS 9971 SPEC FOR LACTIC ACID

23
FRUITS AND FRUIT
PRODUCTS

IS 2860 SAMPLING & TEST FOR PROCESSED

FRUITS & VEGETABLES

IS 13815 F & v-DETERMINATION OF TSS-

REFRACTOMETER

IS 13845 F & V-ASH INSOLUBLE I DIL.HCL

IS 1797 TEST FOR SPICES & CONDIMENTS

IS 7807 METHODS OF TEST FOR ASAFOTEIDA

IS 4941 EXTRACTED HONEY-SPECIFICATION

MILK & DAIRY

PRODUCTS

IS 1479 PART-1 METHODS OF TEST FOR DAIRY

INDUSTRY-RAPID EXAMINATION OF
MILK

IS 1479 PART-2 METHODS OF TEST FOR DAIRY

IDUSTRY-CHEMICAL ANALYSIS

IS 3507 SAMPLING & TEST FOR BUTTER

IS 3508 SAMPLING & TEST FOR GHEE

MEAT AND MARINE

PRODUCTS

IS 5960 PART-1 METHOD OF TEST-NITROGE CONTENT

IS 5960 PART-2 METHOD OF TEST –TOTAL ASH

IS 5960 PART-3 METHOD OF TEST-TOTAL FAT

IS 5960 PART-4 METHOD OF TEST- FREE FAT

CONTENT

IS 5960 PART-5 MOISTURE CONTENT

24
 IS 5960 PART6 SEC1 CHLORIDE CONTENT(VOLHARD
METHOD)

IS 5960 PART6 SEC 2 CHLORIDE CONTENT-

POTENTIOMETRIC METHOD

IS 5960 PART-7 NITRITE CONTENT

IS 5960 PART-10 MEASUREMENT OF PH

IS 5960 PART-13 POLYPHOSPHATES

IS 5960 PART-16 TOTAL PHOSPHOROUS CONTENT

WATER:
METHODS OF SAMPLING AND TEST (PHYSICAL AND CHEMICAL) FOR WATER
AND WASTE WATER:

COLOUR:
Scope - Prescribes the following two methods for the determination of colour.

a) PLATINUM COBALT (VISUAL COMPARISION)

Principle - Colour is measured by visual comparison of the sample with platinum -

cobalt standards. One unit of colour is that produced by 1 mg of platinum per litre in the form of
chloroplatinate ion.

B) APPARATUS:

Nessler cylinders - 50 ml capacity

Preparation of standards: Prepare standards having colours units of 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 60 and 70 water to 100 ml.

ODOUR: [PART 5]

Fill the bottle half full of sample

Stopper insert

Vigorous shaking (2 to 3 sec)

25
 Observe the odour

TASTE RATING: (Part 8)

Principle - Each panelist (tester) is presented with a list of nine statements about the
water, ranging from very favourable to very unfavourable. The tester selects a statement that best
expresses his opinion. The scored rating is the scale number of the statement selected. The panel
rating is the arithmetic mean of the scale numbers of all testers.

The samples are rated as follows:

a) Initially taste about half of the sample by taking the water into the mouth, holding it for
several seconds, and discharging it without swallowing;

b) Form an initial judgement on the rating scale;

c) Make a second tasting the same manner as the first;

d) Make a final- rating for the sample and record the results on the appropriate data form;

e) Rinse the mouth with taste-and odour-free water; and

f) Rest for one minute before repeating steps (a) to (e) on the next sample.

pH: (part 11)

The pH value is determined by measurement of the electromotive force of a cell o

consisting of an indicator electrode (an electrode responsive to hydrogen ions such as a glass Q;
electrode) Immersed in the test solution and a reference electrode (usually mercury/calomel 0:
electrode). Contact between the test solution and the reference electrode is usually achieved by
means of a liquid junction, which forms part of the reference electrode. The electromotive force
is measured with a pH meter, that is, a high impedence voltmeter calibrated In terms of pH .

ELECTRICAL CONDUCTIVITY: (Part 14)

Principle: Ability of solution to conduct an electric current is a function of the

concentration and charge of ions in solution depends on ionic mobility.

TURBIDITY: (Part 10)

Principle: It is based on the comparison of the intensity of light scattered by the sample
under defined conditions with the intensity of light scattered by a standard reference suspension
under the same condition.[If intensity is high it may be highly turbid ]

TOTAL HARDNESS: (Part 21)

26
 Principle: This method depends on ability of ethylenediamine tetraacetic acid
(C10H16O8N2) or its disodium salt to form stable complexes with calcium and magnesium ions.
When the dye eriochrome black T (EBT) is added to a solution containing calcium and
magnesium ions at pH 10.0 a wine red complex is formed. This solution is titrated with standard
solution of disodium salt of EDTA, which extracts calcium and magnesium from the dye
complex and the dye is changed back to its original blue colour.

Procedure:

50 ml sample was taken

Add 2ml ammonium buffer+0.5g EBT

Titrate against EDTA sol(till sky blue colour)

Calculation:

Titre value *M of EDTA *100*1000 / vol of sample.

CALCIUM:

Principle: This method depends on ability of ethylenediamine tetraacetic acid

(C10H16O8N2) or its disodium salt to form stable complexes with calcium and magnesium ions.
Calcium is directly determined with EDTA when the ph is high(12 to 10) and magnesium is
precipitated as the hydroxide and an indicator to form the wine red complex.

Procedure:

Take aliquot amount of sample

Add 2ml NaOH

Add 1-2 drops of patton’s &reader’s indicator (blue dye)

Titrate against EDTA

Purple blue colour (end point)

Calculation:

27
Titre value *M of EDTA *40.08*1000 / vol of sample

TOTAL ALKALINITY:

Principle: Alkalinity of water is the capacity of that water to accept protons. It

may g be defined as the quantitative capacity of an aqueous medium to react with hydrogen Ions
to pH 8.3 (phenolphthalein alkalinity) and then to pH 3.7 (total alkalinity or methyl orange
alkalinity).

Take 100ml sample

Add 5 drops of mixed indicator(sharper end point colour change)

Titrate against std sulphuric acid till light pink colour appears

Calculation:

Titre value *N of H2SO4 *50*1000 / vol of sample

CHLORIDE:

Principle: In a neutral or alkaline solution potassium chromate can indicate the end
point of the silver nitrate, titration of chloride .silver chromate is precipitated before red silver
chromate is formed.

Procedure:

Take req amount of sample

Add few drops of potassium chromate (oxidizing agent)

Titrate against std AgNO3

Pinkish yellow (end point)

Calculation:

Titre value *N of AgNO3 *35.45*1000 / vol of sample

28
MAGNESIUM:

Calculation:

[Total hardness –calcium]

Titre value *M of EDTA*24.31*1000 / vol of sample

NITRATE: APHA METHOD

Principle: This techniques is suitable for samples have low organic matter. Nitrate was
measure at 225&275 nm. 225nm enables is to determine the dissolved organic matter 275 nm
doesn’t absorb the dissolved organic matter.

Procedure:

Take 50 ml of sample

Add 1ml 1N HCL

Wait for 10 mins

Read @220 &275 nm

Calculation:

Sample absorbance*calibration factor*dilution factor*50 / vol of sample

NO3 [IS METHOD]

a)Chromotrphic acid method:

Principle: in chromotrophic acid method, two moles of nitrate nitrogen react with one
mole of chromotrophic acid to form a yellow reaction product.

