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Barbara Jeršek
Ljubljana, 2017
FOOD TOXICOLOGY
AND
CONTAMINATION
INSTRUCTIONS AND WORKBOOK FOR
LABORATORY EXERCISES
FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK
FOR LABORATORY EXERCISES.
1
FOOD TOXICOLOGY AND CONTAMINATION
INSTRUCTIONS AND WORKBOOK FOR LABORATORY EXERCISES
Barbara Jeršek
Instructions and workbook for laboratory exercises
Food toxicology and contamination.
Publisher:
University of Ljubljana
Biotechnical Faculty
Department of Food Science
Ljubljana, October 2017
All rights reserved. No part of this publication may be reproduced or used in any
other way (graphic, electronic or mechanical, including photocopying, recording or
transfer in the database) without the written consent of the copyright holder.
FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK
FOR LABORATORY EXERCISES.
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TABLE OF CONTENTS
MYCOTOXINS ............................................................................................................... 4
PURPOSE .................................................................................................................... 4
COURSE OF EXPERIMENTAL WORK ................................................................ 4
INSTRUCTIONS ........................................................................................................ 4
WORKBOOK .............................................................................................................. 9 PERFORMANCE ................................................................................................................ 9
MATERIAL, WORKFLOW AND RESULTS ................................................................ 10
ALERGENS ................................................................................................................... 14
PURPOSE .................................................................................................................. 14
COURSE OF EXPERIMENTAL WORK .............................................................. 14
INSTRUCTIONS ...................................................................................................... 14
WORKBOOK ............................................................................................................ 21 PERFORMANCE .............................................................................................................. 21
MATERIAL, WORKFLOW AND RESULTS ................................................................ 22
ENVIRONMENTAL CONTAMNANTS .................................................................... 26
PURPOSE .................................................................................................................. 26
COURSE OF EXPERIMENTAL WORK .............................................................. 26
INSTRUCTIONS ...................................................................................................... 26
WORKBOOK ............................................................................................................ 31 PERFORMANCE .............................................................................................................. 31
MATERIAL, WORKFLOW AND RESULTS ................................................................ 32
REFERENCES .............................................................................................................. 39
ANNEX ........................................................................................................................... 40
MYCOTOXINS ......................................................................................................... 40
ALERGENS ............................................................................................................... 43
ENVIRONMENTAL CONTAMINANTS .............................................................. 44
FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK
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Table of figures
Fig. 1: Scheme of media and temperatures for incubation of plates for
moulds identification (1, 2: designations for two moulds strains) ................. 5
Fig. 2: Scheme of TLC ....................................................................................... 7
Fig. 3: Time-temperature program for determining walnut with real-time
PCR .................................................................................................................. 19
Fig. 4: Dependence of the Ct value from the decimal logarithm of the walnut
percentage in the standard walnut biscuit (Terpin, 2010) ........................... 20
Table of tables
Table 1: Composition of nutrient solution……………………..………….27
Table of appendixes
Annex 1: Mould isolation on DRBC (A) and DG18 (B) .................................. 40
Annex 2: Example of identification according to Pitt and Hocking (1985) .. 40
Annex 3: Aspergillus (400x magnification) .................................................... 41
Annex 4: Penicillium (400x magnification) ................................................... 41
Annex 5: Ochratoxin A (OTA) in aflatoxin B1 (AFB1) .................................. 42
Annex 6: TLC-plate for detection of OTA under UV illumination................ 42
Annex 7: The principle of detecting amplicons with real-time PCR by
TaqMan and SybrGreen methods .................................................................. 43
Annex 9: Microscopic images of onion cells: (A) anaphase, (B) telophase, (C)
prophase, (D) metaphase, (E) interphase ...................................................... 44
Annex 10: Microscopic images of onion cells with different injuries in
metaphase and anaphase ............................................................................... 45
FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK
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MYCOTOXINS
PURPOSE
In food sample determine ochratoxin A (OTA) and aflatoxin B1 (AFB1)
through determination of moulds that produce OTA and AFB1.
COURSE OF EXPERIMENTAL WORK
• Determination of mould in/on food
• Identification of selected mould
• Determination of potential mycotoxigenic mould
• Determination of ochratoxin A and aflatoxin B1
• Evaluation of results
INSTRUCTIONS
1. ISOLATION OF MOULDS FROM FOOD WITH CLASSICAL
MICROBIOLOGICAL METHODS
For the determination of moulds on food aseptically cut food on Al-foil
into smaller parts. For the determination of moulds in food, disinfect it
with 70% ethanol (for example, walnut in shell, immerse in ethanol),
then cut food aseptically. Homogenize the entire food sample, or at least
100 g and 50 g depending on the type of food and the type of
microbiological tests. Small parts of food is then aseptically transferred
to appropriate media when qualitative analysis is performed, or basic
solution is prepared when quantitative food analysis.
