This document provides information about various analyses conducted at a food analysis laboratory, including procedures for estimating fat, protein, ash content, moisture content, carbohydrates, and energy in food samples. Specific methods described include rose gotlieb method for fat estimation in milk powder, Kjeldahl method for protein estimation, and calculating carbohydrates by determining all other nutritional parameters. The document also discusses instruments used, including an analytical balance, and analysis of air, soil, hazardous waste and petroleum products.

1. FOOD ANALYSIS LABORATORY
PRIYANKA BAL
HALDIA INSTITUTE
OF TECHNOLOGY
09/06/2014- 12/06/2014

2. NUTRITIONAL PARAMETERS:
For any procedure to be followed we need to go through some specific norms
like sequential steps-
1. Calibration and Verification of all the
instruments that are to be used.
2. Method validation of the procedure that is to be
followed.
3. Sample preparation for the specific analyte that
is to be detected.
4. Detection of the required parameters within the
sample following the SOP(Standard Operation
Procedures)which are validated.
FAT ESTIMATION:
Fat is a nutrient. It is crucial for normal body function since it supplies
maximum calorific value. Chemically they are tri-esters of Glycerol and Fatty
acids. They are soluble in non-polar solvents but insoluble in water. Depending
upon this particular property of solubility and partition co-efficient fat is
estimated in a food sample.
Fat can be estimated by 3 procedures depending upon the nature, chemical
properties as well as its physical properties. They are:
 By Soxhlets Apparatus
 Acid hydrolysis
 Rose gotlieb method
Here we have given special emphasis upon rose gotliebs method for the
estimation of fat in a milk powder sample.
1. DETERMINATION OF FAT IN MILK POWDER SAMPLE BY ROSE
GOTLIEB METHOD:
1.2. Procedure:
Step1: 1gm of milk powder sample was taken. To it 10ml of boiling water or
luke -warm water added (so as to make it soluble). To it 2-2.5 ml of NH4OH
was added. NH4OH differentiates the fat and protein layers by hydrolysing the

3. bond between them. To it Petroleum benzene (BP-60 TO 80) added at a
quantity of 20-25 ml.
Step 2: The total things are taken in a separating funnel (also known as
mozzenors flask) and for 5 minutes shaken and simultaneously pressure
released by opening the lid. The pressure inside the funnel increases due to the
volatile nature of the non polar solvent and due to the variation in vapour
pressure between water and petroleum benzene. if preventions not taken by
releasing the pressure inside it can blast and break the flask.
Step 3: Now while shaking froathing occurred and foams are formed. To avoid
it 10 ml of alchohol was added which dissolves the foam hence act as anti
foaming agent. Now in a separating funnel a cotton plug was kept and upon it
Na2S04 (Sodium Sulphate)was added in required amount and the whole liquid in
the separating funnel was allowed to pass through it. This method was followed
so that if any impurities were present which are soluble in water will get
absorbed in the Na2S04 bed since it’s a good water absorbent. Now after passing
through it the total organic layer was collected in a beaker which contains only
organic layer containing fat within it since fat is soluble in non polar solvent.
Now step 2 and 3 were followed thrice for better extraction. Now the total
organic layer after 3 repeated extraction was taken in a beaker and kept in water
bath and the temperature was set at the boiling point of the organic solvent used.
This is because at that temperature the solvent will vapourize out and will
evaporate keeping behind within the beaker only the fats which was desired to
be estimated.
Step 4: Now the weight of the beaker taken (W2) separately.
1.2.Calculation:
Weight of the empty flask = W1.
Sample weight = SW
Final weight of the flask = W2
Percentage (%) of fat estimated = ×100

4. PROTEIN ESTIMATION:
Proteins are basically long chain compounds made up of amino acid chains
connected by peptide bonds. The amino acids constitute of N-terminal which
comprises the amino group and on the other hand has a C-terminal that

5. constitutes the carboxyl end. Proteins constitute the major portion of our body
since our body is made up of muscles, visceral organs, hormones, enzymes all
comprises proteins.
2.DETERMINATION OF PROTEIN BY AUTO KJELDAHL METHOD:
The SOP that was followed in this method was IS-7219. It is accomplished
basically by protein digestion method.:
2.1. Procedure:
Step 1: 1gm of the sample taken. To it 0.9-1.2 gm of CuS04 added. Then 10gm
of Na2SO4 added to it. To it 25 ml of H2SO4 added and it was given for
digestion for 3 hrs. For first ½ hour temperature was kept at 350 degree than
the temperature was risen slowly to 450 degree.
Observation-
1. It will be observed that the blank and the standard will appear green.
2. Sample 1, 2, 3 will appear brown in colour.
3. Now after digestion fumes that will be evolving were allowed to pass
through the Neutralizer which consists of 33% NaOH and Bromothiomol
as indicator.