Procedure:

Take 2ml sample in 10 ml flask

29
 Add one drop sulphite urea reagent

The flask with sample maintained to 10 -20*c

Add 2ml antimony reagent(wait for 4 mins)

Add 1ml chromotrophic acid(wait for 3 mins)

Make upto 10ml with conc.H2SO4

Mix the solution by inverting the closed flask

Keep it at room temperature for 45 mins

If the sample is reduced, makeup with 10 ml conc.H2SO4

After 15 mins,read @410nm

Calculation:

Absorbance*calibration factor

b)Devardhas alloy method

Principle: In devardhas alloy method nitrate &nitrite are reduced to ammonia under
alkaline condition.

Procedure:

Take 250 ml sample

Add 25 ml borate buffer

30
 Add 1g devardhas alloy salt

Collecting the nitrate

Add boric acid+methyl blue indicator (colour change to green)

Titrate against conc H2SO4

NITRITE (Part 34)

Principle: It is determined through formation of reddish purple azo dye produced at

(ph 2-2.5) by coupling diazotized sulphanic acid with N-1 napthyl 1- thylene diamine
dihydrochloride. The colour obey beer’s law upto 180 micro g with 1cm path length at 543nm.

Procedure:

Take 50ml sample

Add 1ml sulphanilamide(wait for 10 mins)

Add 1ml NEEDA solution (wait for 10 mins)

Read @543nm

FLUORIDE (Part60):
a)Visual comparison:

Principle: The colour (red to yellow with high concn of fluoride) obtained with zirconium
alizarin reagent is matched against that produced with a std fluoride solutions.

Procedure:

Take 100ml sample

Add 5ml zirconium-alizarin reagent

Wait for 1 hour, compare with std fluoride soln

SULPHATE (Part 24)-IS method:

Principle: Sulphate ion is precipitated in HCL medium with barium chloride in such a
manner to form barium sulphate crystals of uniform size. Absorbance of barium sulphate

31
suspension is measured by a spectrophotometer &sulphate ion concn is determined by
comparison of the reading with a std curve.

Procedure:

Take 20 ml sample

Add 1ml 1:9HCL+conditioning reagent

Add 0.5g barium chloride (wait for 10 min)

Read @420nm

Calculation:

Absorbance*calibration factor*dilution factor*20 / vol of sample

SO4 (APHA Method):

Take req amount of sample

Make up with distilled water in 100ml flask

Add 20ml SO4 buffer

Add barium chloride(wait for 10 mins)

Read @ 420nm

IRON (part 53)

Principle: iron in the solution is reduced to ferrous state by boiling with hydrochloric acid
&hydroxylamine &treated with 1,10 phenonthroline at ph 3.3.three molecules of phenonthroline
chelate each atom of ferrous iron to form orange red complex.the coloured solution obey beer’s
law. Colour intensity is independent of ph from 3to 9. A ph between 2.9 to 3.5 insure rapid
colour development in the presence of a high phenonthroline.

Procedure:

Take 100ml sample

Add 1ml hydroxylamine&2ml conc.HCL

Place on hot plate for boiling

Sample reduced to 50ml, cool down the soln

32
 Add 10ml ammonium acetate&10 ml 1,10- phenonthroline soln

Make upto 100ml with distilled water

Read @ 510 nm

Calculation:

Absorbance*calibration factor*dilution factor*100 / vol of sample

SILICA (PART 35):

Principle: Ammonium molybdate at pH 1.2 react with silica & any phosphate present to
produce heteropoly acids. Yellow molybolosilicic acid is reduced by means of aminonapthol
sulphonic acid to heteropoly blue.

Procedure: 50ml sample was taken-then add 1ml of HCl and 2ml ammonium molybdate.
Then after 10 mins, add 2ml of oxalic acid & 2ml of reducing agent. Then after 10 mins, read the
solution @ 815nm in spectrophotometer.

AMMONIA- PART 34:

Procedure: Take 50ml of sample- then add one drop of EDTA reagent. 2ml of neiseler
reagent is added. Wait for 10 minutes, read @ 400nm in spectrophotometer.

Calculation: (B*C*D*50*0.83)/A, A-volume of sample, B-absorbance, C-calibration D-

dilution factor.

HEXAVALENT CHROMIUM PART-52

(Diphenyl carbazide method)

50ml sample-Then add 5drops of orthophosphoric acid & makeup to 100ml. Add 2ml of
diphenyl carbazide solution. After 5-10mins, read@ 540nm.

Calculation: Absorbance *C .F* makeup volume* dilution factor/ volume of sample.

SULPHIDE: (IODOMETRIC METHOD)-PART 29

Principle: sulphides are stripped from the acidified sample with an inert gas & collected in
zinc acetate solution. Excess iodine solution added to the zinc sulphide suspension react with the
sulphide under acidic solution.

Procedure:Take 1-5ml of sample in 10ml flask –then add 0.5ml amine H2SO4, add 0.15ml
of Fecl3.6H2O. In a separate 10ml flask ,take 1.5ml distilled water(blank)-add 0.5ml of 1:1
H2SO4, add 0.15ml of Fecl3.6H2O, wait for 10mins. And then add 1-6ml of diammonium
dihydrogen phosphate for both flasks. Wait for 3mins & read @ 664nm

33
 Calculation: B*C*D

MICROBIOLOGY DEPARTMENT:

In microbiology department, food (Marine), water, environmental (air) samples are

analysed. The analysis is carried out by different methods such as:

 IS
 ISO
 BAM Method
The food samples include:
 Shrimps
 Egg
 Fish varieties – cuttle fish
 Meat samples
 Fish meal
 Fish soluble paste
 Fish oil
 Hotel foods
 Bakery samples – Puff, sweets, jam
 Honey
 Tinned vegetables
 Cocoa powder
 Baby foods – cerelac
 Sugar and salt
 Milk
Water samples:

 STP water
 Domestic water
 Drinking water
 BIS Samples.
Parameters to be analyzed:

1. Food samples:

The contamination of foods from natural sources may take place before the food is
harvested or during handling and processing of food. Additional contamination may come from
equipment coming in contact with foods. The parameters that are analyzed in food samples
include:

34
  TPC
 Escherichia coli
 Coliform
 Bacillus cereus
 Enterobacteriacea
 Staphylococcus aureus
 Listeria monocytogens
 Yeast and mould
 Shigella
 Salmonella
 Vibrio cholera
 Vibrio parahaemolyticus
 Vibrio vulnificus
 Clostridium perfringes

2. Water samples:
Natural water contains not only their natural flora but also microorganisms from soil and
possibly from animals or sewage. APHA have developed some methods for confirmation of
E.coli in water samples. Some of nonfermenting bacteria such as pseudomonas species actually
grow in water lines and are not detected by traditional coliform analysis, so TPC are carried out.

 TPC
 Total coliform
 Escherichia coli
 Faecal streptococci
 Faecal coliform
 Vibrio species
3. Air samples:

Microorganisms in air sample depend on sunshine, humidity, location and amount of

suspended dust. Microorganisms count in air is increased by air currents caused by movement of
people, by ventilation and by breezes.