For mould isolation DRBC (Dichloran Rose-Bengal chloramphenicol;
medium with dicloran red and Bengal dyes and chloramphenicol), OGY
(Oxytetracycline Glucose Yeast Agar, the medium with glucose, yeast
extract and oxytetracycline), MEA (Malt Extract agar medium
containing malt extract) and DG-18 (medium with dicloran and glycerol)
can be used. DRBC and OGY are media used for the isolation and
quantification of moulds from foods that contain a higher concentration
of bacteria, because they contain an antibiotic that inhibits their growth.
MEA is used for isolation and quantification of moulds and yeasts from
the food. It contains malt extract and peptone; it also has the lactic acid,
FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK
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which inhibits the growth of bacteria (pH of 3.5). The medium DG-18 is
used for isolation and quantification xerophilic moulds from dried and
semi-dried foods (dried fruits, candied fruits, spices, cereals, meat and
fish products). DG-18 contains peptone as the nitrogen source, glucose as
a source of energy, and vitamins and minerals required for the growth of
fungi. The medium has also dicloran, which inhibits the spread of
colonies, chloramphenicol and chlor-tetracycline to inhibit bacterial
growth. The medium has 18% (w/w) glycerol, which reduces aw (water
activity) from 0.999 to 0.95. Plates of MEA and DG-18 are incubated at
25 °C for 7 days.
2. MOULDS IDENTIFICATION
After MEA (or OGY or DRBC) and DG-18 plates incubation, describe the
results (qualitative and quantitative) and choose single colony for mould
identification. For mould identification prepare spore suspension in 0.5%
agar with Tween (AT) – with inoculating loop aseptically transfer spores
into AT (in laminar flow cabinet).
Mould spores are then inoculated on following media: CYA (Czapek yeast
extract agar), MEA, G25N (25 % Glycerol nitrate agar) (Pitt in Hocking,
1997) according to the scheme in Figure 1.
Fig. 1: Scheme of media and temperatures for incubation of plates for moulds
identification (1, 2: designations for two moulds strains)
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After 7-day incubation of media shown in Fig. 1 describe macro-
morphological properties for each mould strain (2r of colony, colour and
structure of substrate mycelium, colour and structure of aerial
mycelium, formation of exudate) and micro-morphological properties
seen under stereo magnifier (40x-80x) (formation of cleistotecium,and
ascus) and on native microscopic slide under microscope (100x-1000x)
(septate or non-septate mycelium, formation of sporangiospores or
conidia, kind of conidiogenic cells, properties of conidia or
sporangiospores, apex, metula, phialide,…). Native microscopic slide is
prepared in a drop of water with added lacto-fuchsine. Detailed
description, identification keys and species descriptions are in Pitt and
Hocking (1997) and Samson et al. (2000). After microscopy
decontaminate microscopic slide in NaOCl solution (18%) and clean
microscopes objectives
Depending on identified mould predict from literature if the isolate can
form mycotoxins and which one, ochratoxin A and/or aflatoxin B1. These
isolates are then re-inoculated on YES (Yeast Succrose Agar) and MEA
or CYA or DG-18 as described in 1. Plates are incubated at 25 oC for 7 to
28 days.
3. GENERAL INSTRUCTIONS FOR THIN LAYER
CHROMATOGRAPHY (TLC) DETECTION OF MYCOTOXINS
When working with mycotoxins it is the mandatory to use protective
equipment! All material that comes into contact with mycotoxin throw in
a prepared solution of NaOCl! All solvents must be treated as hazardous
substances and work must be carried out in a fume hood.
1. Depending on type of mycotoxin that will be analysed with TLC, first
prepare mixture of solvents – mobile phase (200 ml for bigger chamber,
20 ml for smaller chamber).
2. As stationary phase use TLC-plate (TLC Silica gel 60 F294), which is
consisted of the support (aluminium plate or glass) with a thin layer of
stationary phase (silica gel and aluminium oxide).
3. On TLC plate label with a pencil (very gently) the line (2.5 cm from
the bottom edge), and mark the samples at intervals of 1.5 cm (Fig. 2).
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4. On the first place is always standard solution of investigated
mycotoxin (S), pipette standard solution (5 µl) very carefully so that
diameter of spot is smaller than 3 mm.
5. Afterword add on places marked as V1, V2, V3, … moulds samples like
plugs obtained from YES medium. Each mould is sampled 3 times and
each plug is added to plate 3 times.
6. When finishing application of plugs, evaporate the solvent in a fume
hood.
7. TLC-plate is then carefully placed in chamber with mobile phase,
cover the chamber and wait until mobile phase is approximately 0.5 cm
from the top of TLC-plate.
8. TLC-plate is then carefully taken from the chamber and live it in the
fume hood until mobile phase is evaporated.
9. Illuminate the TLC-plate with UV light (366 nm) so that spots become
visible spots and mark them. Measure the distances travelled by each
compound and mobile phase (d).
10. Calculate retention factors. The ratio between the distance
travelled by each compound and the one travelled by mobile phase is
called the retention factor (Rf):
Rf = d sample / d mobile phase
Fig. 2: Scheme of TLC
Legend: S: standard solution of OTA or AFB1; V1: mould 1; V2: mould 2; d:distance
FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK
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TLC can be used as quick screening method for assessment of production
of mycotoxin and when appropriately worked it can be semi-quantitative
method.