6. ASH CONTENT: The inorganic residue obtained by igniting a specimen
of pulp, paper, other cellulosic materials in such a way that the
combustible and volatile compounds are removed. The ash content is
usually expressed as the percentage of such residue based on the weight
of the test sample.
3.DETERMINATION OF ASH CONTENT OF ANY FOOD SAMPLE:
3.1. Procedure:
Step1: The sample was taken in a crucible.
Step2: the sample was kept in HOT AIR OVEN at 550 degree such that it gets
ignited completely and become carbon free.
3.5.CALCULATION:
Weight of empty crucible = M1
Final weight of crucible after charing at 500 degree (crucible + ash) = M2.
Sample weight = SW.
Percentage (%) of ash = ×100
Percentage (%) of ash [on dry basis] =
MOISTURE CONTENT: Moisture content or water content is the
quantity of water contained in a material consisting of both monolayer
water as well as capillary water.
4.DETERMINATION OF MOISTURE CONTENT OF ANY FOOD SAMPLE:
4.1. Procedure:
Step1: Weight of the sample and the disc taken and it was dried at a temperature
Step2: After drying final weight was taken.
4.5.CALCULATION:
Weight before drying (dish + sample) = (M1+10).

7. Weight after drying (dish + sample) = M2.
Percentage (%) of moisture = (M2-M1-10) × 100
CARBOHYDRATE ESTIMATION: Large group of organic
compounds occurring in food and living tissues and including sugars,
starch and cellulose. They contain hydrogen and water in the same ratio
as that of water i.e. (2:1) and typically can be broken down to release
energy in body joined by glycosidic bonds.
5.DETERMINATION OF CARBOHYDRATE IN A FOOD SAMPLE:
Carbohydrate cannot be determined directly by any process or experiment. It
was completely calculation based. So for carbohydrate estimation we need to
estimate all other nutritional parameters.
5.1. CALCULATION:
Percentage (%) of carbohydrate = 100-[moisture+ fat+ protein+ ash].
ENERGY CALCULATION:
{[9×Fat] + [4×carbohydrate] + [4×protein]} = total energy in a food sample
expressed in Kcal/100gm

8. ENVIORONMENT LABORATORY
INSTRUMENTS:
Analytical Balance:
It is used to measure the mass of any substance as per the norms of
UACPA or BIS. It can measure a weight from 0.1 – 200 gm precisely. It follows 3
basic properties. They are- 1. Linearity:
If the result follows Y = MX +C. And for
linearity R2
= 0.95 ;( regression co –efficient).
2. Repeatability:
Same instrument giving same reading for number of
times. Here we need to calculate the standard deviation which is equal to 0.1%. if not
then it s erroneous.
RSD = %
3. Reproducibility:
Result given by different analyst for same machine on calibration for which the
value shouldn’t be more than 5%.
4. off centre test:
Checked once in 3 months. The mass is kept at four different positions
within the pan and the mass and its deviation was noted.
Difference shouldn’t be more than 0.002 gm.
AIR:
The quality of air is divided into 3 categories. They are classified as under-
 Ambient air
 Stack emission
 Indoor air quality.
PARAMETERS OF AIR:

9.  AMBIENT AIR:
1. Particulate matter size <10 µ
2. PM 2.5
3. SOX
4. NOX
5. NH3
6. CO
7. O3
8. C6H6
9. BENZO α PYRENE
10. Nickel
11. Argon
12. Lead
 STACK EMISSION:
1. Particulate matter
2. SOX
3. NOX
4. O2
5. CO
6. CO2
7. Velocity of gas flow
8. Temperature
9. Acid
10. Mist
11. Hydrocarbons ( unburnt).
 INDOOR AIR:
1. Particulate matter
2. Formaldehyde
3. VOC (volatile organic compound )
4. SOX
5. NOX
6. Humidity
7. Temperature
8. Co2
 WORK ZONE:
1. Particulate matter.
2. SO2.
3. N02
 HAZARDOUS WASTES:

10. 1. Flash point
2. pH
3. TCLP (toxicity characteristic leaching procedure).
SOIL (EDAHIC FACTORS):
 Mandatory factors for soil:
1. Sodium.
2. Potassium.
3. Calcium.
4. Phosphorous.
5. Magnesium.
 Other parameters:
1. Organic carbon
2. pH.
3. Texture.
4. Porosity.
INSTRUMENTS FOR HAZARDOUS/PETROLEUM TESTING:
1. DEVELANT OPEN CUP FLASH POINT APPARATUS
1. ABEL FLASH POINT APPARATUS

11. 2. PENSKY MORTENS FLASH POINT APPARATUS
Here no water is used as a heating medium in PENSKY MORTENS flash point apparatus.
 Flash Point:
 Fire Point:
BOMB CALORIMETRE:
A Bomb Calorimeter is used to detect the calorific value of any combustible product.

12.  Principle:
Total Heat Gained by the Calorimeter = Total Heat Lost By The substance.
 Calculation:
Calorific value =
ORSAT APPARATUS:
This apparatus is used to measure O2, CO2, CO present in the air. First air
is collected in some TEFLAR bags then connected to the apparatus. All the fluids
within the vessel are marked to auto zero then as per the deviation in the height in the
fluid column due to variation in the pressure the amount is calculated.
Orsat Apparatus.
CANON FLASK VISCOMETER:
CO ANALYZER:

13. This device is used to measure CO present in the air. Teflar bags are connected to it
and the machine is automated. A filter paper present inside that restricts the motion of
all particulate matters.
 It works on the basis of IR technology.
 Calibration is done once in a year.