 Legionella

METHODS:

For packaged drinking water:

ORGAANISMS IS METHODS

35
 AMC-22̊C (72 hours) IS 5402: 2012

AMC-37̊C (24 hours) IS 5402: 2012

Coliforms IS 15185: 2016

E.coli IS 15185: 2016

Faecal streptococci IS 15186: 2002

Pseudomonas aeruginosa IS 13428: 2005

Salmonella IS 15187: 2016

Shigella IS 5887 Part 7:

1999

Sulpite reducing anaerobes IS 13428: 2005

Vibrio cholera IS 5887 Part 5:

1976

Vibrio parahaemolyticus IS 5887 Part 5:

1976

Yeast and mould IS 5403: 1999

Staphylococcus aureus IS 5887 Part 2:

1976

Drinking water: 10500

ORGANISMS METHOD

Coliform IS 15185

E. coli IS 15185

Lipolytic bacteria IS 4251

Proteolytic bacteria IS 4251

36
Food samples:

ORGANISMS ISO METHOD

Bacillus cereus ISO 7932

Clostridium perfringes ISO 7937

Coliform ISO 4832

Enterobactericae ISO 21528 Part 2

E.coli ISO 16649 Part 2

L.monocytogens ISO 11290 Part 1

Salmonella ISO 6579 Part 1

Shigella IS 5887 Part 7

Staphylococcus aureus ISO 6888 Part 1

SRB ISO 15213

TPC ISO 4833

Vibrio cholera ISO 21872 Part 1

Yeast and mould IS 5403

Vibrio parahaemolyticus ISO 21872 Part 1

Vibrio Vulnificus ISO4872 Part1:2017

Air samples: Legionella – ISO 11731 Part 2: 2004

BAM Method:

 E.Coli - Chapter 4
 Coli - Chapter 4
 Staphylococcus aureus - Chapter 12
 Shigella - Chapter 6

37
  Salmonella - Chapter 5
 Vibrio cholera - Chapter 9
 Vibrio parahaemolyticus - Chapter 9
 Vibrio vulnificus - Chapter 9
 Listeria monocytogens - Chapter 9
 Entreobactericae - APHA 5th edition Chapter 9

MEDIA AND ITS USES:

1. Acetamide broth - Confirmation of pseudomonas aeurginosa

2. Agar agar- Adjust desired Ph of medium
3. Alkaline peptone water - Enrichment of Vibrio species
4. Azide dextrose broth - Detection of streptococci in sewage water
5. Alkaline saline peptone water - Enrichment of vibrio species
6. Baird parker agar base - Isolation and enumeration of staphylococci
7. Brilliant green bile broth - Detection and confirmation of coliform
8. Bile esculine azide agar - Isolation and identification of coliform
9. Bile salt agar - Isoaltion of bile tolerant enteric bacteria
10. Bismuth sulphite agar - Isolationof salmonella
11. Brain heart infusion - Isolation of staphylococci
12. Buffered peptone water - Pre enrichment medium for salmonella
13. Buffered charcoal yeast extract agar - Isolation and cultivation of legionella
14. Clostridial agar - Selective isolation of pathogenic clostridia
15. Cooked meat medium - Cultivation of aerobes and anaerobes
16. Deoxycholate citrate agar - Isolation of salmonella and shigella
17. CPC agar - Used for vibrio species
18. Dextrose tryptone agar - Isolation and cultivation of aciduric, thermophilic bacteria
19. Rose Bengal agar – Isolation and enumeration of fungi, yeast and mould
20. DRCM - Cultivation of clostridia from water.
21. Peptone saline\salt solution - Used as nutritive medium, to maintain osmotic pressure

Different media and its PH:

Micro organisms have a minimal, maximal and optimum PH for growth. In general
yeasts and molds are more acid tolerant than bacteria. Foods with low PH (below 4.5) usually are
not readily spoiled by bacteria and are most susceptible to spoilage by yeasts and mold

1. Acetamide broth - 7.0±0.2

2. Alkaline peptone water - 8.4±0.2
3. Alkaline saline peptone water - 8.6±0.2
4. Arginine dihydrolase broth - 6.0±0.2

38
 5. Azide dextrose broth - 7.2±0.2
6. Alkaline saline peptone water - 7.2±0.2
7. Baird parker agar base - 6.8±0.2
8. Bile esculine azide agar – 7.1 ±0.2
9. Bile salt agar - 8.5±0.2
10. Bismuth sulphite agar - 7.7±0.2
11. Brain heart infusion - 7.4±0.2
12. Buffered peptone water - 7.2±0.2
13. Buffered charcoal yeast extract agar - 6.9±0.2
14. Chloramphenical yeast glucose agar - 6.6±0.2
15. Cooked meat medium - 7.8±0.2
16. Deoxycholate citrate agar - 7.5±0.2
17. CPC agar - 7.6±0.2
18. Dextrose tryptone agar -6.7±0.2
19. Rose Bengal agar – 5.6±0.2
20. DRCM -7.2±0.2
Food samples:

ISO METHOD (Enumeration):

1. TPC (Total plate count)

Material required: Peptone saline solution and plate count agar

Procedure: 10 gram of sample should be added to 90 ml of peptone saline solution.Further 1 ml

from first dilution should be added to 9 ml of PSS and its continued upto 10*5 times.Incubation
period - 30±1̊C for 72±3 hours.

2. Escherichia coli

Material required: Peptone saline solution and TBX Agar

Procedure: 10 gram of sample should be added to 90 ml of peptone saline solution.Further 1 ml

from first dilution should be added to 9 ml of PSS and its continued upto 10*5 times.Incubation
period - 44±1̊C for 18-24 hours.If there is presence of E.coli, blue colour colonies will be
observed by pour plate technique.

3. Total coliform:

Material required: Peptone saline solution and VRBLA

Procedure: 10 gram of sample should be added to 90 ml of peptone saline solution. Further 1 ml

from first dilution should be added to 9 ml of PSS and its continued upto 10*5 times. Incubation

39
period - 37̊C for 24 hours. In positive control, pink color colonies with zone of precipitation will
be observed by pour plate technique.

4. Staphylococcus aureus :

Material required: Peptone saline solution

Procedure: 10 gram of sample should be added to 90 ml of peptone saline solution. Further 1 ml

(0.3, 0.3,0.4 ) from first dilution should be added to 9 ml of PSS and its continued upto 10*5
times. Incubation period - 37̊C for 48 hours.

5. Enterobacteriace:

Materials required: Peptone saline solution, Violet red bile glucose agar

Procedure: 10 gram of sample should be added to 90 ml of peptone saline solution. Further 1 ml

from first dilution should be added to 9 ml of PSS and its continued upto 10*5 times. Incubation
period - 37̊C for 24±2 hours.

Positive control: Pink color colonies with white precipitation.

ISO METHOD(Isolation):

1. Listeria monocytogens:
Materials required :Half fraser broth, Fraser broth, L.mono differential agar, PALCAM agar.

Procedure : 25 gram of sample – inoculated into 225 ml of half fraser broth`

Media Inc. time Inc. Positive control

temperature

1 enrichment 30±1̊C 25±1 Growth observed

Half fraser broth

2 enrichment 37±1̊C 24±2 Growth observed

Fraser broth

Selective agar 1 37±1̊C 48±2 Green blue colonies with opaque

halo zone observed.
(1 enrichment)

L.mono differential
agar

Selective agar 2 37±1̊C 48±2 Black colonies with black halo

40
(1 enrichment) zone observed

PALCAM agar

Selective agar 1 37±1̊C 48±2 Green blue colonies with opaque

halo zone observed.
(2 enrichment)

L.mono differential
agar

Selective agar 2 37±1̊C 48±2 Black colonies and black halo

zone observed..
(2 enrichment)

PALCAM agar

2. Salmonella:

Materials required: BPW, RVS, MKTTN, XLDA, BGA.

Procedure: 25 gram of sample – inoculated into 225 ml of Buffered peptone water.