4. Detection of OTA with TLC
For OTA determination with TLC prepare mobile phase as mixture of
solvents – TEF in ratio 5:4:1 (toluene, ethyl acetate, formic acid) in the
fume hood. Continue analysis according to the description in 3.
5. Detection of AFB1 with TLC
For AFB1 determination with TLC prepare mobile phase as mixture of
solvents – KAC in ratio 9:1 (chloroform: acetone) in the fume hood.
Continue analysis according to the description in 3.
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WORKBOOK
PERFORMANCE
Draw a plan of the experimental work!
FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK
FOR LABORATORY EXERCISES.
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MATERIAL, WORKFLOW AND RESULTS
1. Description of food on/in which moulds are determined
2. Description of moulds isolation from food
3. Determination of moulds in food by conventional
microbiological methods - qualitative and quantitative description of
isolated moulds on the isolation medium; selection and designation of
isolates for identification
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4. Moulds identification
In table below write macro-morphological properties of mould with
designation ………………………
Medium Macro-morphological properties
MEA 25 oC
2r:
SM:
ZM:
EK:
CYA 25 oC
2r:
SM:
ZM:
EK:
CYA 37 oC
2r:
SM:
ZM:
EK:
CYA 5 oC
2r:
SM:
ZM:
EK:
G25N
25 oC
2r:
SM:
ZM:
EK:
Legend:2R colony diameter; SM:substrate mycelium; ZM:aerial mycelium; EK: exudate
FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK
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Micro-morphological properties of mould with designation ………….are:
Isolate is identified as mould species …………………….……………………,
Other identified mould species are: ………………………………………….
…………………………………………………………………………………………..
………………………………………………………………………………………..
In table below write all identified moulds and add data from literature
about possible mycotoxin(s) formation.
Mould Mycotoxin(s)
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5. Determination of OTA with TLC – draw TLC-plate with results,
measure distances and calculate retention factors! Describe the results
according to food!
6. Determination of AFB1 with TLC – draw TLC-plate with results,
measure distances and calculate retention factors! Describe the results
according to food!
FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK
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ALERGENS
PURPOSE
Determine nut (Juglans regia) as an allergen in randomly selected foods.
COURSE OF EXPERIMENTAL WORK
• Prepare of standard walnut mixture;
• Make a standard curve for standard walnut mixtures (isolate DNA from
standard walnut mixtures, determine concentration of isolated DNA
spectrophotometrically, prepare a normalized concentration of e.g. 30
ng/µl of isolated DNA, perform real-time PCR, from known DNA
concentrations of walnut (%) and obtained Ct draw a standard curve);
• In randomly selected foods, detect and quantify nut (from food isolate
DNA, determine DNA concentration spectrophotometrically, prepare
normalised DNA e.g.30 ng/µl, perform real-time PCR, determine the
relative walnut content from the standard curve as an allergen)
INSTRUCTIONS
1. Preparation of standard walnut mixtures
Standard walnut mixtures are prepared with milled and degreased walnuts
and flour.
Grinding walnuts:
Purify grinder with a DNA removal agent, wash with water and dry with a
paper towel. We take 10 g of walnuts, transfer them to the mill and crush
them as much as possible.
Degreasing milled nuts with acetone:
Weigh out about 500 mg of crushed nuts in a 12 tubes of 2-ml. Then add
1000 µl of acetone, close tubes and mix the contents well on the table stirrer
(1 min). Then centrifuge samples for 5 min at 14.500 min-1. The upper,
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liquid phase is thrown into table trash. Then, repeat process of washing
walnuts with acetone two times more.
After the last washing, discharge upper phase and transfer crushed walnuts
to filter paper in the Petri dish (note that the filter paper is as little as
possible touching the bottom of the Petri dish!). Add empty 2 ml- tube into
Petri dish, in which we added degreased sample. Then, in semi-open Petri
dishes, samples are dried in the digestory for at least 1 hour. When samples
are dried, carefully transfer them to a 10 ml tube with a lid.
2. Preparation of foods
Preparation of food sample to determine the allergen (walnut) contains two
stages - homogenization and degreasing of the sample.
Homogenization of foods
The sample of food is homogenized by cutting, crushing, grinding and/or
mixing.
Degreasing of food samples with acetone
Weigh out approximately 500 mg of a homogenized food sample in a 2 ml-
test tube. Then 1000 µl of acetone is added, the tube is sealed and the
contents thoroughly mixed on a table mixer (1 min). The sample is
centrifuged for 5 min at 14.500 min-1. The upper, liquid phase is thrown into
the table trash. Then, the process of washing sample with acetone is
repeated 2x.