14. For packed drinking
water procedure
followed is BIS-14543-
2004
And for unpacked
drinking water is IS-
10500-2012
For packaged natural
mineral water rain
water river water
process followed is
given by IS-13428-
2005
Agencies that look for
water are:
 BIS
 WAPCB
 MoEF
Water
analysis
Water is very important part of our life. We cannot ignore water
from our daily life. So its puerility must be analyzed. BIS give
method for checking its quality.
PARAMETERS FOR WATER AND
WASTE WATER:
1. Conductivity
2. Color
3. ph
4. Fixed solid
5. Volatile solid
6. Total solid
7. Total dissolved solid
8. Total suspended solid
9. Turbidity
10. Temperature
11. Flocculation test
12. Odor
13. Salinity
14. Settle able solid
15. Sludge Volume Index (SVI)
INORGANIC PARAMETERS AND NON-
METALLIC)
1. Acidity
2. Alkalinity
3. Ammonical nitrogen
4. Chlorides (as Cl-
)
5. Chloride residue (as Cl2)
6. Dissolve oxygen
7. Fluorides
8. Total hardness (as CaCO3)
9. Total kjeldahl nitrogen (TKN)
10. Nitrate nitrogen (as NO3)
11. Nitrite nitrogen (as NO2)
12. Phosphate (as PO4)

15. 13. Sulphate (as SO4)
14. Bromides
15. Carbon dioxide
16. Chloride demand
17. Silica (as Si)
18. Cyanide (as CN)
19. Sulphides (as H2S)
INORGANIC PARAMETERS (METALS)
1. Boron (B)
2. Cadmium
3. Calcium
4. Chromium total
5. Chromium hexavalent
6. Copper
7. Iron
8. Lead
9. Magnesium
10. Mercury
11. Nickel
12. Potassium
13. Sodium
14. Zink
15. Arsenic
16. Aluminum
17. Beryllium
18. Lithium
19. Manganese
20. Selenium
21. Silver
22. Strontium
23. Tin
24. Antimony

16. 25. Cobalt
26. Vanadium
ORGANIC (GENERAL) AND TRACE ORGANICS
PARAMETERS:
1. Bio chemical oxygen demand (BOD)
2. Chemical oxygen demand (COD)
3. Oil and grease
4. Phenol (as C6H5OH)
5. Pesticides: oregano-nitrogen phosphorus
6. Pesticides: organo-chloride
7. Total organic carbon
8. Surfactants (as MBAS)
9. Tannin and lignin
10. Poly-chlorinated biphenyl (PCB’s) each
11. Polynuclear aromatic hydrocarbon (PAH)
12. Organic carbon (in solid)
13. Carbon/nitrogen ratio
MICROBIAL TEST PARAMETERS:
1. Total coli form
2. Fecal streptococci
3. Total plate count
4. Coli phage
5. Fecal coli form
6. E.coli
7. Enterococcus
LIST OF INSTRUMENTS FOR WATER ANALYSIS:
1. Ph meter
2. Conductivity meter
3. UV visible spectrophotometer
4. Turbidity meter
5. Hot air oven
6. Muffle furnace
7. AAS (Atomic Absorbance spectrophotometer )
8. ICPMS (inductive coupled plasma mass spectrometer)
9. COD digester
10. TOC analyzer
11. Ion chromatography
12. CHNS analyzer

17. ION CHROMATOGHAPH
Total solid can be detected by gravimetric method. Metal detector are AAS, ICPMS.
FIVE IMPORTANT PARAMETERS TO BE DETECTED IN
WATER SAMPLE….
1. pH:
It is a measure of the acidity or basicity of an aqueous solution. Pure water has a pH
very close to 7.
pH is defined as the decimal logarithm of the reciprocal of the hydrogen
ion activity, aH+, in a solution.
2. TSS (total suspended solid) :
It measures the total solid particles present in sample. Here is steps to calculate TSS…
 Take 50 ml sample and pass to preweighted filter paper (w1)(paper dried at 105˚C for
1 hr)
 Again dry the filter paper at 105˚C till constant weight is achieved.
 Take final weight W2
TSS (mg/l)
3. COD (Chemical Oxygen Demand :
It is the no. of ml of oxygen required to chemically oxidize 1l of water.
4. BOD (Biochemical Oxygen Demand):
BOD is number of ml of oxygen required to biologically oxidize 1l of water.

18. 5. Oil and grease:
Steps to find oil and grease in sample:
 Take 1000 ml oil mixed sample in a separating funnel
 Add (1:1) 5ml HCl to maintain pH less than 2.
 Add 30ml n-hexane shake well add another 2 times n-hexane 30ml each. Shake well.
 Wait for layer separation
 On o separating funnel put a filter paper and sodium sulfate and wet it with n-hexane.
 Now drain the water layer.
 Pass the organic layer through sodium sulfate into weighted beaker(W1)
 Evaporate n-hexane in water bath.
 Take final weight(W2)
O&G (mg/l)
PARAMETERS FOR PACKED DRINKING WATER:
1. Color
2. Odor
3. Taste
4. Turbidity
5. Total Dissolved Solid
6. Ph
7. Nitrate
8. Nitrite
9. Fluoride
10.Chloride

19. 11.Sulfate
12.Alkalinity
13.Calcium
14.Magnesium
15.Residual Free
16.E Chloride
17.Phenolic Compounds
18.Anionic Surface Active Agent
19.Sulfides
20.Cyanide.
PACKAGED DRINKING WATER BOTTLES.