Media Inc. Inc. Sample Positive control

time temperature

Pre enrichment medium 18±2 34 to 38̊C Turbidity observed Growth observed

BPW

Selective enrichment 24±3 41.5 to 42.5̊C Turbidity observed Growth observed

A RVS

B MKTTN Broth 24±3 37±1̊C Turbidity observed Growth observed

3. Vibrio cholera:

Materials required: BPW, RVS, MKTTN, XLDA, BGA.

Procedure: 25 gram of sample – inoculated into 225 ml of Alkaline peptone water.

Media Inc. Inc. Sample Positive control

time temperature

1 selective enrichment 6±1 34 to 38̊C Turbidity observed Growth observed

medium – APW

41
2 Selective enrichment 18±1 41.5 to 42.5̊C Turbidity observed Growth observed

APW

Selective agar 1(str. 24±3 37±1̊C Yellow color Growth observed

From 1 enrichment) colonies observed

TCBS Agar

Selective agar 2(str. 24±3 37±1̊C Colorless colonies Growth observed

From 1 enrichment)

Bile salt Agar

Selective agar 1(str. 24±3 37±1̊C Yellow color Growth observed

From 2 enrichment) colonies observed

TCBS Agar

Selective agar 2(str. 24±3 37±1̊C Colorless colonies Growth observed

From 2 enrichment)

Bile salt Agar

IS Methods: (Enumeration)

1. Yeast and mould:

Materials required: Peptone saline solution, CYGA

Procedure: 10 gram of sample should be added to 90 ml of peptone saline solution.Further 1 ml

from first dilution should be added to 9 ml of PSS and its continued upto 10*5 times.Incubation
period - 25̊C±1̊C for 5 days

2. Bacillus cereus:

Materials required: Peptone saline solution,mannitol egg yolk polymxin agar

Procedure: 10 gram of sample should be added to 90 ml of peptone saline solution.Further 1 ml

from first dilution should be added to 9 ml of PSS and its continued upto 10*5 times.Incubation
period - 30̊C for 48 hours.

Positive control: Pink color colonies with white precipitation.

3. LBC:

42
Sample dilution: 0.85 % Saline solution.

Dilution: 1ml 9ml 1ml9ml upto 10*5 diluitons.

Medium used:Tributrin and nutrient agar.

Incubation : 30̊C for 72 hours

Inoculation: 1 ml.

4. PBC:

Sample dilution: 0.85 % Saline solution.

Dilution: 1ml 9ml 1ml9ml upto 10*5 diluitons.

Medium used:Skim milk agar.

Incubation : 37̊C for 48 hours

Inoculation: 1 ml

IS Method(MFM):

1. Pseudomonas aeruginsoa:

Procedure: 250 ml of sample filtered through membrane filter an dfilterpaper(0.45µm and 47mm
diameter) placed on appropriate medium.

Medium Incubation Observation

Selective agar 37̊C – 48 Hour Turbidity observed

APB

Milk agar and 42̊C – 24 Hour Pigmentation and casein

cetrimide hydrolysis observed.

BAM Method:

1. Clostridium perfringens:

Method: Enumeration method

Sample dilution: 0.1 % peptone diluents solution.

43
Dilution: 25 gm/ml of sample in 225 ml of PDS and diluted upto 10*6.

Medium used: TSC agar.

Incubation: 35̊C for 20-24 hours

Inoculation: 1 ml.

In positive control, black color colonies with white opaque zone will be observed.

2. Bacillus cereus:

Method: Enumeration method

Sample dilution: Butterfields phosphate buffered solution.

Dilution: 50 gm/ml of sample in 450 ml of BPBS and diluted upto 10*6.

Medium used: Mannitol egg yolk polymixin agar.

Incubation: 30̊C for 48 hours

Inoculation: 0.1 ml.

In positive control, pink color colonies with white precipitation will be observed.

3. Enterobacteriace:

Test method: APHA 5th edition Chapter 9

Method: Enumeration method

Sample dilution: 0.1 % peptone diluents solution.

Dilution: 25 gm/ml of sample in 225 ml of PDS and diluted upto 10*6.

Medium used: Violet red bile glucose agar.

Incubation : 35̊C for 18-24 hours

Inoculation: 1 ml.

In positive control, pink color colonies with zone of precipitation will be observed

4. Shigella:

Materials required: Shigella broth base

Procedure:25 gm/ ml of sample in 225 ml of shigella broth base

44
 Medium Incubation Positive control

Enrichment medium 42̊C – 20 Hour Growth observed

(SBB)

Incub. anaerobically

selective agar 35̊C – 20 Hour Colonies are slightly pink,

translucent, rough edges
(MaC) observed.

Nutrient agar 35̊C – 24 Hour Growth observed.

Further, biochemical test are carried to confirm the presence of particular organisms.

5. Vibrio alginolyticus:

Materials required: Alkaline peptone water.

Procedure:25 gm/ ml of sample in 225 ml of Alkaline peptone water.

Medium Incubation Positive control

Enrichment medium 35±2̊C – 6 to 8 Hour Turbidity observed

(APW)

Incub. anaerobically

Selective agar 35±2̊C –18 – 24 Yellow colony

Hour
(TCBS agar)

Selective agar 35±2̊C – 18 – 24 Green to purple

Hour colony.
(CPC agar )

T1N1 Agar 35±2̊C – 18 - 24 Growth observed.

Hour

INSTRUMENTATION DEPARTMENT:

LC EXTRACTION DIVISION:

45
 The LC extraction division deals with the extraction process of seven antibiotics and
pesticides which are then quantified using LCMS-MS(Liquid chromatography – mass
spectrometry).

EXTRACTION:

Extraction in chemistry is the separation process which involves separation of target

substance from the sample or matrix. There are four types of extraction which is as follows,

 Solid-liquid extraction: When the sample is a solid and the required phase for analysis is a
liquid, the process is called solid-liquid extraction.
 Liquid-liquid extraction: In liquid -liquid extraction compounds separate according to their
relative solubility in two different immiscible liquid phases.
 Solid phase extraction: solid phase extraction is a sample preparation process by which
compounds that are dissolved or suspended in a liquid matrix are separated from the other
compounds in the mixture according to physical and chemical properties.
 Acid-base extraction: Acid -base extraction is typically used to separate organic compound
from each other based on their acid base interaction properties. Acid-base extraction is a type
of liquid- liquid extraction.

ANTIBIOTICS EXTRACTION:

The antibiotic parameters extracted here are done by the solid – liquid extraction process and
solid phase extraction process. The antibiotics extracted here includes,

 NFM(Nitrofuran metabolite)
 NFP(Nitrofuran parent)
 CAP(Chloramphenicol)
 Tetracycline compounds
 Dyes
 Sulphonamide compounds
 Quinolone compounds

NFM & NFP COMPOUNDS:

These are synthetic board spectrum antibiotics having a furan group. They are
heterocyclic compounds. It is a carcinogenic substance. The NFM compounds are the
metabolites of NFP compounds which are having a short life of about 63 mins. So the NFP
extraction is done as soon as the sample is received. For NFM extraction the sample is added
with 0.2M HCl and 2 NBA (2-Nitrobenzaldehyde) and incubated at 37±20C for 16hrs.