After the last washing, the upper phase is discarded again, and sample is
transferred to filter paper in Petri dish (note that the filter paper is as little
as possible touching the bottom of the petri dish!). Place an empty 2 ml-tube
in the Petri dish, in which the sample was degreased. Then, in a semi-doped
petri dish, the sample is dried in the digestory for 1 hour. When the sample
is dried, carefully transfer it to a 10 ml tube with a lid
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3. Preparation of standard mixtures of flour and walnut
Prepare the following standard mixtures from degreased walnuts and flour:
Mixture
designation % of nut Preparation
A 0 200 mg flour
B 0.001 Weigh out in a 50 ml-plastic tube 9 g of flour
and add 1 g 0.01 % mixture (D)
C 0.005 Weigh out in a 50 ml-plastic tube 9 g of flour
and add 1 g 0.05 % mixture (E)
D 0.01 Weigh out in a 50 ml-plastic tube 9 g of flour
and add 1 g 0.1 % mixture (F)
E 0.05 Weigh out in a 50 ml-plastic tube 9 g of flour
and add 1 g 0.5 % mixture (G)
F 0.1 Weigh out in a 50 ml-plastic tube 9 g of flour
and add 1 g 1 % mixture (H)
G 0.5 Weigh out in a 50 ml-plastic tube 9 g of flour
and add 1 g 5 % mixture (I)
H 1 Weigh out in a 50 ml-plastic tube 9 g of flour
and add 1 g 10 % mixture (J)
I 5 Weigh out in a 50 ml-plastic tube 9.5 g of flour
and add 0.5 g of walnut
J 10 Weigh out in a 50 ml-plastic tube 9 g of flour
and add 1 g of walnut
K 100 200 mg walnut
Prepare first a sample with designation A, then a sample labelled K, and
then samples from J to B. Mix each mixture very well - homogenize before
preparing the next mixture! Afterwords weigh 200 mg from the standard
mixtures J-B into 2 ml-test tubes.
4. Isolation of DNA from standard walnut mixtures and food
samples
DNA is isolated by DNA Nucleospin Food DNA Kit (Macherey-Nagel,
Germany) containing CF buffer, proteinase K, C2 buffer, C3 buffer, rinsing
CE buffer, rinsing columns and collecting tubes. If the sample is high-fat
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food, it is degreased with acetone before the isolation of the DNA using the
same procedure as described above.
Procedure of DNA isolation:
1. Add 1100 µl CF buffer (heated to 65 ºC) to 200 mg of sample, mix 15 s on
a table stirrer, add 20 µl proteinase K (10 mg/ml) and mix on a table
mixer; incubate for 30 min in a water bath at 65 ºC (mix every 5 minutes
on a table mixer).
2. The sample is centrifuged for 10 min at a speed of 14,500 rpm.
3. Transfer 300 µl of absolute ethanol (-20 oC) to the fresh 2 ml-test tube,
add 300 µl of C4 buffer and 300 µl of the supernatant from 2.
4. A sample of is mixed 30 s on a table mixer.
5. Carefully transfer 700 µl of the sample to the rinse column inserted in
the collection tube.
6. The sample is centrifuged for 1 min at 14,500 rpm and the liquid phase
is discarded.
7. Washing and drying of silicone membrane with binded DNA:
1. washing: add 400 µl of CQW buffer to the rinse column, centrifuge for
1 min at 12,000 rpm and discard the liquid phase,
2. washing: add 700 µl buffer C5 to the rinse column, centrifuge 1 min at
12,000 rpm and discard the liquid phase,
3. washing: add 200 µl buffer C5 to the flushing column, centrifuge for 2
minutes at 12,000 rpm and discard the liquid phase.
8. Put the column into new collection tube.
9. Add 100 µl buffer CE (heated to 70 ºC), incubate mixture for 5 min at
room temperature and centrifuge for 1 min at 12,000 rpm.
10. Transfer the whole liquid (100 µl) to a new 1.5 ml-test tube and store the
DNA solutions at -20 oC
5. Spectrophotometric determination of isolated DNA concentration
Dilute previously isolated DNA by adding 15 µl DNA (dilution 10-1) in a 200
µl test tube to 135 µl of H2OKEM. From this diluted DNA, 100 µl of the DNA
is transferred into a special microtiter plate to measure the absorbance in
the UV range. 100 µl of H2OKEM is added to the plate as a blank pattern. The
absorbance was measured at 260 nm (maximum for DNA) and at 280 nm
(protein maximum). Based on the measured absorbance at 260 nm, the
concentration of DNA is calculated:
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C (DNA) = (A260 sample – A260 blank sample) x 50 (ng / µl)
Purity of isolated DNA = (A260 sample – A260 blank sample) / (A280 sample – A280 blank
sample)
Pure DNA has A260 / A280 between 1.7 and 2.0.
6. DNA normalisation
DNA normalization means the preparation of a specific concentration of
DNA of all samples (standard samples and samples of foods) for real-time
PCR. The choice of concentration is empirically determined according to the
effectiveness of DNA isolation.
For the determination of the walnut as an allergen in the food, the PCR
reaction mixture should always be e.g. 30 ng DNA / µl and consequently
150 ng / 25 µl in real-time PCR with 5 of DNA.