20. PRIYANKA
13/06/2014 – 17/O6/2014
RESIDUE ANALYSIS
LABORATORY

21. to vials for detecti
1. ROTA EVAPORATOR:
This instrument is used for evaporating the liquid
that is obtained after extraction. It works on the principle of BOILING POINT.
The boiling point of any liquid is that particular temperature at which its vapour
pressure becomes equal to the atmospheric pressure and the liquid starts boiling
and under goes phase change. In rota evaporator at time one sample is being used.
There is a water bath which provides the heating medium. The temperature is set
as per the boiling point of the solvent that is to be vaporized. The flask is attached
to the condenser and kept rotating in the water bath clockwise. As soon as the
temperature rises, the liquid starts passing through the condenser where it gets
concentrated and free from any impurity. And thereby what was left after
completion of the process was collected by taking out the round bottom flask.
Precaution:
 Pressure should be gently released while opening otherwise it could
crack the flask.
 The instrument should be always calibrated and verified.
2. SYNCHORE EVAPORATOR:
Principle similar as that of rota evaporator. Mainly used if the amount
of sample that is to be vaporised is more than 100-200 ml.
Advantage
 At time more than 1 sample can be worked together because it is having
capacity of 6 samples.
 Saves time and labour saving service.
3. pH METER:
Principle: Used for measuring pH of any solution. pH is actually negative
log[H+
] ions. It ranges from 0 – 14.
 pH = 7 (neutral)
 pH > 7 (acidic)
 pH < 7 (basic)

22. Calibration: pH meter is calibrated by using several buffers like acidic buffer,
basic buffer.
4. CENTRIFUGATION CHAMBER:
In centrifugation chamber we use to centrifuge any analyte or
commodity. There is a pot inside fitted within the machine inside. Within it
remain centrifugation tubes where we need to put our samples. Always they are
balanced with equal amount of weight diametrically opposite. Then by means of
software the speed was adjusted in terms of RPM (revolution per minute) with the
time which indicates how long it would centrifuge. Then after the revolution
completes machine automatically indicates and it is stopped and opened. Then the
tubes were taken out slowly. The analyte within it was separated into two part
supernatant and
5. MULTI REAX VORTEX:
It is basically a shaking agent used for thorough mixing of
the components in solution.
6. ULTRA SONIC BATH SONICATOR:
Used to de-gas the mobile phase in any process.
7. SILENT CRUSHER:
It allows taking out or extracting trace elements from inside the
cell matrix which enters into the cell components by means of endosmosis.
8. TURBO VAP EVAPORATOR:
It too works the same as that of rota evaporator or synch-ore
evaporator. But the difference lies in the volume of the sample evaporated.
Generally ria –vials are vaporized using turbo evaporator.
9. FUME HOOD:
Generally when we deal with acids it’s better to use fume hood.
Because it absorbs all the toxic, harmful, poisonous fumes that generates after
addition of corrosive acids in the analyte. As a result of which volatile matters
also oozes out with the fumes so for precaution they should be performed within
the fume hood.
10.MICRO BALANCE AND ANALYTICAL BALANCE:

23. Micro balance is mainly used to weigh the samples ranging from 1mg - 6mg.
Whereas analytical balance is used to measure weight from 5mg – 220 mg.
11.REFRIGERATOR:
The refrigerator is used to store standards for preservation at low
temperature for long time. Temperature maintained inside it is (2-8) ᵒC. Mother
Stock are kept within the refrigerator.
12.GRINDER:
Basically in EFRAC we used 2 grinders. One of 20 kg and the other of 50kg.
When it was asked to measure the amount of pesticide present in a trench or a
block of farming plot the plot was divided into 4 quadrants and from each corner
samples are taken and one from centre and grinded in the respective grinders as
per weight. Then after crushing they were taken for sample preparation and
further detection.
SAMPLE PREPARATION
Basically three steps are followed while sample preparation. They are as
follows-
1. Extraction ,
2. Clean up’ and
3. Concentrate the sample.
For extraction basically we follow QUECHERS METHOD, which means Quick
Easy Cheap Effective Rugger and Safe. Extraction is mainly of 3 types-
 SOLID LIQUID EXTRACTION.
 LIQUID LIQUID EXTRACTION.
 SOLID PHASE EXTRACTION.
Now brief ideas of the above topics are given below.
 PRACTICAL PROCEDURES OF SAMPLE PREPARATION:

24.  Solid Liquid Extraction:
Step1- Grind the sample in a mixer grinder. To detect the compounds
some standards will be provided. From them working standards will be
prepared of known concentration by means of serial dilution.
Step2- We will prepare two samples. One control sample and the other
spike sample. To the spike sample we will add 1 ml of the spike.
 What Is Spike?
Ans- Spike is a known concentration of a mother stock bearing
similar compounds as that of the elements that are going to be
detected within the commodity so as to check whether the process
followed is correct or not and to see the percentage of recovery.
Spike calculation-
Step3- Now 10 ml of ACN added to the sample (Control + spike).
Step4- We vortex the sample to homogenize the solution properly.
Step5- After that 1gm of NaCl and 4gm of Na2SO4 was added to it.
Step6- It was further centrifuged at 8000 RPM for 10 minutes.
Step 7- Then from it 4 ml of the solution after centrifugation was taken in
Ria- Vials.
Step8 – To it now 50mg PSA (Primary Secondary Amine), 100 mg C18,
2-5mg of GCB (Graphite Carbon Black) was added.
Step9- Again the vials were centrifuged at 5000 RPM for 5 minutes.
Step10- Then from them 1ml of the sample was taken in a fresh Ria-Vial
and allowed to vaporize via Turbo vaporizer.