The compounds includes,

46
 NFP Compounds NFM Compounds

Furazolidone AOZ(Aminooxazolidone)

Furaltadone AMOZ(Aminomorpholinomethyloxazolidone)

Nitrofurazone SCA(semicarbazide hydrochloride)

Nitrofurantoin AHD(Aminohydrotoinhydrochloride)

NFM extraction principle:

Nitrofuran metabolite is derivatised and extracted with ethyl acetate and then it is
delipidated using hexane and then quantified in the LCMS-MS.

CAP (CHLORAMPHENICOL):

Chloramphenicol is an antibiotic useful for the treatment of a number of bacterial

infections. The chloramphenicol is a carcinogenic compounds and has many health effects. The
common side effects include bone marrow suppression, nausea, and diarrhea. The bone marrow
suppression may results in death. The extraction process of this single compound involves
addition of sodium chloride salt and Ethylacetate to the known quantity of sample and then it is
stabilized using Diethyl glycol. Then it is delipidated using Isooctane.

Action of reagents:

 Sodium chloride: for salting out (i.e., to prevent precipitation)

 Ethylacetate: for easy evaporation
 Diethyl glycol: to prevent degradization of CAP
 Isooctane: for lipid and other unwanted compound removal

TETRACYCLINE COMPOUNDS:

There are seven compounds which are included under this tetracycline group.
These are broad spectrum antibiotic .This includes,

 Oxytetracycline
 4-Epitetracycliine
 Tetracycline
 Chlortetracycline
 Deoxycycline
 4-Epioxytetracycline
 4-Epichlortetracycline

47
Extraction procedure:

The known amount of sample is extracted using Trichloroacetic acid and

McValline Buffer which is separated and eluted using the C18 cartridge after it is conditioned by
passing it with methanol and water.

Action of reagent:

 Trichloroacetic acid: for loosening the tissue bonds in the sample and releasing the target
compound
 McValline Buffer: for pH maintenance
 C18 cartridge: to remove non polar interference (i.e., lipid removal)
 Methanol: to elute the tetracycline compounds (as it is soluble in methanol) from the
cartridge

DYES:

Dyes are coloured organic compounds that are used to impart colour to various
substance. Each dye has unique compounds. Usually the dyes are classified into two groups
which include natural dyes and synthetic dyes. The major source of dyes in the sea food sample
is the antifungal and other antibiotic substances given to the shrimp during its rearing. These
dyes usually quantified as it has many toxic effects. The dyes compounds that are extracted here
are synthetic basic dyes which includes,

 Malachite green (coloured dye)

 Leucomalachite green (colourless dye)
 Crystalviolet (coloured dye)
 Leucocrystal violet(colourless dye

All these dyes are Diphenyl methane dyes group.

Extraction procedure:

The dyes are extracted by adding formic acid, salts (Magnesium sulphate and sodium
acetate), C18 and PSA(Primary and secondary amines) to the known amount of sample.

Reagent purpose:

 Formic acid: for loosening the tissue bond and releasing the target analyze
 Magnesium sulphate: for excess moisture removal
 PSA: for fat removal
 C18 salt: for removal of non polar interference

SULFONAMIDES AND QUINOLONE COMPOUNDS:

48
 There are 12 sulfonamide compounds and 11 quinolone compounds which are
extracted using the single integrated method.

SULFONAMIDES:

The sulfonamides are the first synthetic antibiotic agents. It has a good antibacterial activity
mainly on gram positive bacteria. These compounds has sulfur group attached with 2 analyze
atoms and amide group at one end and carboxylic group at other end. The 12 compounds
includes,

 Sulfanilamide
 Sulfamethoxy pyridazine
 Trimethoprim
 Sulfamethazin
 Sulfamethoxazole
 Sulfamethiozole
 Sulfapyridine
 Sulfadimethoxine
 Sulfathiazole
 Sulfachloropyridazine
 Sulfadiazine and
 Sulfamerazine

QUINOLONE COMPOUNDS:

The Nalidizic acid is considered to be the first quinolone drug. It was introduced in
the year 1962. These compounds contain fluorine atom attached to its structure and are effective
against both gram positive and gram negative bacteria by its activity of preventing the DNA
unwinding and duplication. The 11 quinolone Compounds includes,

 Ciprofloxacine
 Difloracine
 Enrofloxacine
 Flumequine
 Nalidizic acid
 Oxalinic acid
 Sarafloxacin
 Danofloxacin
49
  Norfloxacin
 Oflaxacin
 Marbofloxacin

PESTICIDE EXTRACTION:

Pesticides are chemical compounds that are used to kill pests, including insects,
rodents, fungi and unwanted plants (weeds). Pesticides are used in public health to kill vectors of
disease, such as mosquitoes, and in agriculture, to kill pests that damage crops. Based on the
chemical composition the pesticides are classified into four types

 Organochlorine compounds
 Organophosphorus compounds
 Carbamates
 Pyrethrin and pyrethroids

More than 90 pesticides are extracted from various food samples and are quantified in
LSMS-MS. The extraction process for pesticides differs depending upon the matrix from which
the pesticide is extracted. The most common method of extraction of pesticide is the Quechers
method.

STANDARDS:

Standards  are materials containing a precisely known concentration of a

substance with suitable purity for use in the quantitative analysis. It has a specific life period.
A standard provides a reference that can be used to determine unknown concentrations of that
substance in the matrix to which it is compared and it is also used to
calibrate analytical instruments.

A certified reference material (CRM) is a material which has been certified by

some trusted organisation to be of a consistent quality and composition for a particular period as
mentioned by the organisation. This certified reference material is termed to be mother standard
here. From this mother the standard stock solution is prepared which usually have a
concentration near 1000 ppm, it has a life period of about one year. From this standard stock
solution the intermediate standard-1 of concentration 100ppm is prepared which has the life
period of about 3 months. From this the intermediate standard-2 of concentration 10ppm is
prepared which has the life period of about one month.From this the intermediate standard -3 of
concentration 1ppm is prepared From this the working standard of required concentration is
prepared during the extraction process. This working standard is used to compare the
concentration of substance in the matrix.

INSTRUMENTS:

50
 The instruments used in extraction includes

1. Centrifuge:

It is almost used in all the extraction process done here. The centrifuge works using the
sedimentation principle, where the centrifugal acceleration causes denser substances and
particles to move outward in the radial direction. At the same time, objects that are less dense are
displaced and move to the center. So it separates the target analyte into the extraction liquid
based on its density.

2. Nitrogen evaporator:

The nitrogen evaporator system consist of chamber filled with water that resembles a
water bath , then stand which is designed for holding the evaporation tubes is fixed to the
system .Here the inert gas nitrogen is passed into samples( where the target compound is
dissolved in the extraction liquid) in the tubes. The heat from the water bath makes the
evaporation process easier. The extraction solvent gets evaporated at the end of this process
leaving only the analyte residue along with the fatty substances present in the matrix which is
then removed using some non polar solvents like hexane , isooctane etc. This evaporation is done
for tetracycline, NFM, CAP and sulfa-quinolone extraction.

3. Sonicator:

Sonicator uses the sound waves to agitate the particles in the solution. It converts the
electrical signal into a physical vibration to break the particles apart. This vibration is used for
complete dissolution of reagents or salts in a particular solution. So it used for preparing buffer
and some analytical reagents.

4. vacuum manifold chamber:

It is a chamber connected to pump and a large conical flask covered with a rubber cork .It
is mostly used for tetracycline and some pesticides extraction like melamine. Here the target
compound that dissolved in the extraction solvents are passed through the C18 cartridge fixed at
the top of the chamber after it is conditioned with methanol and water to loosen the C18 layer.
The solvents and the sample solution which is passed through the cartridge is filled inside the
chamber .Upon the application pressure it is collected in the large conical flask. The target
compound gets bound to the C18 column which is diluted using methanol and collected in
evaporation tube.