Depending on the spectrophotometrically determined DNA concentration,
for each sample, calculate the volume of DNA that has to be diluted for 100
µl of a solution at a concentration of 30 ng / µl (C1 x V1 = C2 x V2).
For example, if the concentration of isolated DNA was found to be 125 ng /
µl then prepare 100 µl of a solution with a concentration of 30 ng / µl in a
new 1.5 ml-test tube by adding 24 µl of a DNA solution with a concentration
of 125 ng / µl to 76 µl of H2O.
7. Real-time PCR for detection and relative quantification of
wallnut
7.1 Preparation of the reaction mixture for real-time PCR:
The reaction mixture is prepared for standard samples, food samples,
negative template control sample (NTC), and so on. and spare sample (due
to pipetting) - total for N samples. The reaction mixture contains all the
components necessary for the enzyme reaction (without DNA!) And it is
always prepared as a single mixture.
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Depending on the total number of samples (N), the volumes of the individual
constituents of the enzyme reaction are calculated, taking into account that
in the reaction mixture for 1 sample consists of:
• Universal master mix (TaqMan Universal Master Mix 2x): 12.5 µl
• Pair of primers for walnut:
• JuglR: 2.,25 µl (900 nmol)
• JuglF: 2.25 µl (900 nmol)
• Probe for walnut: JuglP: 0.125 µl (50 nmol)
• H2OPCR: should be calculated (total volume is 25 µl, volume of DNA is 5
µl)
The reaction mixture for real-time PCR is then divided into 20 µl aliquots
into the reaction tubes or stripes. Then, samples of normalized DNA
(concentration of 30 ng/µl) (samples of standard walnut mixtures, food
samples, and NTC) are added to the so-divided reaction mixture of 5 µl.
Mark the samples in the table between the materials - point 5.3.
7.2 Real-time PCR
Real-time PCR is performed with standard programme in PCR-cycler (ABI
Prism 7500) (Fig. 3).
1 PHASE
1 cycle
3 PHASE
60 cycles
50 oC
95 oC 95
oC
60 oC
2 min
10 min 15 s
1 min
2 PHASE
1 cycle
Fig. 3: Time-temperature program for determining walnut with real-time PCR
7.3 Evaluation of results real-time PCR
After completing the enzyme reaction to evaluate the results, we set the
following parameters on PCR-cycler:
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• threshold: manually set to 0.02
• starting point: automatic setting
so that we can compare the results of different real-time PCR runs.
Then, for each sample, we determine the Ct value. The Ct value means how
many cycles is necessary to detect a normalized fluorescent signal (∆Rn),
and means that under certain conditions the fluorescence of the sample
increases over the background fluorescence
7.4 Standard curve
From the obtained Ct values for standard walnut mixtures, draw the
standard curve as the dependence of the Ct value on the relative walnut
concentration in the walnut mixture expressed as log (% walnut) (example
of Fig. 4). Calculate all parameters (e.g. correlation coefficient, limit of
detection, efficiency, range of quantification).
Fig. 4: Dependence of the Ct value from the decimal logarithm of the walnut
percentage in the standard walnut biscuit (Terpin, 2010)
7.5 Relative quantification of nut in foods
Depending on the specific Ct values of each food sample (point 7.3) the
concentration of walnut in food samples is achieved from the standard curve
(point 7.4).
C (log % of walnut in standard mixture)
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WORKBOOK
PERFORMANCE
Draw a plan of the experimental work!
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FOR LABORATORY EXERCISES.
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MATERIAL, WORKFLOW AND RESULTS
1. Preparation of standard walnut mixtures
2. Preparation of foods
3. Spectrophotometric determination of DNA concentration and
preparation of normalised DNA (30 ng/µl) for real-time PCR
3.1 Samples of standard walnut mixtures
Sample
designation
DNA
concentration
C1 (ng/µl)
DNA
purity
A260/A280
Normalised DNA with C2 30 ng/µl
V1 DNA (µl ) V H20 (µl )
A
B
C
D
E
F
G
H
I
J
K
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3.2 Food samples
Sample
designation
DNA
concentration
C1 (ng/µl)
DNA
purity
A260/A280
Normalised DNA with C2 30 ng/µl
V1 DNA (µl ) V H20 (µl )
1
2
3
4
5
6
7
8
9
10
4. Preparation of real-time PCR reaction mixture - calculation
No. of samples N:
COMPOSITION V FOR 1
SAMPLE (µl)
V FOR N
SAMPLES (µl)
UMM
12,5 µl
JuglR 2,25 µl
JuglF 2,25 µl
JuglP 0,125 µl
H2OPCR
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5. Ct values of standard walnut mixtures are determined by real-
time PCR
Sample
designation % of walnut Ct
A
0
B
0.001
C
0.005
D
0.01
E
0.05
F
0.1
G
0.5
H
1
I 5
J 10
K 100
6. Standard curve as dependence of Ct value on relative concentration
of walnut in walnut mixture expressed as log (% walnut)
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7. Ct values of food samples determined by real-time PCR
Sample
designation
Sample
name Description of sample Ct % walnut
8. Comment results according to the purpose of exercise
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ENVIRONMENTAL CONTAMNANTS
PURPOSE
In the water sample (for example, drinking water, wastewater), pure
chemical substances or mixtures (for example, plant extract), assess the
overall toxicity and level of genotoxicity.