25. Step11- Now if the sample is to be detected by GC (Gas
Chromatography), then to it Ethyl Acetate added and if by UPLC then
DCM (Di Chloro Methane) was to be added.
 Liquid Liquid Separation:
Liquid liquid extraction was mainly performed on the
basis of immiscibility nature of the solvent separated on the basis of-
 Partition Co-efficient.
 Distribution CO-efficient.
The solvent which is mainly used here for extraction is DCM (Di
Chloro Methane). Now the sample preparation for this process as per
in-house validation is described below-
Step1- 1lt of the sample taken in a measuring cylinder and poured into
separating funnel or Mozzeners Flask.
Step2 – pH of the sample was tested using pH probe (calibrated) or by
pH strip.
Step3- To it 80- 120 gm NaCl was added.
Step3- Now DCM was added to it for 3 times 60 ml each and the
process was followed so that for better extractions.
Step 4- Now the separating funnel is allowed to shake for 5 minutes
for complete mixing of the polar and non polar solvents for fine
extraction.
Step5- After some time we will notice that the two layers got departed.
The organic layer goes down due less specific gravity where as the
aqueous layer remains up due much higher density.
Step6- Now the solution was allowed to pass through a funnel
containing Na2SO4, which is guarded by a cotton plug at the bottom.
Na2SO4 is good water absorbent. It absorbs moisture and the impurity
that are water soluble.
Step 7- Now the extracted sample was allowed to vaporize by means
of rota evaporator.

26. Step8- After evaporation the concentrated form that will stick to the
wall of the round bottom flask will be extracted out with hexane (4- 5
ml).
Step9- Then from it 4- 4.5ml of the sample taken in Ria- vials and
vaporized out through turbo evaporator and finally the remaining
extract was treated with either Ethyl Acetate for running through GC
or ACN for running through UPLC.
 Solid Phase Extraction:
In case of solid phase extraction the analyte is extracted
in solid belt (C18, silica) cartridges for getting higher recovery. The
analyte was collected in belt and negative pressure applied to it.
Basically the phenomenon depends upon the principle of ionisation i.e.
hydrophilic lyophilic balanced reverse phase. The procedures that are
followed for solid phase extraction is as follows-
Step1- Conditioning [CH3OH or Methanol is mainly used to activate the
functional groups].
Step2- Equilibrate [generally water is used].
Step3- Loading [passing the sample].
Step4- Washing [to remove the impurities].
Step5- Illusion [solvent used to remove the analyte].
 No evaporation is required for the above process.
 This process is mainly used for extraction of antibiotics.
 Types Of Cartridges used-
I. Weak Cation Exchange [WCE].
II. Strong Cation Exchange [SCE].
III. Mixed Cation Exchange [MCE].
IV. Weak Anion Exchange [WAE].
V. Strong Anion Exchange [SAE].

27. INSTRUMENTS USED FOR DETECTION:
1. UPLC (MS/MS)[ULTRA HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY USING MASS SPECTROMETRY
DETECTOR]:
 Principle:
The basic principle upon which UPLC (MS/MS) works is the
variation in there stationary and mobile phases. Generally in the
early decades chromatography was performed normally using
NON POLAR MOBILE PHASE (hexane, DCM) and POLAR
STATIONARY PHASE that is Water. But nowadays Reverse
phases are used. STATIONARY PHASE is considered to be NON
POLAR and MOBILE PHASE to be POLAR.
Why do we need to change the phase?
a) Since all the drugs are soluble in water.
b) In human body 98% is water and it is mobile in
nature since it takes part in basic circulatory system
mixed with blood.
 Detector used:
In MS we analyze by mass: charge ratio of any element, so we
need to ionize the compounds. Generally we use FORMIC ACID,
BUFFER contents to ionise the compounds.

28. 2. GC- MS/FID/ECD [GAS CHROMATOGRAPHY using MASS
SPECTROMETRY,FLAME IONISATION DETECTOR OR
ELECTRON CAPTURE DETECTOR]:
 Principle :
The basic principle is same since here also reverse phase
chromatography is used. Generally thermally stable compounds are
detected by GC. Working temperature of this instrument is 300ᵒC.
Three types of injectors are used here-
a. Split mode- When the sample quantity is less and we have to
assure whether our sample is ok or not. It splits the injecting
mechanism in 2 or 3 subdivisions. For e.g.- suppose 5ml of
sample to be taken, it can be taken in twice as 3ml + 2ml.
b. Split less mode- When the sample quantity is less we can
directly inject 5 ml in the vials for detection.
c. Head space mode- When we are working will alcohols,
generally we need to check the purity of the sample we use this
mode. The samples are taken within the vials and kept in auto
injector where high temperature is provided. Alcohols are
volatile by nature so they quickly get converted into vapour