INSTRUMENT CALIBRATION:

Calibration of instrument is the process of determining its accuracy. The process

involves obtaining a reading from the instrument and measuring its variation from the standard

51
value prescribed for the particular instrument. These calibration processes is done based on the
standards and specification given by ISO (International organization for standards).

Significance of calibration:

The calibration process will ensure the accuracy of readings from the instrument and also
to establish the reliability and consistency of the instrument.

Calibration process:

Here all the instruments are calibrated periodically which involves,

 Daily calibration
 Monthly calibration and
 Yearly external calibration

The daily and monthly calibration is done within in the lab by the instrumental person
working in the lab. In case of any deviation or abnormality in the working of the instrument is
found in daily or monthly calibration then the instrument service person will be called for
examination and repair of the instrument.

The monthly calibration will take into other considerations like temperature, Relative
humidity and Air pressure .This calibration process is done for all the instruments even for the
micro pipette, volumetric glassware, temperature monitoring meter etc.

INSTRUMENTATION DEPARTMENT:

The instrumentation department has the following instruments:

 HPLC

 FTIR

 IC

 ICPMS/AAS

 LCMSMS

 GCMSMS

HPLC

High-performance liquid chromatography (high-pressure liquid chromatography)HPLC is

a technique in analytical chemistry used to separate, identify, and quantify each component in a

52
mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture
through a column filled with a solid adsorbent material.

Each component in the sample interacts slightly differently with the adsorbent material,
causing different flow rates for the different components and leading to the separation of the
components as they flow out of the column.

Chromatography can be described as a mass transfer process involving adsorption. HPLC

relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with
adsorbent, leading to the separation of the sample components.

The active component of the column, the adsorbent, is typically a granular material made
of solid particles ,silica, 2–50 μm in size. The components of the sample mixture are separated
from each other due to their different degrees of interaction with the adsorbent particles. The
pressurized liquid is typically a mixture of solvents (e.g., water, acetonitrile and/or methanol)
and is referred to as a "mobile phase".

Its composition and temperature play a major role in the separation process by

influencing the interactions taking place between sample components and adsorbent. These
interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic,
most often a combination..

The schematic of a HPLC instrument typically includes a

 Degasser
 Sampler
 Pumps
 Detector

The sampler brings the sample mixture into the mobile phase stream which carries it into
the column. The pumps deliver the desired flow and composition of the mobile phase through the
column. The detector generates a signal proportional to the amount of sample component
emerging from the column, hence allowing for quantitative analysis of the sample components.

A digital microprocessor and user software control the HPLC instrument and provide

data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple
solvents together in ratios changing in time, generating a composition gradient in the mobile
phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based
on mass spectrometry. Most HPLC instruments also have a column oven that allows for
adjusting the temperature at which the separation is performed.

53
OPERATION:
The sample mixture to be separated and analyzed is introduced, in a discrete small
volume (typically microliters), into the stream of mobile phase percolating through the column.

The components of the sample move through the column at different velocities, which are a
function of specific physical interactions with the adsorbent (also called stationary phase).

The velocity of each component depends on its chemical nature, on the nature of the
stationary phase (column) and on the composition of the mobile phase.

The time at which a specific analyte elutes (emerges from the column) is called its
retention time. The retention time measured under particular conditions is an identifying
characteristic of a given analyte.

Common mobile phases used include any miscible combination of water with various
organic solvents (the most common are acetonitrile and methanol). Some HPLC techniques use
water-free mobile phases (see normal-phase chromatography below).

The chosen composition of the mobile phase (also called eluent) depends on the intensity
of interactions between various sample components ("analytes") and stationary phase (e.g.,
hydrophobic interactions in reversed-phase HPLC).

Depending on their affinity for the stationary and mobile phasesanalytes partition
between the two during the separation process taking place in the column. This partitioning
process is similar to that which occurs during a liquid–liquid extraction but is continuous, not
step-wise. In this example, using a water/acetonitrile gradient, more hydrophobic components
will elute (come off the column) late, once the mobile phase gets more concentrated in
acetonitrile (i.e., in a mobile phase of higher eluting strength).

ION CHROMOTOGRAPHY

In ion exchange chromatography the ions such as bromate,nitrate ,are analyzed in water
samples .

PRINCIPLE:

54
 The most popular method for the purification of proteins and other charged molecules is
ion exchange chromotography. Conversely in anion exchange chromotgraphy,negatively charged
molecules are attracted towards a positively charged solid support.

INTRODUCTION:

Ion chromotgraphy is a chromatography process that separates ions and polar molecules
based on their affinity to the ion exchange .it works on almost any kind of charged molecule -
including large proteins ,small nucleotides and amnio acids.

As positively chargedactions flow across action resin beads, the actions are exchanged
for hydrogen (H+) . Likewise, as negatively charged anions flow across anion resin beads ,the
anions are exchanged for hydroxyl( OH-).

Ion chromotgraphy is commonly used to separate charged biological molecules such as

proteins, amino acids .the amino acids that make up proteins are zwitterionic compounds that
contain both positively and negatively charged chemical groups. .

Ion chromatography is used for water chemistry analysis .ion chromatographs are able to
measure concentration of major anions such as fluoride, chloride, nitrate and sulfate as well as
major actions such as lithium, sodium, ammonium, potassium , calcium, magnesium in the ppb
range .

SUPPRESSOR:

The thermo scientific dionex ERS 500 electrolytically regenerated suppressor exhibits
back pressure tolerance up to 900 psi, has peak efficiencies optimized for 4micrometer resin
bead based columns and has very high current efficiency and static capacity

Additionally, the suppressor is anintergal part of RFIC system, where the samples is
determined using supperssed conductivity detection in the lowest possible background of high
purity water. Autosuppression means ease of use; the supperssor is constantly regenerated by the
continuous electrolysis of water derived from the cell effluent.

FTIR:

The concentration of dispersed oil and grease (OG) is an important parameter for water
quality and safety.
The absorbance measurements were performed using the PerkinElmer Spectrum 400 FT-
IR/FT-NIR spectrophotometer in mid-IR mode and equipped with a DTGS detector . Other
instrument models with similar configurations1 such as the Spectrum One or Spectrum 100 can
also be used. The software used to acquire the spectra was Spectrum version 6.3. Spectra were

55
collected in transmission mode using a glass cell with 10 mm pathlength. Spectra were acquired
over the range 3200 - 2700cm-1 at 4 cm-1 resolution with ~1 minute acquisition time, and
ratioed against a spectrum of pure solvent. The peak maximum between 2930 and 2926 cm-1
was determined and used in the linear regression described below. A linear baseline fit through
the points at 3100 and 2800 cm-1 was subtracted before measuring the peak height.

Procedure:

The calibration reference oil was prepared by mixing iso-octane, hexadecane and benzene in the
ratio 3:3:2, and stored in a sealed container to avoid evaporative loss. The calibration stock
solution was prepared by weighing about 1 gm of calibration reference oil into a clean and dry
100 mL volumetric flask and diluting up to the mark with solvent, i.e., carbon tetrachloride.
From the calibration stock solution, a series of standard solutions were prepared using the
volumetric techniques in the range 1-40 mg/L with 9 calibration points.