COURSE OF EXPERIMENTAL WORK
• Preparation of onions
• Preparation of control solutions
• Exposure of onions to control solutions and test specimens,
incubation
• Preparation of microscopic preparations
• Determination of micro-morphological properties
• Determination of macro-morphological properties
• Evaluation of results
INSTRUCTIONS
1. Preparation of onions
To carry out the onion test (Allium test) we need 12 onions (Allium cepa
L) for each sample of water. For negative control (negative control
implies no toxic response), 12 onions and 12 onions for positive control
are used (positive control means that a toxic response is known in a
particular proportion); in each batch, 20% of the bad onions can be
discarded. Onions must be of the same size (1.5 to 2.0 cm in diameter),
up to 6 months old (stored at 10-14 ° C in dry and airy spaces up to 50%
RH). Onions that are dried, moldy or have green shoots are not suitable
for the onion test. The external damaged leaves are removed from
onions, also the brownish lower part (carefully not to damage the
primordial root ring). When we need large number of onions, they are
stored in clean water until use.
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2. Preparation of control solutions
As a negative control nutrient solution is used. Nutrient solution (Table
1) is a drinking water with the addition of nutrients that enable optimal
grow of onions. From the stock solution, prepare a nutrient solution and
divide it into 10 tubes. The stock solutions are prepared with distilled
water, then the nutrient solution is prepared by diluting the basic
solutions with distilled water. If drinking water is used instead of the
nutrient solution, we must verify that it has a neutral pH, it can be
relatively hard water (Ca + Mg: 50 -70 mg/l) and does not contain toxic
substances (for example, Cu2+ <0.05 mg/l). The water is filtered through
the filter (0.2 µm) before the test. If water-insoluble chemical compounds
are tested in water, they must be dissolved in a suitable solvent (for
example, ethanol, methanol, acetone), and in such a case, the solvent
content must be added to the nutrient solution. Please note that the final
solvent content does not exceed 1% vol.
Table 1: Composition of nutrient solution
Nutrient Concentration in
stock
Concentration in
control solution Ca(N03)2 . 4H20 1.0 mM 0.1 mM
KNO3 2.0 mM 0.2 mM
MgS04. 7H20 1.0 mM 0.1 mM
KH2P04 1.0 mM 0.1 mM
Fe-EDTA 3H20 0.2 mM 0.02 mM
Elementi v sledovih
MnS04 3.64 µM 0.364 µM
CuCl2 0.48 µM 0.048 µM
Na2Mo04 0.0078 µM 0.00078 µM
ZnSO4 0.0042 µM 0.00042 µM
H3BO3 3.7 µM 0.37 µM
As a positive control, a solution of methyl methane sulfonate (MMS;
Sigma M4016; 1 mg/l or 10 mg/l) is alkylating agents whose cytotoxic and
/ or mutagenic effect is due primarily to methylation of DNA). For each
test, use 10 tubes of MMS solution and add onions.
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3. Exposing onions to control samples and test water samples
The onions are quickly and carefully dried on a paper towel and placed in
the tubes into which the nutrient solution (negative control) is added - 10
tubes, MMS solution (positive control) - 10 tubes and test tubes - 10
tubes so that the lower part of onions touching the nutrient solution,
MMS solution or test sample. Incubation is up to 6 days at room
temperature (20 oC) so that the samples are protected from direct
sunlight.
4. Preparation of microscopic slides
After 2 days of incubation a microscopic slide is prepared from the roots,.
Sampling for microscopic slides include 5 onions (another 5 onions are
left on further incubation) from the negative control group, 5 onions from
the positive control group and 5 onions from the total test sample.
Procedure for preparing a microscopic slide:
• Fixing: Add approximately 0.5 ml fixation solution (9 vol. parts of
CH3COOH (45%) and 1 vol. of 1 M HCl) in the Al- dish (each bulb,
its Al- dish)! Then with sharp scissors cut 2 to 4 root tips (up to 2
mm) and immediately transfer them to the Al-dish and heat in a
water bath at 55 oC for 10 min.
• Staining: add one drop of orcein (2% orcein in CH3COOH (45%)) on
slide. Root peaks (without fixation solution!) are transferred to
orcein on the slide and carefully cut and smashed with scalpel
Than cover the slide with a cover glass, and gently squeeze cover
slide to arrange the cells into one plane, and wipe excess of dye
with a piece of paper towels. If the cover slide is glued, microscopis
slide can be stored for 2 months at 4 oC
5. Determination of micro-morphological properties
Microscopic slides are examined under microscope under 400x
magnification (100x, 1000x). First, we examine microscopic preparations
of control onions (negative control, positive control) and then the
preparations of onions that were exposed to test water samples. We
determine:
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• Mitotic index (MI, %): We examine 50 cells and determine the
number of cells in each stage of mitosis: prophase (P), metaphase
(M), anaphase (A), and telophase (T), and in the interphase (I).