29. phase and then this vapour is directly taken by the injector as
mobile phase.
 Other parameters used:
Nearly 250- 350ev high voltage is applied at high temperature to
vaporize as well as ionise the samples and pass through the
columns
Columns are long cylindrical rods of varying diameter and length
made up of packed C18 or polysiloxane performs the function of
stationary phase
Methanol/ACN/Acetone can’t be used directly because they can
choke the columns.
3. GC MS/MS [GAS CHROMATOGRAPHY USING MASS
SPECTROPHOTOMETRY(Quantitative)/MASS
SPECTROPHOTOMETRY(Qualitative)]:

30.  Principle- The process works step by step. Firstly we use to scan
and tune any analyte so as to make it acquainted with the machine
and see whether the particular analyte is present in the sample or
not. For this we take 50- 550mg of sample and provide it oven
temperature.
Now we will detect its RETENTION TIME (time for which the
analyte will remain bound to the stationary phase) and the parent
ions to see which gives the maximum response. This gives the
qualitative response.
 Calibration- GC MS/MS is calibrated by PFTBA.
 Generally pesticides residues are detected by it which is not
thermally stable.
4. HRGC/HRMS [HIGH RESOLUTION GAS
CHROMATOGRAPHY/HIGH RESOLUTION MASS
SPECTROSCOPY]:
 Principle- Electromagnets are there with columns from which the
compounds after passing through the electromagnetic field in
vacuum splits up into spectral bands where they are detected. The
whole instrument is kept in air not in tough with the ground so as

31. to avoid minimum vibration that may cause fluctuation in the flow
of electromagnetic radiations.
 Can give result up to 5 points after decimal so as to verify
accuracy.
 Used to detect DIOXIMES, FURANS.
 For verification reference standards are used like PFK (per fluoro
kerosene) which is injected in between the mid of the run at
anytime for double verification.
DETERMINATION OF OC COMPOUNDS IN
EGG SAMPLE AND SAMPLE ANALYSIS:
The commodity that was provided to us was an egg sample. The method
of extraction that was followed is solid liquid extraction procedure by
means of Quenchers dye.
 Preparation Of Calibration Curve-
10 ppm of known standard was provided. From it 5 working
standards formed namely that of concentration 200ppb, 100ppb,
50ppb, 10ppb so as to plot the calibration curve.
 Methods Followed-
A. 2.0351 gm of the control sample and 2.0344 gm of spike
sample taken.
B. To it 1ml of spike standard inoculated.
C. 10 ml of ethyl acetate added to it.
D. 2gm NaCl and 3 gm Na2SO4 added.
E. Allowed to vortex it.
F. Then it was centrifuged for 10000 RPM for 10 minutes.

32. G. Then the debris was discarded and the supernatant was taken
by my micro pipette of 4 ml and put into Ria- vials.
H. Then to it 70 mg of PSA, 120 mg of C18 and 200 mg of
MgSO4 was added.
I. Kept for some time and then again centrifuged at 5000 RPM
for 5mins.
J. Then from it 1 ml of the sample was taken in fresh Ria –
vials and allowed to evaporate by the turbo evaporator.
Finally to it EA was added and transferred on in GC-
MS/ECD/FID

33. HALDIA INSTITUTE OF TECHNOLOGY.
18/06/2014 - 21/06/2014.

34. RESIDUE ANALYSIS LABORATORY

35. REAL TIME PCR
A real-time PCR, also called as quantitative polymerase chain reaction (qPCR) is a
laboratory technique of molecular biology based on the polymerase chain reaction
(PCR) which is used to amplify and simultaneously quantify a targeted DNA
molecule. Basically it is used to detect strands of particular DNAs so as to judge
whether the substance is genetically modified organism or not.
The procedure followed was the general principle of polymerase chain reaction; its
key feature is that the amplification of DNA was detected at the instantaneous
reaction progress so called as Real Time.
Gene expression analysis can be done using 1. TaqMan® chemistry.
2. SYBR® Green I dye chemistry.
Basically here we use to detect 2 particular strands-
 35S(Cauliflower Mosaic Virus)
 N0S (Agrobacterium tumificans)
The polymerase chain reaction proceeds in 3 subsequent ways-
A. Denaturation of template (95ᵒC)
B. Annealing of primer to template (60ᵒC)
C. Extension of primer (60ᵒC)
BIOLOGICAL SIGNIFICANCE: It involves TaqMan chemistry which uses a fluorogenic
probe to enable the detection of a specific PCR product as it accumulates during the PCR
cycle.

36. Initially, intercalator dyes were used to measure a real time PCR product. The primary
disadvantage of these dyes is that they detect accumulation of both specific and non specific
dyes.
 How TaqMan sequence detection chemistry works:
Step1: An oligonucleotide probe is constructed containing a reporter fluorescent dye on the
5’end and a quencher dye on the 3’end.While the probe is intact the proximity of the
quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence
resonance transfer (FRET) through space.
Step2: If the target sequence is present, the probe anneals downstream from 1 of the primer
sites and is cleaved by the 5’nuclease activity of Taq DNA polymerase as this primer is
extended.
Step 3: Cleavage of the probe separates the reporter dye from the quencher dye, increasing
the reporter dye signal
On the other hand removal of the probe from the target strand, allowing primer extension to
continue to the end of the template strand. Thus inclusion of the probe does not inhibit the
overall PCR process.

