food toxicology and contamination - bf.uni-lj.si · food toxicology and contamination. instructions...

Barbara Jeršek

Ljubljana, 2017

FOOD TOXICOLOGY

AND

CONTAMINATION

INSTRUCTIONS AND WORKBOOK FOR

LABORATORY EXERCISES

FOOD TOXICOLOGY AND CONTAMINATION. INSTRUCTIONS AND WORKBOOK

FOR LABORATORY EXERCISES.

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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.



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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


ENVIRONMENTAL CONTAMNANTS .................................................................... 26

PURPOSE .................................................................................................................. 26

COURSE OF EXPERIMENTAL WORK .............................................................. 26

INSTRUCTIONS ...................................................................................................... 26

WORKBOOK ............................................................................................................ 31 PERFORMANCE .............................................................................................................. 31


REFERENCES .............................................................................................................. 39

ANNEX ........................................................................................................................... 40

MYCOTOXINS ......................................................................................................... 40

ALERGENS ............................................................................................................... 43

ENVIRONMENTAL CONTAMINANTS .............................................................. 44



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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



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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,



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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



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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!



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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



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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!



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ALERGENS

PURPOSE

Determine nut (Juglans regia) as an allergen in randomly selected foods.


• 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




22


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.


• 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:



29

• 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.



30

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.



31

WORKBOOK

PERFORMANCE




32


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):



33

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



34

5. Micro-morphological properties of positive control onions -

drawings


6. Micro-morphological properties of positive control onions


Designation

of onion

No. of

cells



Inter-

phase

Pro-

phase

Meta-

phase

Ana-

phase

Telo-

phase

MI

(%)

Percentage

of injured

cells (%)

PK1

PK2

PK3

PK4

PK5



35

7. Micro-morphological properties of positive control onions -

drawings


8. Micro-morphological properties of tested onions


Designation

of onion

No. of

cells



Inter-

phase

Pro-

phase

Meta-

phase

Ana-

phase

Telo-

phase

MI

(%)

Percentage

of injured

cells (%)



36

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


PK1

PK2

PK3

PK4

PK5

Average



37

11. Macro-morphological properties of test onions

Time of incubation:

Designation

of onion

Length of


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



38

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



39

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.



40

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)



41

Annex 3: Aspergillus (400x magnification)

Annex 4: Penicillium (400x magnification)



42

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



43

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



44

ENVIRONMENTAL CONTAMINANTS

Annex 8: Microscopic images of onion cells: (A) anaphase, (B) telophase,

(C) prophase, (D) metaphase, (E) interphase

(A) (B)

(C) (D)

(E)



45

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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