Then we calculate the mitotic index:
�� = (��. ���� � + ��. ���� � + ��. ���� � + ��. ���� �)
��. ����� ���������� (50)�100
• On onion preparations exposed to different samples (negative
control, positive control, investigated water sample), changes in
chromosomes in anaphase and metaphase are observed, and the
number and type of injuries are described:
o The occurrence of chromosomal adhesion - an indicator of a
very toxic, mostly irreversible action leading to cell death
o o Clastogenic effect: a genotoxic effect on chromosomes - the
acquisition, alteration or loss of chromosome part (best seen
in metaphase as chromosomal fragments or bridges) - is an
indicator of mutagenic activity
o Extended and separated chromosomes
According on the obtained results, the percentage of damaged cells is
calculated and the level of genotoxicity is assessed. Based on the
obtained results, we calculate the percentage of damaged cells and assess
the level of genotoxicity.
6. Determination of macro-morphological properties
After 4 days of incubation, measure the length of the roots and describe
the appearance of the roots in the control onions and onions exposed to
the test water on the remaining five onions in each group (positive and
negative control, test water). Accurate measurements of length are done
with roots that are cut and measured, but in this case the onion is
unusable for further incubation. Depending on the measurements, we
calculate the average root length of each onion and the proportion of
length relative to the control onions. By describing the roots, the colour
is especially important, because it can change, for example, if it is dead
tissue (a strong toxic effect), the roots become more or less brown or if
the ions of copper sulphate are blue green in color.
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By using ANOVA test comparisons of lengths of roots of test samples and
control samples significant differences are determined and the level of
general toxicity could be estimated.
7. Modifications of the basic method
Reproducibility - reversibility of injuries:
For this testing we need 20 onions, and from the first 10 we proceed as
described from 1 to 6. After four days of incubation, tested liquid is
replaced with the nutrient solution in five onions, and then again replace
the nutrient solution on the fifth day, and measure the length of the
roots on the sixth day. If toxicity has been reversible over the first four
days, these onions will have new roots or will be longer than test bulbs
exposed to test liquids.
Treatment with colchicine
For more detailed studies of chromosomal injuries (breaks), instead of 10
bulbs, 12-13 onions are tested. These additional 2 - 3 onions are
incubated in a 0.1% solution of colchicine prior to the preparation of the
microscopic preparation, and further investigations are carried out as
described in 1-6.
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WORKBOOK
PERFORMANCE
Draw a plan of the experimental work!
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MATERIAL, WORKFLOW AND RESULTS
1. Describe water sample (appearance, preparation, number of
parallels, labels) that is under investigation
2. Describe control samples (preparation, no. of parallels,
designations)
Negative controls (NK):
Positive controls (PK):
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3. Micro-morphological properties of negative control onions -
drawings
Time of incubation: Designation of sample: Magnification:
4. Micro-morphological properties of negative control onions
Time of incubation: Designation of sample:
Designation
of onion
No. of
cells
No. of cells in each phase of
mitosis / No. of injured cells Mitotic index
Inter-
phase
Pro-
phase
Meta-
phase
Ana-
phase
Telo-
phase
MI
(%)
Percentage
of injured
cells (%)
NK1
NK2
NK3
NK4
NK5
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5. Micro-morphological properties of positive control onions -
drawings
Time of incubation: Designation of sample: Magnification:
6. Micro-morphological properties of positive control onions
Time of incubation: Designation of sample:
Designation
of onion
No. of
cells
No. of cells in each phase of
mitosis / No. of injured cells Mitotic index
Inter-
phase
Pro-
phase
Meta-
phase
Ana-
phase
Telo-
phase
MI
(%)
Percentage
of injured
cells (%)
PK1
PK2
PK3
PK4
PK5
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7. Micro-morphological properties of positive control onions -
drawings
Time of incubation: Designation of sample: Magnification:
8. Micro-morphological properties of tested onions
Time of incubation: Designation of sample:
Designation
of onion
No. of
cells
No. of cells in each phase of
mitosis / No. of injured cells Mitotic index
Inter-
phase
Pro-
phase
Meta-
phase
Ana-
phase
Telo-
phase
MI
(%)
Percentage
of injured
cells (%)
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9. Macro-morphological properties of negative control onions
Time of incubation:
Designation
of onion
Length of
roots (mm) Description of roots
NK1
NK2
NK3
NK4
NK5
Average
10. Macro-morphological properties of positive control onions
Time of incubation:
Designation
of onion
Length of
roots (mm) Description of roots
PK1
PK2
PK3
PK4
PK5
Average
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11. Macro-morphological properties of test onions
Time of incubation:
Designation
of onion
Length of
roots (mm) Description of roots
Average
12. Summary of results - general toxicity - calculation with ANOVA
test
Onions Average root length General toxicity
Negative control /
Positive control
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
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13. Summary of results - level of genotoxicity - calculation with
ANOVA test
Onions Average no. of cells in metaphase Level of risk - risk
assessment (%) * All Injured
Positive control
Negative control
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
*Risk assesment: <2 %: /; 3-5%: low; 6-11%: medium; 12-15%: high; >16%: critical
14. Comment the results according to the purpose of the exercise
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REFERENCES
Edwards, K. Logan, J., Saunders, N. 2004. Real-time PCR An Essential Guide.