37. Step 4: Additional reporter dye molecules are cleaved from their respective probes with each
cycle resulting in an increase in the fluorescence intensity proportional to the amount of
amplification.
 ADVANTAGES OF TaqMan CHEMISTRY:
1. Specific hybridization between probe and target is required to generate fluorescent
signal.
2. Probes may be labelled with different, distinguishable reporter dyes, which allow
amplification of two distinct sequences in one reaction tube.
3. Post PCR process was eliminated which reduces assay labour and material cost.
 DISADVANTAGES:
1. Primary disadvantage was the synthesis of different probes was required for different
sequences.
MACHINE HANDELING:
In a real time PCR everything is automated. Just what was required was that there are some
wells which need to be filled with sample that to be detected.
Firstly DNA s was isolated using kit needed to add as per SOP.
Secondly PRIME and PROBE were added.
Within the kit there were master mix (DNTP with Taq polymerase, negative control (RNS
free water), ROx dye that gives fluorescence’s.
Prime probe mix contains positive control with it in some different coloured vials.
Taking 130µl of master mix + 5.2µl of dye + 10µl of prime probe mix it was fed to the wells.
Then they were covered with a plastic polymeric strip so as to avoid contamination from 1
well to another consecutive well

38. Within the PCR there are Thermal Cyclic Block at the base which provides heat to the
inoculum , a Halogen detector which catches the fluorescence and a camera at the top that
detects and captures and detects the images.
MEDIA PREPARATION:
Medias are mainly prepared to provide enrichment to the bacteria’s for their growth.
Basically specific Medias are prepared for specific organisms.
1. PCA – Total bacterial count.
2. YEDCA – Total yeast and mould count.
3. BPA - Staphylococcus sp.
4. MB (DS/SS) – E.coli
5. PDA – Fungus.
PLATING TECHNIQUES:
Plating is mainly of three types-
1. Pour plate
Here the organisms are inoculated before and then the medium is poured and rotated
clockwise and anti clockwise.
2. Spread plate
Here the medium is already made and the organisms are spread upon the plate by
means of a bent glass rod.
3. Streak plate
Here the culture is taken in a nichrome loop and taken in solid media and parallel
lines are drawn to isolate pure culture.

39. STAINING TECHNIQUES:
 THE GRAM STAIN
This is a DIFFERENTIAL STAIN. It requires a PRIMARY STAIN and a
COUNTERSTAIN. It divides most of the EUBACTERIA into two large groups: GRAM
POSITIVE bacteria and GRAM NEGATIVE bacteria.

40. The basic procedure goes like this:
1. Take a heat fixed bacterial smear.
2. Flood the smear with CRYSTAL VIOLET, 1 minute, then wash with water. [PRIMARY
STAIN]
3. Flood the smear with IODINE, 1 minute, and then wash with water. [MORDANT]
4. Flood the smear with ETHANOL-ACETONE, quickly, and then wash with water.
[DECOLORIZE]
5. Flood the smear with SAFRANIN, 1 minute, and then wash with water.
[COUNTERSTAIN]
6. Blot the smear, air dry and observe.
 EXPERIMENT:
o 1.)Perform the gram stain on smears made form the following organisms:
 Staphylococcus aureus
 Escherichia coli
2. ) Use a sterile applicator stick to obtain a sample of the "crud" on your teeth
at the gum line.
Mix the sample with a loopful of water on a microscope slide and allow
the specimen to air dry.
Heat fixes and performs the gram stain on this sample.
3.) Observe all of your specimens under oil immersion and record cell
morphology and arrangement and gram reaction.
GRAM (+) organisms holds onto the crystal violet-iodine complex more tightly than the
GRAM (-) bacteria do. However this is not an absolute phenomenon! Sometimes gram (+)
cultures will appear gram (-) or GRAM VARIABLE (a mixture of gram (+) and gram (-)
cells). What are some of the reasons for this?
1. Overdecolorization
2. Too harsh heat fixation
3. Too thick a smear
4. Improper washing between steps
5. Too old a culture
6. Impure or mixed culture

41. It is believed that the gram staining characteristics of an organism is a function of its cell
wall. Therefore any time the cell wall is damaged the gram stain characteristics of gram
positive cells will change. On the other hand, gram (-) cells never become gram (+) if the
stain procedure is done correctly.
1. Gram (+) cells have thicker cell walls - more peptidoglycan and teichoic acid.
2. Gram (-) cells have Lip polysaccharide (Endotoxin) in the outer membrane of their cell
walls.
3. Gram (+) cells is generally more sensitive to those antibiotics which interfere with cell
wall.
4. Gram (+) cells is more sensitive to lysozyme - a peptidoglycan digesting enzyme.
The gram morphology of some common bacteria:
Gram (+) = Staphylococcus, Streptococcus, Bacillus, Clostridium
Gram (-) = the coliforms: (Escherichia, Klebsiella, Serratia), the enteric pathogens:
Salmonella, Shigella, Campylobacter
 THE CAPSULE STAIN -
Most bacteria have some kind of CAPSULE. This viscous surface layer is also known as the
SLIME LAYER, the GLYCOCALYX or the EXTRACELLULAR POLYMERIC
SUBSTANCE (EPS). Most bacterial capsules are composed of polysaccharide however
some genera produce polypeptide capsules. Capsular material is very moist (slimy) and any
heating will cause it to shrink - it is for this reason that we will not heat fix the slide before
staining. Also, heating may cause the bacterial cell to shrink resulting in a clear zone around
the cell - which may cause cells which don't have capsules to appear as if they do.
The polymers which make up the capsule tend to be uncharged and as such they are not
easily stained. For this reason we use a NEGATIVE STAIN to visualize them. That is, we
use a stain which stains the background against which the uncolored capsule can be seen.
Our procedure, the Gin's Method, uses india ink to color the background and crystal violet to
stain the bacterial cell "body"