Sample Preparation:
Samples were prepared as follows:
1. Acidification of 1 liter of sample using hydrochloric acid to pH 2.0.
2. E xtraction of above sample with 30 mL of carbontetrachloride three times (i.e., 1 x 3 times)
3. Filtration of extract through 10 g of sodium sulfate and dilution of combined collected extract
up to 100 mL with solvent.
4. Measurement of the solution at the absorbance maximum near 2930 /cm
Calibration – Linearity:
Over the calibration range excellent linearity was observed; with a correlation coefficient
(R2) of 0.9997. A standard error of prediction of 4 mg/L was obtained.
Spike recovery studies:
A recovery study has been performed at 6 mg/L concentration in three replicates. the
recoveries are excellent, ranging from 90 to 95 percent. This indicates that the solvent extraction
recovers nearly all of the OG and introduces only a small negative bias to the reported result.

AAS AND ICPMS:

In AAS and ICPMS department, heavy metal analysis is carried out, where
minerals are analyzed in AAS and trace elements are analyzed in ICPMS. In AAS, the elements
are determined in ppm levels and in ICPMS, the elements are analyzed in ppb levels.

Whereas, elements like magnesium oxide, methyl mercury, and other oxide
forms are first analysed as basic elements and then they are converted to oxide forms using
formulas.

The department receives-

56
  Food samples (FD,FS)
 Soil samples (EN)
 Water samples (EN)
 Container samples (CT)
 Air samples (Ambient air quality-AAQ)
Both the instruments present are sensitive and hence the samples are converted
to liquid, digested and then they are fed into the instrument.

SAMPLE PREPARATION

Water sample-

If water samples are clear i.e., raw water, borewell water, following method is used-

 50ml of sample is taken in a beaker.

 Add 1.5ml conc.HNO3 to acidify them.
 Then they are vortexed and feed into the system.
If water is unclear i.e., sludges, treated water from various plants and other, the
following method is used.

 100 ml of sample is taken in beaker

 5ml of conc.HNO3 is added with it.
 It is then digested in hotplate until the volume of sample comes down slightly
below 50ml.
 After digestion,the samples are made upto 100ml using double distilled water in
SMF.
 Then they are filtered using Sartorius filter paper number 393 and are placed in
little cans.
 During feeding into instrument, they are transferred to centrifuge tubes.
Soil samples-

1g of soil sample is taken and 100ml of water is added to it. Then it follows the
procedure same as procedure for unclear water samples.

Food samples-

The food samples when in solid form, they are grinded to smaller fine particles. If animal foods,
like shrimps they are deveined, peeled and homogenized.

 For food,0.5g of sample is taken in polytetrafluoroethylene tubes.

 Then they are added with 5ml of deionised water and 5ml of conc.HNO3 and
allowed to digest for 5 minutes in open condition.

57
  Then they are digested in microwave digestor for 1:30 hour.
 After which, they are made upto 25ml in centrifuge tubes and filtered and then
they are fed into the system.

ATOMIC ABSORPTION SPECTROPHOTOMETER:

PRINCIPLE:

The instrument is based on beer-lambert law. It makes use of the atomic absorption
spectrum of a sample to assess the concentration of analytes in it. It requires standards with
known analyte content to establish the relation between the measured absorbance and the analyte
concentration.

INSTRUMENTATION:

To analyze a sample for its atomic constituents, it has to be atomized. There are two
types of atomizers -flames and electrothermal (graphite tube) atomizers. The atoms are irradiated
by optical radiation, and the radiation source could be an element-specific line radiation source
or a continuum radiation source. The radiation then passes through a monochromator in order to
separate the element-specific radiation from any other radiation emitted by the radiation source,
which is finally measured by a detector.

HOLLOW CATHODE LAMP: It is changed according to the element analysed.

FLAME: Which is of air and acetylene (reaches 2300 degree celcius) or nitrous and acetylene
(reaches 3000 degree celcius).

FURNACE: In furnace, tubes, caps and holder are present. Cap and holder holds the tube
between them and the tube is heated when electricity applied and atomization occurs.

COMPRESSOR: The gases used are compressed and are sent to the instrument.

NEBULISER: Which converts liquid particles to aerosols.

MONOCHROMATOR: Which allows element-specific light needed to analyze.

DETECTOR: Photo multiplier tube detector detects the remaining wavelengths which was not
absorbed by the element.

The liquid sample which are fed to the instrument through nebulizer are converted to
aerosols which when reaching the flame gets atomized and absorbs the light which are send from
the hollow cathode lamp and gets excited and absorbs some wavelengths and the remaining light
reaches the detector.

58
 The concentration of the sample is analyzed with the help of various standards which
are prepared earlier.

INDUCTIVELY COUPLED PLASMA-MASS SPECTROMETRY:

Inductively coupled plasma mass spectrometry (ICP-MS) is a type of mass

spectrometry that uses an Inductively coupled plasma to ionize the sample. It atomizes the
sample and creates atomic and small polyatomic ions, which are then detected. It is known and
used for its ability to detect metals and several non-metals in liquid samples at very low
concentrations. It can detect different isotopes of the same element, which makes it a versatile
tool in Isotopic labeling.
Compared to atomic absorption spectroscopy, ICP-MS has greater speed, precision, and
sensitivity. However, compared with other types of mass spectrometry, such as thermal
ionization mass spectrometry (TIMS) and glow discharge mass spectrometry (GD-MS), ICP-MS
introduces many interfering species: argon from the plasma, component gases of air that leak
through the cone orifices, and contamination from glassware and the cones.
Unlike atomic absorption spectroscopy, which can only measure a single element at a
time, ICP-MS has the capability to scan for all elements simultaneously. This allows rapid
sample processing. A simultaneous ICP-MS that can record the entire analytical spectrum from
lithium to uranium in every analysis.
An ICP-MS may use multiple scan modes, each one striking a different balance between
speed and precision. Using the magnet alone to scan is slow, due to hysteresis, but is precise.
Electrostatic plates are used in addition to the magnet to increase the speed, and this, combined
with multiple collectors, can allow a scan of every element from Lithium 6 to Uranium Oxide
256 in less than a quarter of a second. For low detection limits, interfering species and high
precision, the counting time can increase substantially. The rapid scanning, large dynamic range
and large mass range is ideally suited to measuring multiple unknown concentrations and isotope
ratios in samples that have had minimal preparation.
INSTRUMENTATION:
AUTO SAMPLER: Which automatically collects sample which when placed in the vials
provided.
SAMPLE COLLECTING TUBE AND PERISTALTIC PUMP: The tube collects the sample
to the nebulizer with the pressure of the peristaltic pump.
NEBULISER: The liquid sample is converted to aerosols with it.
TORCH: Where the samples are cooled and the heavy items which are not digested are drain
back and the needed aerosols reaches the plasma through the connecting tube.

59
PLASMA: The plasma is produced by electomagnetive coil. It is coated with platinum.
DETECTOR: Which is filled with vacuum to prevent interferences.
The sample injected into the instrument is collected by peristaltic pump and reaches
nebulizer where it is converted into aerosols and then reaches torch and enters plasma and gets
ionized and are detected based on their mass to charge ratio.
CONES- For coupling to mass spectrometry, the ions from the plasma are extracted through a
series of cones into a mass spectrometer, usually a quadrupole.
MASS SPECTOMETRY-
The ions are separated on the basis of their mass-to-charge ratio and a detector receives
an ion signal proportional to the concentration. The concentration of a sample can be determined
through calibration with certified reference material such as single or multi-element reference
standards. ICP-MS also lends itself to quantitative determinations through isotope dilution, a
single point method based on an isotopically enriched standard.
COMMON INTERFERENCES:
 ISOBARIC INTERFERENCE-same mass.
 Doubly charged.
 POLYATOMIC INTERFERENCE-two or more atoms combined.
To prevent polyatomic interference, collision cell is used, where Helium gas in the cell
collides with measurement ion and polyatomic ion. The polyatomic ion overcomes the kinetic
energy barrier (KED). Polyatomic ion having a higher energy loss because of larger cross
section, the energy loss of it increases and is separated in the energy barrier.
FORMULA FOR CALCULATION:
(Sample concentration-blank concentration) x dilution factor x volume made up
Sample weight x 1000
Dilution factor - how many times it has been diluted.
Volume makeup - usually 25ml/100ml.
1000 - for converting ppb into ppm
Sample weight - 0.5g/1g
MECHANICAL:

This department tests the container, films and other packaging materials. The parameters
analyzed are design, shape, dimension, finish & appearance, colour, odour, transparency,
thickness, capacity, brimful capacity, migration, overall migration, colour migration, closure

60
leakage, vibration leakage, air pressure leakage, water potability, water portability, drop test,
stack load test, dart impact resistance test, ink adhesion test, elongation, tensile strength, etc.

QUALITY CONTROL:

QC carries out the activities of ensuring the quality of analysis held in the lab. The major
activities are IQC, Internal audit, Vertical audit, Proficiency testing, Checklist preparation,
feasibility checking of parameters with the NABL scope, putting down the specification for the
parameters based on the specified regulations and comparing it with the obtained results, sorting
out the problems and finding the root cause for it, Complaints maintenance, Amendment of
forms, registers and methods, following GFLP and deals with the outsourcing labs for the non-
feasible parameters.

This lab has accreditation from the following organizations.

 NABL :
National Accreditation Board for testing and calibration Laboratories. This is a
constituent part of Quality Council of India. It is founded in 1988 with the headquarters
in Guargon. It is an autonomous body providing accreditation of technical competence of
testing, calibration, medical testing laboratories, Proficiency Testing Providers (PTP) &
Reference Material Providers (RMP). This follows ISO standards and has mutual
agreement with ILAC and APLAC (Asia Pacific Laboratory Accreditation Cooperation).
It provides Accreditation in all major fields of science and engineering. NABL also
conducts many training courses.
 FSSAI :
It has a three tier system of labs in India- notified food lab, referral lab and
reference lab. The reference lab develops method, procedure and testing food across the
country. This is responsible for evaluating the performance of other notified laboratories.
Also, it is a resource center for the provision of CRM (Certified Reference Material). The
authority may conduct surprise audit to monitor or review the functioning of a food
laboratory. Lab should submit monthly/quarterly/annual statement on number of samples
received for testing, number of samples tested, and number of samples failed, specifying
the parameters or test and other details.
 ISO-9001:2008:
International Organization for Standardization provides the standard procedure for
testing of all kinds of samples and the calibration procedures for the instruments.
 ISO OHSAS-18001:2007:
Occupational Health & Safety Management This ensures the workplace safety for
the employees.
 TEA BOARD:
This is responsible for the assignment of certification numbers to exports of tea.
The board is responsible for reducing the fraudulent exports on tea.

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  MOEF
Ministry Of Environment and Forest accreditation is necessary to test the
environmental samples like air, water.
 BIS:
Bureau of Indian Standards is established under PFA. This is mandatory for water.
The standards are IS 14543:2005 for Packaged Drinking Water and IS 13428:2005 for
Packaged Natural Mineral water. There are totally 18 BIS licenses for Packaged Natural
Mineral water, 2354 licenses for packed drinking water through RO and 633 licenses for
bottling Packaged Drinking water from natural sources.
 EIC:
Export Inspection Council is the official export-certification body of India which
ensures quality and safety of products exported from India. It provides mandatory
certification for food items for fish & fishery products, dairy products, honey, egg
products, meat and meat products, animal casing, gelatin, ossein and crushed bones, feed
additive and pre-mixtures while other food and non-food products are certified on
voluntary basis.
 AGMARK:
This is a mandatory certification mark for Agricultural Produce by DMI in 1937.
Totally 222 agricultural commodities are notified. Though it is voluntary for others,
under FSSA it is mandatory for Blended vegetable oils, fat spread, oils, honey, ghee, etc.
There are 11regional and a Central AGMARK laboratory. Of these 12 labs, 9 are NABL
accredited.

IQC:

The IQC (Internal Quality Check) is done to test the competency of the analyst. These
results are then documented and are done every month for every department in the lab. The IQC
tests by 3ways:

 Spike and recovery

 Retest
 Recovery
 Spike and recovery: QC person would spike an unknown concentration of the sample and
the analyst would analyze the spiked value and give results. The observed value is compared
with the spiked value and the recovery percentage is calculated. The recovery percent must
lie between 80 and 120. A value below 80 or above 120 would show that the analyst is not
competent enough.
 Retest and replicate: The samples are retested to check the reproducibility of the results.
But the replicates are done along while analysis to ensure the accuracy of results.

INTERNAL AUDIT:

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 This helps to evaluate the company’s internal controls ensuring the compliance with laws
and regulation. Horizontal and vertical audit falls under this category.

 VERTICAL AUDIT:

The vertical audit is to check the traceability of a sample at all stages of analysis in the
lab. A sample code is taken and checked for its traceability from booking of samples to analysis
till the report section. In case of any non-traceability in any stage of the process, the root cause
for it is found and documented.

PROFICIENCY TESTING:

The proficiency testing is done to test the competency of the lab. This is conducted by
either NABL or NABL accredited PT providers. A sample of unknown concentration is given to
the lab for testing and the observed valves are sent to the providers. They would assign a Z score
based on the variation between the values sent by the lab and their values. The score must lie
between 0 and 2. A value greater than this is unacceptable.

CHECKLIST PREPARATION:

A checklist for every department is prepared each month. Then each department is
checked for all the things in the checklist and if anything is found wrong, necessary corrective
actions must be taken.

FEASIBLITY AND OUTSOURCING:

The parameters required for the client must be checked whether they are feasible to be
analyzed by the laboratory. If any parameter is found non-feasible, it must be outsourced.

SPECIFICATIONS:

The specification for the parameters is taken from the standards required by the client.
Then the results are checked whether they fall under the limits specified. The specifications
mostly followed here are

 FSSAI: It has released three compendiums for additives, contaminants and fortification. The
food additive compendium covers the physical, wet, microbial parameters and also additives
for all the food categories. The food contaminant compendium covers the heavy metals, crop
contaminants, pesticide residues and antibiotic residues for all food categories. The
fortification compendium covers the level of fortification of nutrients in the food products
like atta, maida, milk, oil, salt, etc.

 CAC: Codex Alimentarius Commission has set the standard limits for the trade and export
practices.

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 EU (European Union): RASFF- Rapid Alert System for Food and Feed and EFSA- European
Food Safety Authority are the two major governing bodies in EU. It is established in Italy in
2002. It covers animal health and welfare, Plant Protection, Plant health and Nutrition. Risk
Assessment is the major work carried out. These standards are highly stringent.

 EIC

 IS standards

 USFDA(United states Food and Drug Administration): Guidelines are followed for
Nutritional Labeling such as RDA
INFERENCE:
Thus we have learnt both the technical and managerial skills. We had a great and
inspiring time with the lab. We have gained more knowledge from this lab. This lab maintains
quality in all aspects.

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