Horizon Bioscience, Wymondham. 239 str.
Firbas P. 2013. Uporaba Allium metafaznega testa za določanje kakovosti
okolja. Laboratorij za rastlinsko aplikativno citogenetiko. 5 str.
Fiskesjo G. Allium test. 1995. V: In vitro Toxicity Testing Protocols. OHare S.
in Atterwill C. K. (Ur.), New Jersey, Humana Press, 119-128
Jeršek, B., Poklar Ulrih, N., Skrt, M., Gavrič, N., Božin, B., Smole Možina, S.
2014 Effects of selected essential oils on the growth and production of
ochratoxin A by Penicillium verrucosum. Arhiv za higijenu rada i
toksikologiju, 65, 2, 199-208
Klančnik, A., Kovač, M., Toplak, N., Piskernik, S., Jeršek, B. 2012. PCR in food
analysis. V: Hernandez-Rodrigues, P. (ur.), Ramirez Gomez, A. P. (ur.).
Polymerase chain reaction. Rijeka: Intech, cop., str. 195-220
Kuchta, T., Drahovska, H., Pangallo, D., Siekel, P.2006. Application of
polymerase chain reaction to food analysis. Bratislava, Vyskumny ustav
potravinarsky,107 str.
Sampson, H. A., Simon, R. A. 2008. Food allergy: adverse reactions to foods and
food additives. 4th ed., Malden (Mass.): Blackwell Publishing, 613 str.
Slovenski standard. SIST EN ISO 6888-1:1999, Mikrobiologija živil in krme -
Horizontalna metoda štetja koagulaza pozitivnih stafilokokov
(Staphylococcus aureus in drugih vrst ) - 1. del: Tehnika uporabe Baird -
Parkerjevega agarja (ISO 6888-1:1999), Ljubljana, Slovenski inštitut za
standardizacijo, 1999, 11 str.
Pitt, J.I., Hocking, A. D. 1985. Fungi and food spoilage. 1st ed. London [etc.] :
Blackie Academic & Professional, cop. 1997. 445 str.
Samson, R. A., Hoekstra, E. S., Frisvad, J. C., Filtenborg, O. 2000. Introduction
to food- and airborne fungi. 6th ed., Utrecht, Centraalbureau voor
Schimmelcultures, cop., 389 str.
Terpin P. 2010. Določanje oreha kot alergena v živilih s PCR v realnem času:
diplomsko delo, univerzitetni študij. Biotehniška fakulteta, Ljubljana, 66
str.
Trnčíkova, T., Piskernik, S., Kaclíkova, E., Smole Možina, S., Kuchta, T.,
Jeršek, B. 2010. Characterization of Staphylococcus aureus strains isolated
from food produced in Slovakia and Slovenia with regard to the presence of
genes encoding for enterotoxins. Journal of food and nutrition research. 49,
4, 215-220.
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ANNEX
MYCOTOXINS
Annex 1: Mould isolation on DRBC (A) and DG18 (B)
Annex 2: Example of identification according to Pitt and Hocking (1985)
(A) (B)
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Annex 3: Aspergillus (400x magnification)
Annex 4: Penicillium (400x magnification)
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Annex 5: Ochratoxin A (OTA) in aflatoxin B1 (AFB1)
(http://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs+hsdb:@term+@DOCNO+4305;
http://toxnet.nlm.nih.gov/cpdb/chempages/AFLATOXIN%20B1.html)
Annex 6: TLC-plate for detection of OTA under UV illumination
Legend: S: standard OTA (1 µg/ml), V: sample of mould
S V
OTA AFB1
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ALERGENS
Annex 7: The principle of detecting amplicons with real-time PCR by
TaqMan and SybrGreen methods
http://www.lifetechnologies.com/si/en/home/life-science/pcr/real-time-pcr/qpcr-
education/taqman-assays-vs-sybr-green-dye-for-qpcr.html
http://www.lifetechnologies.com/si/en/home/life-science/pcr/real-time-pcr/qpcr-
education/taqman-assays-vs-sybr-green-dye-for-qpcr.html
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ENVIRONMENTAL CONTAMINANTS
Annex 8: Microscopic images of onion cells: (A) anaphase, (B) telophase,
(C) prophase, (D) metaphase, (E) interphase
(A) (B)
(C) (D)
(E)
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Annex 9: Microscopic images of onion cells with different injuries in
metaphase and anaphase
(A) irregular metaphase with incorrectly distributed chromosomes and
breaks, (B) prolonged metaphase chromosomes, (C) anaphase micro-bridges,
(D) anaphase bridges, (E) breaks ofchromosomes, (F) incorrect classification,
adhesion of chromosomes
(A) (B)
(C) (D)
(E) (F)
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