42. This structure helps the bacterial cell to ATTACH TO SURFACES and to AVOID BEING
PHAGOCYTOSED. For instance, the oral streptococci produce a glucan based EPS which
helps them to attach to the teeth. When this material begins to accumulate on the teeth it is
referred to as dental plaque. As a general phenomenon, organisms with capsules tend to be
more virulent presumably because of their resistance to phagocytosis and killing.
Streptococcus pneumoniae exists in a smooth form (encapsulated) and a rough form (non-
encapsulated). Only the smooth form is lethal for mice.
 EXPERIMENT
o a. Use a loop to mix a drop of water, a drop of india ink and a small amount of
Klebsiella pneumoniae together at the end of a slide.
b. Use another slide to spread the smear like a blood smear. (As the instructor
will demonstrate.) Allow the smear to air dry. DON'T HEAT FIX!
c. Flood the smear with crystal violet, 1 minute. Wash with water, blot, dry,
observe. Compare your observations with the illustrations on page 30 of
Leboffe and Pierce.
 ENDOSPORES - The Schaeffer - Fulton Stain
These are very resistant structures made by only a few genera of
bacteria. The two genera which we will study are:
 Clostridium is an anaerobic organism that forms spores. Tetanus, botulism, gas
gangrene and pseudomembranous colitis are diseases caused by different species in
this genus.
Bacillus is a common aerobic genus whose species can form endospores. Anthrax and
Bacillus cereus food poisoning are two diseases caused by members of this genus.

43. Spores are extremely resistant structures, difficult to destroy with heat or other physical and
chemical disinfecting agents. Endospore destruction is the standard for testing the operation
of an autoclave.
 EXPERIMENT
o Prepare a smear of Bacillus megaterium, allow the smear to dry and then heat-
fix.
o Place the slide on the staining rack in the sink and flood the smear with
malachite green stain.
o Heat the stain to steaming by passing a lit bunsen burner over the smear.
Don't overheat the stain! Once the steaming stops, pass the bunsen burner
over the slide again. As the stain evaporates add more stain. Continue this
procedure for 5-10 minutes.
 (Safety Note: Please remember that the acetone-alcohol decolorizer
from the gram stain experiment is extremely flammable. Do not
perform this flaming step while people are gram staining!)
o Wash the smear gently and thoroughly with running water.
o Counterstain with aqueous safranin for 1 minute.
o Wash the slide with water, blot gently and allow the smear to air dry.
 SIMPLE STAINING
 Purpose: To recognize the three basic shapes of bacterial cells.
 Principle: In order to observe most bacterial cells using bright field microscopy
the cells must be dark enough to see, that is they must have contrast to the light.
To create contrast a simple stain can be used. Simple stains use basic dyes which
are positively charged. These positive dyes interact with the slightly negatively
charged bacterial cell wall thus lending the colour of the dye to the cell wall.
FUNGAL STAINING:
1. 1st
lactophenol cotton blue added
2. Then culture added
3. Then separated
4. Observed under microscope.

44. BIOCHEMICAL TEST:
This test is done to check whether bacteria is present or not. We can say them as conformity
test too. They are as follows:
 Oxidase test
The oxidase test is a test used in microbiology to determine if
a bacterium produces certain cytochrome c oxidases. It uses reagent such as N,N,N′,N′-
tetramethyl-p-phenylenediamine (TMPD) or N,N-dim ethyl-p- phenylenediamine (DMPD),
which is also a redox indicator. The reagent is a dark-blue to maroon colour when oxidized,
and colourless when reduced.
e.g: Pseudomonaus aeruginosa
 Catalytic test
Some bacteria have catalyzed enzyme. Which decompose hydrogen peroxide to water and
oxygen. Catalytic enzyme protect the bacteria from oxidative damage.
Eg- E.coli
 Coagulase test
Certain bacteria can coagulate fibrin plasma into fibrinogen.
 Indol test

45. It is biochemical test performed on a bacterial species to determine
the ability of organism to convert tryptophan into the indol. Positive indol test give red ring
on the surface. E.g -E.coli
CLASSIFICATION OF AREA:
Viable(cfu/2hr) Non viable
*
Gloves
Per hand
Air limit
Per m3
Contact plate limit
Class A < 1 <1 <1 <1
Class B <5 <5 <10 <5
Class C <50 ND <100 <25
Class D <100 ND <200 <50
*non viable (particle count)
.5µl diameter 5 l diameter
Class A
Class B
Class C
Class D
At rest At op.
350 3500
3500 350000
350000 3500000
3500000 nd
At rest At op.
0 0
0 2000
2000 20000
20000 Nd
CULTURE MAINTANCE:

46. ELISA READER( enzyme-linked immunosorbent assay)
ELISA is a test that uses antibodies and colour change to identify a substance.
 Use to determine
1. Food allergens
2. Hormone
3. Toxins
 Types of elisa
 DIRECT ELISA:
 INDIRECT ELISA

47.  SANDWICH ELISA TECHNIQUE: