food hygiene 2017 - university of ljubljana · 1. exercise: legislation in field of food hygiene...

University of Ljubljana Biotechnical Faculty

Department of Food Science and Technology

FOOD HYGIENE

Barbara Jeršek

Ljubljana, 2017

LABORATORY EXCERCISES FOR STUDENTS OF FOOD SCIENCE AND NUTRITION

2. updated and revised edition

Reviewers: prof. dr. Janez Marinšek prof. dr. Sonja Smole Možina Publisher: University of Ljubljana Biotechnical Faculty Department of Food Science and Technology Translation of Jeršek Barbara Higiena živil: laboratorijske vaje za študente živilstva in prehrane, 2. dopolnjena izd., El. knjiga., Ljubljana: Biotehniška fakulteta, Oddelek za živilstvo, 2013; 2017 According to the decision of the Dean of the Biotechnical Faculty on January 21, 2009, Food Hygiene is a textbook for laboratory exercises at subject of Food Hygiene. 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 permission of the copyright owner..

Jeršek B. Food Hyg iene : Labora tory exerc ises for students o f Food Science

and Nutr i t ion . Un ivers it y o f L jub l jana , B iotechn ica l Facul ty , 2017

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TABLE OF CONTENTS

1. EXERCISE: LEGISLATION IN FIELD OF FOOD HYGIENE ................................... 4

INTRODUCTION................................................................................................................... 4

NATIONAL LEGISLATION – THE MAIN REGULATIONS ......................................... 4

EU LEGISLATION ............................................................................................................. 6

PRACTICAL WORK ............................................................................................................... 7

2. EXERCISE: CHROMOGENIC AND FLUOROGENIC MEDIA .................................. 9

INTRODUCTION................................................................................................................... 9

PRINCIPLES OF DETERMINING MICROORGANISMS WITH CHROMOGENIC

AND FLUOROGENIC MEDIA.............................................................................................. 9

PRACTICAL WORK ............................................................................................................. 17

3. EXERCISE: PETRIFILMS ................................................................................................ 19

INTRODUCTION................................................................................................................. 19

PRINCIPLE OF MICROORGANISMS DETECTION WITH PETRIFILMS ..................... 19

PRACTICAL WORK ............................................................................................................. 21

4. EXERCISE: POLYMERASE CHAIN REACTION ........................................................ 23

INTRODUCTION................................................................................................................. 23

PRINCIPLE AND APPLICATIONS OF PCR ................................................................... 24

PRACTICAL WORK ............................................................................................................. 28

5. EXERCISE: REAL-TIME PCR ......................................................................................... 33

INTRODUCTION................................................................................................................. 33

PRINCIP IN LASTNOSTI PCR V REALNEM ČASU ..................................................... 33

PRACTICAL WORK ............................................................................................................. 37

6. EXERCISE: CONTAMINANTS: DETECTION OF ANTIBIOTICS RESIDUES IN

FOOD ....................................................................................................................................... 41

INTRODUCTION................................................................................................................. 41

DISC DIFFUSION METHOD FOR DETERMINATION OF ANTIBIOTICS IN FOODS 43

PRACTICAL WORK ............................................................................................................. 46

7. EXERCISE: CLEANING: DETERMINATION OF WORKING SURFACE

CLEANILINESS ..................................................................................................................... 48

INTRODUCTION................................................................................................................. 48

CLEANING ....................................................................................................................... 48

CONTROL OF CLEANING EFFICIENCY ...................................................................... 50

SAMPLING WITH SWAB AND MICROBIOLOGICAL INVESTIGATION ........................ 50

SAMPLING WITH WASHESAND MICROBIOLOGICAL INVESTIGATION ................... 51

MEASUREMENT OF ATP-BIOLUMINISCENCE ................................................................ 51

MEASURMENT OF NAD AND NADP .................................................................................... 52

PRACTICAL WORK ............................................................................................................. 54



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8. EXERCISE: DISINFECTION .......................................................................................... 56

INTRODUCTION................................................................................................................. 56

DILUTION-NEUTRALIZATION METHOD ................................................................... 57

PRACTICAL WORK ............................................................................................................. 59

9. EXERCISE: ANTIMICROBIAL ACTIVITY OF DISINFECTANT ............................ 62

INTRODUCTION................................................................................................................. 62

DETERMINATION OF DISINFECTANT EFFECTIVENESS ....................................... 62

AGAR DIFUSSION METHOD ................................................................................................... 63

BROTH DILUTION METHOD ................................................................................................ 63

DIRECT TESTING WITH SURFACE SwAB ........................................................................... 64

PRACTICAL WORK ............................................................................................................. 65

10. EXERCISE: PERSONAL HYGIENE ............................................................................. 70

INTRODUCTION................................................................................................................. 70

NORMAL MICROBIAL POPULATION .............................................................................. 70

PRACTICAL WORK ............................................................................................................. 73

NOTES ..................................................................................................................................... 76

REFERENCES ....................................................................................................................... 77



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1. EXERCISE: LEGISLATION IN FIELD OF FOOD HYGIENE

INTRODUCTION

ccording to the Law on the health suitability of foodstuffs and products and food contact materials (ZZUZIS, 2000, 2002, 2004) food hygiene includes all requirements and measures that are necessary for ensuring health and/food safety at all stages of their production and transport.

Food production and trade, food control, food quality and health suitability, and measures in the event of a health threat, are regulated and controlled by a number of statutory requirements (laws, regulations, ...). The main legal regulations are laws and they must be in compliance with the regulations, lists and other implementing regulations. The rules can be classified into the main groups that contain: • Regulations that determine quality requirements for food • Regulations laying down the microbiological requirements for foods • Regulations defining additives, pesticides, veterinary medicines and other

contaminants and their residues in foods • Regulations laying down hygienic and sanitary technical conditions for production,

sales, warehousing and other food establishments • Regulations that determine who and how to control food handling • Regulations laying down specific measures to protect against infectious diseases

A summary of the main legal regulations:

NATIONAL LEGISLATION – THE MAIN REGULATIONS

• Law on the health suitability of foodstuffs and products and food contact materials (ZZUIZS) (Uradni list RS 52/2000; 42/2002, 47/2004)

• Veterinary law (Zvet-1) (Ur. list RS 33/2001; 62/2004) • Law on veterinary compliance criteria (ZVMS) (Uradni list RS 93/2005) • Health inspection law (ZZdrl) (Uradni list 36/2004, 39/2006, 59/2006) • Law on agriculture (ZKme) (Uradni list RS 45/2008) • Law on infectious diseases (ZNB) (Ur. list RS 65/1995, 119/2005, 33/2006) • Law on plant protection products (ZFfS) (Uradni list RS 11/2001, 37/2004, 98/2004,

14/2007, 35/2007)

A

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• Law on management of genetically modified organisms (ZRGSO) (Uradni list RS 67/2002, 73/2004, 23/2005)

The Law on the health suitability of foodstuffs and products and food contact materials (ZZUIZS, 2000, 2002, 2004) and the Veterinary law Veterinary law (Zvet-1, 2001, 2004) and the relevant implementing regulations define the food, foodstuffs of animal origin, food production and food trade. In order to ensure the health suitability of foods, control methods are defined both in production and in food trade. The Law on agriculture (ZKme, 2008) and the relevant implementing regulations determine the minimum quality of agricultural products or foodstuffs, the manner of production, the ingredients and their content, the requirements for achieving and maintaining quality, classification and labeling. In addition to the minimum quality of products or foods, ecological agricultural products or foodstuffs, integrated agricultural products or foodstuffs as well as agricultural products or traditional foods are defined.

Food is safe and health suitable (ZZUZIS, 2000, 2002) when: 1. food does not contain microorganisms or parasites or their developmental forms or

excretions which may adversely affect human health;

2. residues of pesticides and veterinary medicinal products do not exceed the maximum permitted concentration;

3. food does not contain toxic metals, non-metals, other chemical pollutants from the environment and toxic and other substances in concentrations that may adversely affect human health;

4. food does not contain additives that are not authorized for food production or do not fulfil the conditions of purity or if the quantity of additive does not exceed the permitted quantity;

5. the residues of processing utilities or other substances used in the production of foodstuffs do not exceed the maximum permitted concentration, or do not have a detrimental effect on human health;

6. food does not contain radionuclides above the permitted limit or the food is not irradiated beyond the limit specified by the regulations;

7. food is not mechanically contaminated with admixtures or foreign matter which may be harmful to human health, give rise to resistance or directly endanger human health;

8. food composition, which may affect the biological and energy value of the food, is in accordance with the approved conditions;



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9. food composition or organoleptic characteristics (taste, odor, appearance) due to

physical, chemical, microbiological or other processes is so modified that the food is intentionally useless;

10. the shelf life of food is legible and not expired;

11. food of animal origin is marked with a health mark.

EU LEGISLATION The production, processing, distribution, retail, packaging and labelling of foods are governed by a mass of laws, regulations, practices and guidance. Examples:

REGULATIONS • Commission Regulation (EC) 852/2004 on the hygiene of foodstuffs. Corrigendum

to Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs

• Commission Regulation (EC) No 322/2006 of 23 February 2006 amending Regulation (EC) No 1043/2005 by reason of the provisions on the hygiene of foodstuffs and for food of animal origin provided for by Regulation (EC) No 852/2004 of the European Parliament and of the Council and by Regulation (EC) No 853/2004 of the European Parliament and of the Council

• Commission Regulation (EC) No 37/2005 of 12 January 2005 on the monitoring of temperatures in the means of transport, warehousing and storage of quick-frozen foodstuffs intended for human consumption

• Commission Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for food of animal origin and Corrigendum

All appropriate legal regulations are on websites: https://www.uradni-list.si/ http://eur-lex.europa.eu/homepage.html?locale=en .



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PRACTICAL WORK

Purpose: 1. Review the legal regulations and answer the questions! 1.

2.

3.

4.

Material: 1. Write sources / literature



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Results:



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2. EXERCISE: CHROMOGENIC AND FLUOROGENIC MEDIA

INTRODUCTION

oods are a favourable environment for growth of many bacteria and fungi. If the handling of food is inadequate in course of production or transport, the food may be contaminated with microorganisms and/or their toxins. Consumption of contaminated foods may, in certain circumstances, lead to infections or poisoning

of individuals or major groups (epidemics). Food poisoning with foods which are contaminated with microorganisms and/or their toxins is abbreviated as alimentary toxoinfections. Given the fact that infections and intoxications with foods contaminated with microbes or their toxins are among the most common causes of food poisoning, it is important that the methods by which microbes are detected in foods are sensitive, specific and rapid. A classical microbiological examination for the detection of microorganisms in foods involves the cultivation of microorganisms in a laboratory. Depending on the purpose of the investigation, different media are used for cultivation, such as basic, enrichment, differential, selective media and media for the detection of biochemical properties. In classical identification of bacteria, it is necessary to isolate the pure culture on the primary isolation medium, and then carry out a series of biochemical and serological tests. The procedure is usually very lengthy. Among the various modifications of conventional microbiological cultivation methods, chromogenic and fluorogenic media are often mentioned

PRINCIPLES OF DETERMINING MICROORGANISMS WITH

CHROMOGENIC AND FLUOROGENIC MEDIA

n the last 10-20 years microbial metabolism studies have led to the development of new microbial media. One of the newer group of chromogenic media and fluorogenic media contain artificial chromogenic or fluorogenic enzyme substrates. Chromogenic and fluorogenic media can be basic, differential or selective depending

on the ingredients.

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Fluorogenic and chromogenic enzyme substrates are components of microbiological media and various systems for rapid, direct identification of microorganisms. If fluorogenic and chromogenic substrates are the constituents of selective media, they can be used as primary isolation media. Thus, the identification of isolates does not require new cultivation and a series of biochemical tests, which means that the use of chromogenic and fluorogenic media reduces the time of investigation and saves material and work. Interactions between microorganisms and chemical ingredients of the media are different and mostly depend on the structure of chromophores or fluorophores. The substance formed after the enzyme reaction is: • directly binds to the microbial cells and thus colonies on the medium are specifically

coloured or • diffuses into the medium and gives it a characteristic color or fluorescence

Chromogenic enzyme substrates are substances for specific enzymes with an added chromogenic ingredient that changes colour in the enzyme reaction. Most chromogenic enzyme substrates contain phenol derivatives, for example:

• o- in p-nitrophenols (ONP, PNP) • p-nitroanilins (PNA) • indoxyl (Y) • 5-bromo-4-chloro-3-indolyl (X)

Chromogenic enzyme substrates are water-soluble, insensitive to temperature and not diffused into solid media.

The principle of Escherichia coli detection with chromogenic enzyme substrate: Most of E. coli have an β-D-glucuronidase (GUD) enzyme. As a chromogen substrate, X-β-D-glucuronide (X-GLUC or BCIG) is present in the medium. If E. coli are in/on culture medium, they will break down X-GLUC substrate with GUD enzyme. The released chromophore X will colours E. coli colonies markedly blue-green (Fig. 1). Figure 1: Principle of E. coli detection with chromogenic enzyme substrate

X-GLUC = X-β-D- glucuronide

β-D- glucuronidase from E. coli

Glucuronic acid (colourless)

X (blue-green

coloured colonies of E. coli)

+



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Fluorogenic enzyme substrates consist of a specific substance (sugar or amino acid) and a fluorogen. Most fluorogenic substrates are coumarin derivatives, for example:

• 2H-1 benzopyran-2-1-, 4-methyl (4-MU), • 7-amino-4-methylcoumarin (7-AMC), • β-naphthylamine (β-NAP).

After enzymatic reaction, fluorogen is released which, when illuminated by UV fluorescence. Fluorogenic substrates are water-soluble, highly sensitive and specific. The use of fluorogenic substrates is limited by certain properties such as: strong diffusion into solid media, pH dependence, and UV light. The principle of Escherichia coli detection with fluorogenic enzyme substrate: Most E. coli bacteria have β-D-glucuronidase (GUD) enzyme. As a fluorogen substrate, 4-MU-β-D-glucuronide (MUG) is present in the medium. If E. coli bacteria are in/on medium, they will break down MUG substrate thewith GUD enzyme. The released 4-MU will fluoresce fluorescently when illuminated with UV light (366 nm). Fig.2: Principle of E. coli detection with fluorogenic enzyme substrate

MUG = 4-MUX-β-D- glucuronide

β-D- glucuronidase from E. coli

Glucuronic acid (colourless)

4-MU (blue fluorescence of

E. col colony and medium) +



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EXAMPLES: DETECTION OF Salmonella The detection of Salmonella with conventional microbial media is very difficult because the colonies of bacteria from the genera Salmonella, Proteus and Citrobacter are very similar. Poor specificity or selectivity of media means many false positives (Proteus and Citrobacter) among rare, truly positive Salmonella colonies. Typical colonies of Salmonella on conventional microbiological media: - Salmonella-Shigela medium:

• Salmonella: translucent, mucous colony with or without a dark black center, • Proteus: translucent, mucous colony with or without a dark black center, • Shigella: transparent or slightly pink, may be irregular shape; • enterobacteria that ferment lactose: pink.

- Bismuth sulphide medium:

• Salmonella: black colony, with metallic shine, and a zone of black-colored medium;

• Proteus: brown to brown-black colonies; • E. coli: inhibited growth.

- Brilliant green agar: • Salmonella and some other species that do not ferment lactose: red violet colonies,

similarly colored medium around colonies (S. Typhi in S. Paratyphi are inhibited); • Proteus: pink, mucous colonies, 2 - 3 mm in diameter, medium around colonies

turns red because of a basic reaction; release of ammonium compounds resulting from the degradation of proteins;

• E. coli: yellow to yellow green colonies, the coloring around the colonies is similar to the pH of the lactose fermentation; brilliant green agar becomes yellow.

The use of chromogenic and fluorogenic media therefore makes it easier and faster to determine salmonella-specific colonies. Typical colonies of Salmonella on chromogenic media: - SM-ID medium (bio-Merieux, Francija) In the medium there are two chromogenic substrates X-GAL for β-galactosidase and X-GLU for β-glucosidase (other ingredients: glucuronate, neutral red indicator and selective ingredients brilliant green and bile salts).



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• Salmonella does not exploit chromogenic substrates (X-GAL and X-GLU) and form glucuronate acid. Therefore, the indicator changes to red and salmonella colonies are red.

• Other enterobacteria use X-GLU and X-GAL and form blue or colorless colonies. (Enterobacteria are bacteria from the genera Escherichia, Salmonella, Shigella, Citrobacter, Klebsiella, Erwinia, Serratia, Hafnia, Edwardsiella, Proteus, Providencia, Morganella, Yersinia).

- Rambach medium (CHROMagar, Francija; Merck, Nemčija) Rambach medium contains chromogenic substrate X-GAL for β-galactosidase (other ingredients: propylene glycol, peptone, yeast extract, sodium deoxycholate and neutral red indicator).

• Salmonella form acid from propylene glycol, and therefore the neutral red indicator changes color and salmonella colonies are bright red to violet.

• Coliform bacteria that have β-galactosidase form blue colonies because they degrade X-gal (blue).

• Some coliform bacteria form colonies of purple in order to form acid from propylene glycol and β-galactosidase activity.

• Proteus bacteria are inhibited or form in colourless colonies. MUCAP Test (Biolife, Italija) MUCAP test is used to determine Salmonella on conventional selective media. The basis of the test is the reagent containing the fluorogen enzyme substrate 4-MU-caprylate. The reagent is added to the colonies on the selective medium. Salmonella has a C8 esterase enzyme that cleaves 4-MU-caprylate and releases 4-MU. 4-MU after 1 - 5 minutes under UV illumination (366 nm) is seen as a strong blue fluorescence. DETECTION OF Escherichia coli Typical colonies of E. coli on conventional microbiological media: - Violet red bile agar with lactose (VRBL) is selective medium for isolation of coliforms.

• E. coli forms dark red colonies with a purple red centre. • Other coliform bacteria also grow, for example Enterobacter aerogenes form dark

red colonies, Proteus mirabilis form colourless colonies.

- Eosin methylene blue medium (EMB): • E. coli form purple colonies with a darker centre and a greenish metallic shine. • Enterobacter aerogenes: a red colony with a dark centre with a transparent zone. • Salmonella and Shigella form colourless or pink colonies.



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Typical colonies of E. coli on chromogenic media: About 94-96% of E. coli have an β-D-glucuronidase (GUD) enzyme. GUD enzyme has no other species of the genus of Escherichia and also E. coli strains O157: H7. The activity of the GUD enzyme is determined by E. coli in selective media in which p-nitrophenol β-D-glucuronide (PNPG), X-β-D-glucuronide (X-GLUC or BCIG) and, as chromogens and fluorogenic enzyme substrates, are 4-MU-β-D-glucuronide (MUG). One of the chromogenic media is TBX Agar (Biokar Diagnostics), which has a chromogenic substrate BCIG (X-GLUC). The GUD enzyme uses BCIG and releases the chromophore blue, which colours colonies of E. coli intensely blue green (Fig. 1). Fluorocult Lauryl Sulfate Broth (Merck) and solid media (Fluorocult ECD Agar, Merck); Fluorocult VRB Agar (Merck); Fluorocult MacConkey Agar, (Merck) contain fluorogene enzyme substrate (MUG). After hydrolysis of the fluorogenous substrate MUG, 4-MU is released which blue fluoresces under UV illumination at 366 nm (Fig. 2). The fluorescence of 4-MU is known to depend on the pH value. It is optimally neutral to a slightly alkaline environment, otherwise, after incubation, the basic solution is added. The disadvantage of the MUG substrate is that the 4-MU relatively good diffuses into the medium around the colonies, and the reaction readings are best at mediums after 18 hours or shorter incubation Simultaneous identification of coliform bacteria and Escherichia coli As E. coli and other coliform bacteria are important as indicators of faecal contamination of foods and water, there are many media that allow their simultaneous determination. Coliform bacteria are defined as bacteria having an β-D-galactosidase enzyme that cleaves lactose into glucose and galactose. According to the classic definition, coliform bacteria are defined as Gram-negative rods, non-sporogenic, aerobic or facultative anaerobic bacteria that form acid and gas from lactose within 48 hours at 35 ° C. They are determined by the MPN method or the membrane filtration method and confirmatory tests. The entire investigation may take two to four days. If we define them as bacteria that have a β-D-galactosidase enzyme, we can get a positive result for some non-lactose-resistant enterobacteria. Commercial media for the simultaneous detection of coliform bacteria and E. coli contain various enzyme substrates for the detection of β-D-galactosidase (for all coliform bacteria) and β-D-glucuronidase (GUD) for E. coli). - Chromcult Coliform Agar (Merck) and ROSE-GAL BCIG media (Biokar Diagnostics) contain two chromogenic enzyme substrates, Salmon-gal and X-GLUC, and the characteristic appearance of the colonies is:



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• 6-chloro-3-indolyl β-D-galactopyranoside (Salmon-gal): coliform bacteria form dark blue violet and red colonies,

• X-β-D-glucuronic acid (X-GLUC or BCIG): E. coli form dark blue violet colonies,

• other enterobacteria form colorless colonies

DETECTION OF Clostridium perfringens Typical colonies of Clostridium perfringens on conventional microbiological media: - SICA medium (egg yolk free tryptose sulphite iron citrate cycloserine agar): • Cl. perfringens black colonies, • Other sulphite-reducing clostridia such as Cl. bifermentans, Cl. botulinum, Cl.

paraperfringens, Cl. sardinese in Cl. sporogens forms also black colonies - SPS medium (sulphite polymyxin sulfadiazine agar): • Cl. perfringens: black colonies, • Cl. sporogens: black colonies, • Streptococcus faecalis and Staphylococcus aureus: good growth Typical colonies of Clostridium perfringens on fluorogenic media: To identify Cl. perfringens in the Fluorocult TSC (Merck) medium, a 4-MU-phosphate (4-MUP) fluorogenic enzyme substrate is added. Cl. perfringens with an acid phosphatase enzyme hydrolyze the 4-MUP. After illumination with UV colony of Cl. perfringens light blue fluorescence due to blue 4-MU fluorescence DETECTION OF Listeria monocytogenes Typical colonies of Listeria on conventional microbiological media: Classical isolation and identification of L. monocytogenes is difficult because the colonies of all species of the genus Listeria on the classical selective media like Oxford and Palcam are the same. - Oxford medium: After 24 hours, the listeria colony is small with a diameter of 1 mm, grey with a black zone, after 48 hours they become darker, they can have a green shine, a larger diameter of up to 2 mm, have a black zone and are in the middle of the inverted.



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- PALCAM medium: After 24 hours of incubation, the listeria colonies are small (1.5 to 2 mm in diameter) greyish green or olive green with a black zone, sometimes having a black centre. After 48 hours there are green colonies with a black zone The identification of L. monocytogenes requires confirmation of the genus Listeria and further identification of each species. Typical colonies of Listeria on chromogenic media: Therefore, recent identification media include enzyme substrates containing the phosphatidyl andositol phospholipase C (PI-PLC) enzyme, which is characteristic of L. monocytogenes and L. ivanovii specific chromogen-related substrates. In the chromogenous BCM-LMDS medium (Biosynth, Switzerland) X-mio-inositol-1-phosphate was added as an enzyme chromogenic substrate. L. monocytogenes and L. ivanovii form turquoise colonies. Likewise, L. monocytogenes and L. ivanovii colonies on CHROMagar Listeria (CHROMagar, France) and ALOA (Oxoid, Italy) are blue with cloudy translucent zone, while colonies of other listeria are blue without zonal or white. Rapid L. mono medium (Sanofi, France) contains ingredients needed to determine the activity of the PI-PLC enzyme and xylose. Colonies of L. ivanovii are as blue in the yellow medium (they exploit xylose), and L. monocytogenes colonies are blue in unchanged medium of red (do not exploit xylose).



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PRACTICAL WORK

Purpose: 1. In the sample of mixed bacterial cultures marked as A determine if Salmonella are

present by using appropriate classical and appropriate chromogenic media! After incubation perform MUCAP test on classical medium.

2. In the sample of mixed bacterial cultures marked as B determine if Escherichia coli are present by using appropriate classical and appropriate chromogenic media!

3. In the sample of mixed bacterial cultures marked as C determine if L. monocytogenes are present by using appropriate classical and appropriate chromogenic media!

Material and practical work: 1. Determine appropriate classical and chromogenic media for each examination

Sample Examination Classical medium Chromogenic medium

A

B

C

2. Draw a plan of the experimental work:



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Results 1. For A, B and C samples describe colonies on the classical selective media and

chromogenic media! According to morphological properties of colonies, determine whether A sample contain Salmonella, B sample Escherichia coli and C sample Listeria monocytogenes! Write down on which media you have identified individual bacteria and specific enzymatic reactions specific to bacteria on a particular chromogenic media! For sample A, perform also MUCAP test.

2. Sample A

MUCAP test:

3. Sample B

4. Sample C



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3. EXERCISE: PETRIFILMS

INTRODUCTION

implicity, quick performance, precision, specificity and sensitivity are requirements for microbiological examination, which in practice are difficult to achieve. The rapid development over the past 20 years has helped to make some of the above parameters feasible in practical work. Thus, one of the alternatives to classic

microbial media is the use of pre-prepared media or the use of Petrifilm. In the laboratory, therefore, it is not necessary to prepare media and thus time required to carry out the investigation is shortened.

PRINCIPLE OF MICROORGANISMS DETECTION WITH PETRIFILMS

etrifilms (3MTM, Microbiology Products, Sante) are one of the alternatives to classical methods of microbiological examination of foods. These are commercially prepared sterile, dry media that are rehydrated before the investigation. The media contain similar ingredients like classic microbiological media and chromogenic enzyme substrates.

Using Petrifilms, the time needed to prepare classic microbial media is saved and the costs of investigations are lowered. We do not need additional equipment for equipment in laboratory, nor do we need additional education for employees.

Petrifilm consists of a base and a roof film, and a two-layered film system that is covered with nutrients and a water-soluble gelling agent. Different indicators can be added for specific colouring of colonies. Depending on the type of Petrifilm, the media also contain various selective additives such as chromogenic or fluorogenic enzyme substrates. On the lower film there is a network that facilitates the counting of colonies.

Petrifilms can be used for sampling surface or air, for taking swabs of surfaces. Working with Petrifilm is easy, for example, a food sample: The sample is aseptically prepared for a microbiological examination. Place the petrifilm on a flat horizontal surface, aseptically lift the top foil and pipette 1 ml of the sample of the food or appropriate dilution in the middle of the surface with the medium. Carefully lay the upper film of Petrifilm on the surface so that no air bubbles are formed. Then distribute the inoculum over the entire surface of the medium with a plastic cap - dispenser (in Petrifilms for the determination of yeasts and molds and the total number of microorganisms with a crushed side, for other Petrifilms with a smooth side). After 1 minute, Petrifilm is incubated under specific conditions that allow the growth of the

S

P

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investigated microorganisms. The results of microbiological investigations obtained with Petrifilms are comparable to results obtained with classical microbiological media.

The main advantages of using Petrifilms compared to conventional microbiological media are:

• Accurate results after three simple stages of microbiological examination: sample inoculation, incubation and reading of results

• Standardized methodology • Small, flat media • Petrifilm is already pre-coated with nutrients and gelling agents • Petrifilms can be used for testing raw materials, intermediate products, finished

food products and for environmental samples • Petrifilms are suitable for the HACCP system • The results obtained with Petrifilms are comparable between individual

examinations, between contractors and between laboratories Table 1: Types and properties of some Petrifilms3MTM

Type of Petrifilm 3MTM

Medium Chromogenic substrate

Incubation

Colonies

Aerobic Count Plates

PCA1 TTC2 30o C /

48 h Red colonies

Enterobacteriaceae Count Plates VRBG1

TTC, pH indicator

35o / 37o C, 24 h

Red colonies with yellow zone and/or

bubbles of gas Coliform Count

Plate VRBL1 TTC

30o / 37o C, 24 h

Red colonies with bubbles of gas

Select E. coli Count Plates

VRBL1 TTC, BCIG3 42o / 44o C, 24 h

Blue-green colonies

Escherichia Coli and Coliform Count

Plates VRBL1

TTC, BCIG

35o / 37o C, 24 h

E. coli: blue, other coliforms: red with

bubbles of gas

Yeast and Mold Count Plates

Saboraud with

antibiotic X-phosphate

25o C, 3 – 5 days

Yeasts: small, blue, green, mould: big

Staph Express Count Plate

Modified BP4 TTC 37 oC / 24 urh

Staphylococcus aureus red violet

Legend: 1 modified media: PCA (Plate Count Agar), VRBG (Violet Red Bile Galactose), VRBL

(Violet Red Bile Lactose) 2 TTC: chromogen component 2,3,5-triphenyl tetrazolium chloride, which colored

colonies red 3 BCIG: chromogenic compound 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-

GLUC) which coloured colonies blue 4 Baird- Parker medium



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PRACTICAL WORK

Purpose: In the sample marked as MK (mixed culture) determine with appropriate Petrifilm the number of … … total bacteria … coliform bacteria … Escherichia coli … Staphylococcus aureus Material and practical work: 1. For each investigation select appropriate type of Petrifilm

Investigation: Type of Petrifilm: Total bacteria Coliform bacteria Escherichia coli Staphylococcus aureus

2. Draw a plan of the experimental work:



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Results 1. Describe the appearance of typical colonies on the selected Petrifilm and quantify the

results! Bacteria Petrifilm Colony description N (cfu/ml)

Total bacteria

Coliform bacteria

Escherichia coli

Staphylococcus aureus



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4. EXERCISE: POLYMERASE CHAIN REACTION

INTRODUCTION

nfections or food poisoning are most commonly caused by pathogenic bacteria that are transmitted to humans via animals (zoonosis) or contamination of foods in technological process. The detection of pathogenic bacteria in foods is often difficult because their number is relatively low compared to a high number of other

microorganisms in the food. Most traditional microbiological methods therefore involve more or less selective enrichment, sometimes also the pre-enrichment, isolation and identification of microbes. Therefore, the entire microbiological examination process is often time-consuming and work-consuming. Over the past 20-30 years, many technologies and methods have been developed to identify pathogenic bacteria in foods (raw materials, semi-products, products). Fast, specific and sensitive methods of bacterial detection are also necessary in the process control, determination of purity and hygiene in the production and marketing of food. Some examples of newer methods are shown in Table 2.

Table 2: Examples of novel methods for detection of pathogenic bacteria in foods

PRINCIPLE METHOD

Modifications and automation of methods included in classical microbiological cultivation methods

Spiral plater Chromogenic and fluorogenic media Petrifilms HGMF (Hidrophobic Grid-Membrane Filter technique)

Bioluminescence ATP measurement

Microscopy DEFT (Direct Epifluorescent Filter Technique) Flow cytometry

Measurements of electrical parameters

Impedance methods

The specific reaction of antibody with antigen

Automated immunological methods (Vidas) Immunomagnetic separation ELISA

DNA analysis Hybridization techniques PCR (polymerase chain reaction) Technology of DNA-chip

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24

PRINCIPLE AND APPLICATIONS OF PCR

In addition to various hybridization techniques and newer DNA-chip technology, polymerase chain reaction (PCR) is one of the molecular methods used to identify and detect microorganisms in foods and various other samples. Steps involved in the investigation of sample with PCR are: • preparation of DNA • preparation and implementation of PCR • determination of amplicons

Preparation of DNA: DNA preparation involves various physicochemical and/or enzymatic methods to obtain a more or less pure suspension of DNA for PCR. DNA molecules that are inside the bacterial cells are released from the cells after lysis of cell wall (with thermal, chemical, mechanical or enzymatic action). For PCR, a suspension of all cellular components can be used by cell lysis, or the DNA can be isolated and purified (for example, DNA extraction with phenol-chloroform). Principle of PCR: The principle of PCR is an in vitro amplification of the target DNA with a DNA-polymerase in a cyclic thermostat. Thermostable DNA-polymerase is an enzyme isolated from thermophilic bacteria Thermus aquaticus. The enzyme has an optimum temperature of 72 ° C and maintains its activity even if for shorter time is at higher temperatures required to denaturate the double-stranded DNA. A special thermostat- PCR cycler provides a continuous temperature change in a cycle covering three main phases (Fig. 3): • denaturation / separation of the two-strand DNA (for example: 95 ° C, 30 s) • annealing of primers (for example: 55-72 ° C, 5-60 s), • amplification of DNA (for example: 72o C, 60s). In each cycle, the number of target DNA copies is doubled. With 25 to 35 repeated cycles, the concentration of amplified DNA is exponentially raised to more then 109 copies. In order to multiply part of the DNA, a reaction mixture is needed which, in addition to the target DNA molecules, contains a thermostable DNA-polymerase, deoxynucleotide triphosphate (dNTP: dNTA, dNTT, dNTG, dNTC), Mg2+ ions, reaction buffer and a pair of primers. Each PCR has to be optimized according to its performance and purpose



25

(determination of temperatures and times in each cycle, the number of cycles and composition of reaction mixture). The choice of primers depends on the type of PCR and the type of microorganisms investigated. The sequences of A, T, G, C bases in primers are complementary to the target DNA, and most often, they are selected from known data on the virulent genes of isolates. With PCR target DNA among the selected primers are amplified. The number of amplicons whose size is determined by the distance between oligonucleotide beginners is doubled in each cycle.

Fig. 2: Principle of PCR

PCR amplicons are most easily detected by agarose gel electrophoresis by comparing their size with a molecular marker with known sizes of fragments. The specificity of the multiplication must be controlled by hybridization with an internal DNA probe, by restriction or by the sequencing of the amplitude. The advantages of detecting and identifying microorganisms with PCR versus conventional cultivation methods are better specificity and sensitivity, and above all a

3`

5`

C A T G A A G T

G T A C T T C A

T A T C C A T C

A T A G G T A G

3`

5`

C A T G A A G T

G T A C T T C A

T A T C C A T C

A T A G G T A G

1. Denaturacija DNK, 95o C, 30 s

3`

5`

C A T G A A G T

G T A C T T C A

T A T C C A T C

A T A G G T A G

2. Prileganje, 55 -72o C, 5-60 s

C A T G 5` 3` G T A G 5`

DNK-polimeraza

5` 3`

3` 5`

C A T G A A G T

G T A C T T C A

T A T C C A T C

A T A G G T A G

3. Podaljševanje, 72o C, 60 s

C A T G A A G T 5` A T A G G T A G 5` 3`

3`

3`

Dvoverižna DNK Double stranded DNA

2. Primers annealing, 55 -72 oC, 5-60 s

3. DNA amplification, 72 oC, 60 s

1. DNA denaturation, 95 oC, 30 s

DNA-polymerase



26

shorter time required to obtain a result, as indicated by numerous publications in the literature over the past 25 years. Depending on the type of limitations that occur in the investigation of samples with PCR, in practice, various combinations of classic microbiological methods and PCR are still commonly used in practice. The main limitations in the detection of microorganisms with PCR directly in the sample are:

• Sensitivity of PCR:

For enzyme reaction, theoretically 1 bacterial cell or 1 DNA molecule is sufficient. To the PCR reaction mixture, 1 to 10 µl of prepared DNA is usually added from a sample whose volume (or weight) is in the range of 0.1 ml (g) up to 50 ml (g). For samples, the PCR sensitivity is typically between 103 and 104 cfu/g. Since target microorganisms are usually present at a much lower concentration, it is impossible to directly detect target microorganisms without special preparation of the sample (Fig. 4). Therefore, we use different sample enrichment or different techniques for concentrating the cells from the samples. One of the most promising methods is the immunomagnetic separation/concentration of bacterial cells. • Inhibition of PCR:

Inhibition of the enzyme reaction may result in false negative results. Inadequate amplification conditions (for example, an irregular temperature regime, an inadequate Mg2+ concentration) may result in contamination of the sample (for example: food ingredients or ingredients of the media are inhibited by PCR, DNase and RNase enzymes degrade target DNA and/or primers; disposable gloves or Mg2+ binding substances). Special attention must therefore be paid to contamination with foreign DNA, since it can also lead to false positive results. • The problem of dead and living cells:

In PCR, a specific part of the DNA is amplified regardless of whether the DNA was prepared from live or dead bacterial cells - there is therefore the possibility of false positive results. This can be avoided by enrichment of sample before preparing the DNA, thereby increasing the sensitivity of PCR. Another option is the isolation of mRNA or rRNA, which is technically and practically more demanding procedures.



27

Fig. 3: Possibilities of using PCR for detection of microorganisms in the sample

PCR

SAMPLE

ENRICHMENT

ISOLATION ON SELECTIVE MEDIA

IDENTIFICATION OF BACTERIAL ISOLATE (BIOCHEMICAL AND/OR SEROLOGICAL

TESTS)

ISOLATION OF BACTERIA ON NON-SELECTIVE MEDIA



28

PRACTICAL WORK

Purpose: 1. In a sample of mixed bacterial culture (or from Oxford or Palcam agar) find out if

there are L. monocytogenes by using PCR with specific primers (LM4 and LM5)! Sequences of LM4 and LM5 are chosen from known sequences of hlyA gen (1.6 kb), which code for L. monocytogenes specific listeriolizin, and amplified part of DNA is 429 bp long.

Material and practical work: 1. Preparation of DNA: alkaline lysis of bacterial cells by NaOH in SDS

• Transfer typical colony of Listeria to 100 µl of H2OKEM in 1.5-ml tube, • Centrifuge 5 min at 13000xg, • Remove supernatant • Add 100 µl H2OKEM and mix • Centrifuge 5 min at 13000xg, • Remove supernatant, • Add 100 µl freshly prepared mixture of 0.05 M NaOH and 0.125 % SDS (1 :1) to

pelet and mix, • Heat the mixture for 5 min in water bath at 90 °C, • Store lysate in ice and use 2.5 µl for PCR

2. Preparation of PCR reaction mixture PCR reaction mixture is made in a special room in which we never work with microbial cultures and solutions or suspensions of DNA. Work in the PCR chamber and always wear fresh disposable gloves. All chemicals must be on ice all the time. Depending on the number of samples (N), calculate the volumes of the individual ingredients. N stands for the number of samples for which DNAs were isolated + 1 negative control + additional sample because of pipetting. Then, pipette water into 1.5 ml-tube and then add appropriate PCR 10x buffer and MgCl2, dNTP, primers (LM4, LM5), Tween 20, and finally DNA-polymerase solution. Stir the reaction mixture and distribute it in 0.2 ml-PCR tubes of 47.5 µl. In the second room, add 2.5 µl of prepared DNA to PCR-tubes.



29

Example of calculations for PCR reaction mixture: Component Volume for N samples PCR buffer 10x 5 µl x N MgCl2 (25 mM) 6 µl x N dNTP (2 mM) 5 µl x N LM4 (1 µM) 0.5 µl x N LM5 (1 µM) 0.5 µl x N Tween 20 (0,5 g/ml) 0.2 µl x N DNA-polymerase (5 U/µl) 0.1 µl x N Suspension of DNA 2.5 µl x N Total volume of chemicals and DNA 19.8 µl x N H2OPCR (50 µl x N) – Total volume

3. PCR

Put PCR-tubes in PCR-cycler and run following programme for LM4/LM5: 1 cycel: initial denaturation 95o C 5 min 30 cycles: denaturation 94o C 60s annealing 57o C 60 s amplification 72o C 120 s 1 cikel final amplification 72o C 5 min cooling 4 o C ∞ 4. Agarose electrophoresis Preparation of 1.5 % agarose gel: Weigh 0.96 g of ME agarose, add 60 ml of TEA 0,5x buffer (TEA 10x: 0.4 M Tris, 0.2 M acetic acid, 0.02 M Na2EDTA, pH 8,1), heat agarose in microwave oven until boiling to become clear, then cool to 60 o C. Add the model and pour the gel into electrophoretic model and add electrophoretic comb to the gel. Wait 15 - 30 min, so that the gel polymerise. Preparation of amplicons: Pipette 2 µl of the loading buffer into 1.5 ml-tubes, then add 8 µl of amplicon and mix carefully so that bubbles do not form. Prepare a molecular marker (Invitrogen, 100 bp DNA Ladder) by pipping 2 µl of loading buffer in a 1.5 ml-tube and then add 2 µl of molecular marker (Fig. 5) and mix carefully.



30

Fig. 4: Molecular marker (100bp DNA Ladder ,15628-019 Invitrogen, ZDA)

Electrophoresis: Place the agarose gel in electrophoresis bath with TEA 0.5x buffer, pipette 4 µl of the molecular marker solution into the first place, then pipette 9.5 µl of the prepared amplicons into the next holes. Applying the samples to the gel should be as fast as possible. Electrophoresis takes place at 14 V / cm Staining of agarose gel and documenting: After the electrophoresis is completed, the gel is dyed with ethyl bromide solution. Ethidium bromide is a toxic, mutagenic and teratogenic chemical, therefore dyeing takes place in a special room. The use of protective clothing is mandatory. After 10 minutes of staining, transfer the gel for several minutes to water and then to the transilluminator. Under UV light take a picture of it.

100 bp

200 bp

300 bp 400 bp 500 bp 600 bp



31

Results

1. Describe samples from which you prepared DNA suspensions!

2. Calculate the volumes necessary for PCR mixture preparation!

Calculation of PCR reaction mixture:

N =

Compound Volume for 1 sample (µl) Volume for N samples (µl) Pufer PCR 10x MgCl2 (25 mM) dNTP (2 mM) LM4 (1 µM) LM5 (1 µM) Tween 20 (0.5 g/ml) DNK-polymerase (5U / µl) DNA suspension V total + DNA) H2OPCR

3. Describe preparation of agarose gel, amplicons and conditions of electrophoresis!



32

4. Add figure of agarose gel and describe the results of PCR!



33

5. EXERCISE: REAL-TIME PCR

INTRODUCTION

ethods that based on DNA examination, as for example different performance of PCR, have great meaning in food analytical methods as they are very sensitive and very specific. These two properties allow determination of very small amounts of DNA (or RNA) in the examined sample. Thus, in addition to

pathogenic microbes, we can also determine spoilage microorganisms, genetically modified organisms and allergens in foods, as well as authenticity or food. In recent years, real-time PCR has become one of the methods that can serve different investigations and different purposes and it can also be standardized and automated PRINCIP IN LASTNOSTI PCR V REALNEM ČASU

eal-time PCR is in the improvement of classic PCR. The reaction takes place in a cyclic thermostat, where the temperature is continuously changing in recurrent cycles as in conventional PCR, except that each cycle consists of two phases (instead of tree phases as by classic PCR):

• DNA denaturation at a temperature higher than 90 °C, • primers annealing (and oligonucleotide probe annealing) and amplification of DNA

at temperature of 50-60 °C with DNA-polymerase Amplicons are determined during the enzyme reaction alone, thus saving work and material for determining them by gel electrophoresis. This is also one of the differences between the classic PCR and real-time PCR, as it allows continuous monitoring of amplicons formation. The amount of amplification is measured by fluorogenic labelling of primers, probes or amplicons. An increase in the amount of amplicon is detected as an increase in fluorescence signal resulting from connection between the fluororogenic dye and the amplification or their hybridization. Fluorescence is measured as the reaction progresses, in real time. Due to fluorogen, which are linked to primers, specific probes or amplicons, the specificity of real-time PCR is greater than that of conventional PCR. Real-time PCR results can also be quantified. Real-time PCR is a closed system, and therefore the potential for contamination from environment and cross-contamination of samples is much lower than for conventional PCR.

M

R

5



34

Principle of real-time PCR: The principle of one cycle of real-time PCR is shown in Fig. 5.

1. Annealing of primers and probe to single stranded DNA

2. FRET (Fluorescence Resonance Energy Transfer) - energy transfer between reporter and quencher fluorogen molecules

3. Exonuclease activity of DNA-polymerase

4. Separation or reporter fluorogen and fluorescence emission

5. Disintegration of probe Fig. 5: Principle of real-time PCR with TaqMan-probe (Real-Time PCR Systems, 2004)

3′ 5′

5′ 3′

5′

5′

Q R

5′

3′ 5′

5′ 3′

5′

Q R

5′

3′ 5′

5′ 3′

5′

Q R

5′

3′ 5′

5′ 3′

5′

Q

R

3′ 5′

5′ 3′ 5′

5′

Q R



35

As with conventional PCR, in real-time PCR DNA is doubled in each cycle. With 30 to 40 repeats of cycles, the concentration of amplicons of DNA is exponentially increased by up to 109 copies. Determination of amplicons: By real-time PCR amplicons are detected in real time with fluorescence measurements and basically two methods can be used:

• Non-specific methods: The principle is in binding of fluorogenic dyes, such as SYBRGreen I, to double-stranded DNA and emission of fluorescence at a certain wavelength. The SYBRGreen I bind to every double-stranded DNA and fluoresce only when it is bound to DNA. With amplification there are more amplicons and more SYBRGreen I is bound and the intensity of fluorescence is increased. In these cases, real-time PCR require similar to conventional PCR only two primers and no probe. The disadvantage of non-specific methods is the binding of fluorogens to all amplicons, both to specific and to non-specific amplicons. Results might be inaccurate interpreted or even false positives. Thus melting temperature of amplicons need to be determined.

• Specific methods: Specific methods for determining amplicons are based on the FRET process (fluorescent resonance energy transfer). In these methods, two fluorogenic dyes are bounded to the oligonucleotide probe, primer or amplification molecule: reporter (R) and quencher (Q) fluorogen. The result of amplification of the specific part of DNA and exonuclease activity of DNA-polymerase is in addition to multiplying, also the separation of the reporter and quencher fluorogen, which leads to an increase in the intensity of fluorescence. Depending on the binding of the reporter and the quencher fluorogen, we distinguish several methods and among them is the most well-known method uses double-labelled oligonucleotide probes (TaqMan). The oligonucleotide probe is labelled with a reporter fluorogen at the 5'-end, and the quencher fluorogen is bound at the 3'-end of the probe (Fig. 5). Among the various fluorogenic dyes, 6-carboxy-fluorescein (6-FAM) is most often used as a reporter fluorogen, and 6-carboxy-tetramethyl-rhodamine (TAMRA) as the quencher fluorogen. The quencher fluorogen inhibits the release of the fluorescence of reporter until both dyes are bounded to the probe. In amplification of a particular DNA sequence, the exonuclease activity of the DNA-polymerase degrades the probe, thereby separating reporter and quencher fluorogen, and reporter fluorogen starts to emit fluorescence. By introducing a fluorescently labelled oligonucleotide probe that binds to a portion of the DNA sequence between the two primers, the specificity of real-time PCR is increased, since in addition to specific oligonucleotide primers, a specific probe is also used.



36

The main advantages of real-time PCR:

• amplification and determination of amplicons in the same system • fluorescent dyes and probes allow for continuous monitoring of results of

enzyme reaction • quantification of amplicons is possible • increased specificity due to use of specific probes • range of detection between 10 - 1010 copies • concurrent analysis of a large number of samples • amplification and determination of amplicons are carried out in a fully enclosed

system, and likelihood of contamination of samples and environment is reduced to minimum

Fig 6: Example of real-time PCR results

1: 106 cfu/ml, Ct: 12,8 2: 104 cfu/ml, Ct:16,9 3: 102 cfu/ml: Ct: 21 4: 101 cfu/ml : Ct: 27 5: negative control

No. of cycle

ΔR

n



37

PRACTICAL WORK

Purpose: 1. Determine in different samples if they contain Staphylococcus aureus by using real-time

PCR for gene coding a surface protein of femB. Use primers: • femB-fw 5′-AATTAACGAAATGGGCAGAAACA • femB-rv 5′-TGCGCAACACCCTGAACTT

Specific amplicon is 93 bp.

Material and practical work: 1. Preparation of DNA: Lysis of bacterial cells by heating

• 1 ml of 24-h broth culture transfer (or resuspend 1 colony from agar in 100 µl H2OKEM) in 1.5 ml-micro-tube centrifuge 5 min at 13000 RPM

• Remove supernatant • Add 100 µl H2OCHEM and centrifuge 5 min at 13000 RPM • µl H2OCHEM • Add 100 µl H2OCHEM and heat the suspension 5 min at 94 °C • Store lysate in ice and use 2.5 µl for PCR

2. Preparation of PCR reaction mixture Real-time PCR reaction mixture is prepared in a special room, where microbial cultures and DNA solutions or suspensions are not allowed. Work in PCR-laminar and always wear fresh disposable gloves. All chemicals must be on ice all the time. Depending on the number of samples (N), calculate the volumes of the individual ingredients. N means the number of samples for which DNA is prepared (each sample is worked in two parallels) + 2 negative controls + an additional sample (for pipetting). Then pipette firstly the volume of water into the 1.5 ml sample tube, add appropriate volume of SYBR® Green PCR Master Mix and the solutions of femB-fw and femB-rv primers. Mix the reaction mixture and distribute it into 0.2 ml-PCR tubes (or strips) of 22.5 µl. In the second room, add 2.5 µl of prepared DNA to the PCR tubes.



38

Example of real-time PCR reaction mixture for one sample: Component Volume for 1 sample SYBR® Green PCR Master Mix 12.5 µl femB-fw (10 pmol/µl) 0.75 µl femB-rv (10 pmol/µl) 0.75 µl Suspension of DNA 2.5 µl Vchemicals and DNA 16.5 µl H2OPCR 25 µl – Vchemicals and DNA

3. Real-time PCR PCR-tubes or strips put in real-time PCR apparat and run universal programme as follows: 1 cycle DNA-polymerase activation 95o C 10 min 40 cycles: denaturation 95o C 15s annealing and amplification 57o C 60 s 1 cycle amplicon dissociation 95o C 15 s, 60 oC 1 min, 95 oC 15 s 4. Reading / evaluation of results

Results/amplicon by real-time PCR is a fluorescent signal measured in real-time during enzyme reaction. The fluorescent signal is evaluated after completion of real-time PCR as the number of cycles needed to achieve linear doubling phase. The measured intensity of fluorescence signal is expressed as Ct value (example in Fig. 6). The Ct value is the number of cycles in which intensity of fluorescent signal increases over 10x standard deviation of the base-line fluorescence or background. As more is target DNA, faster the fluorescence intensity will increase, and Ct will be lower. The accuracy of the Ct value depends on the fluorogen and its concentration, the initial amounts of DNA, the sensitivity of the system, and the ability to measure in the system to distinguish between a specific fluorescence signal and a fluorescent background signal



39

Results 1. Describe initial samples from which DNA suspensions were prepared!

2. Prepare reaction mixture for real-time PCR for determination of Staphylococcus aureus (femB surface protein). Calculate volumes of each chemicals for reaction mixture!

Calculaton of reaction mixture volumes for real-time PCR: N =

Reaction mixture: Component Volume for 1 sample Volume for N samples

SYBR® Green PCR Master Mix

femB-fw (10 pmol/µl)

femB-rv (10 pmol/µl)

Suspension of DNA

Vchemicals and DNA

H2OPCR



40

3. Add figure with real-time PCR results and determine Ct ! Evaluate the results.



41

6. EXERCISE: CONTAMINANTS: DETECTION OF ANTIBIOTICS

RESIDUES IN FOOD

INTRODUCTION

n general the risks and dangers which, depending on the raw materials, technological processes and type of foods and storage conditions, should be controlled and measured, are following:

• Microbiological risks: this group includes mainly pathogenic bacteria (for example, Clostridium botulinum, Salmonella, Listeria monocytogenes) and specific types of microorganisms that cause spoilage of foods (for example, Pseudomonas spp., Xanthomonas, molds);

• Chemical risks: toxic substances are naturally present in foodstuffs (for example: mycotoxins, phytotoxins) and artificial contaminants (for example, polychlorinated biphenyls, pesticides, insecticides, fungicides, metals and non-metals, veterinary medicinal preparations);

• Physical risks: this group includes mechanical contamination in foods, for example particles of glass, lash, plastic or metal, sand, insects, hair, bristles, bone fragments, and others.

According to the Law on the health suitability of foodstuffs and products and food contact materials (ZZUIZS) (Uradni list RS 52/2000; 42/2002, 47/2004) a contaminant is any biological, chemical, physical or radiological agent that is unintentionally present in food as a result of the agricultural production methods and raw materials of animal origin, or production and trade of foods, or as a result of environmental pollution. The contaminant in a food can therefore be any substance that has not been intentionally added to food and is the result of food contamination in the process of production, processing, packaging, transport or storage or the consequence of environmental contaminants. Residue of a particular contaminant such as a pesticide is one or more substances present in or on foods resulting from the use of the pesticide, including its metabolites and products resulting from its degradation or reaction, as well as impurities. The primary cause of most food-borne diseases is of microbiological food contamination. Serious food poisoning caused by chemical contaminants is not common, and most often

I

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these are individual cases limited to a particular area. The potentially great danger to man is made up of chemical pollutants because they are taken in small quantities practically all their lives. Examples of chemical contaminants are shown in Table 3. Consequences occur gradually or only after a long time, or maybe with insignificant signs.

Table 3: Chemical contaminants in foods (O`Keeffe, 2000)

Category Examples

a) Natural contaminants

Usual food ingredients phytoestrogens, glycoalkaloids

Natural food contaminants mycotoxins, phytotoxins, poisoning with mushrooms

b) Environmental contaminants

Agricultural chemicals pesticides, fertilizers Veterinary medicines antibiotics, biostimulators Food additives preservatives, antioxidants Chemicals from packaging vinyl monomers, oligomers Chemicals from the technological process

nitrosamines, polycyclic aromatic hydrocarbons (PAHs)

Environmental pollutants dioxins, polychlorinated biphenyls (PCBs), metals and non-metals

Given the problem of contaminants in foods, both international organizations (FAO, WHO) and individual countries have established legal regulations to protect consumer health. For different type of food, maximum quantities are prescribed - the limit values for individual residues of contaminants in individual foods. The maximum residue level (MRL) is the maximum residue level specified in mg/kg of the food with the regulations. Foods that exceed these limit values are assessed as inadequate. Within the framework of systematic monitoring, checking is carried out by the health and veterinary inspection control of residues in foods of plant origin and animal origin

In controlling the content of residues in foods, different techniques and methods are used, which are generally divided into:

• Qualitative or semi-quantitative "screening" methods that are relatively fast, simple and allow for the examination of a large number of samples (for example microbiological testing, enzyme-immune tests); they identify samples that do not comply with existing regulations and need to be further analyzed by quantitative and specific methods

• Different chromatographic and spectroscopic methods for determining a definite residue, qualitative and quantitative (for example, gas chromatography (GC), mass spectroscopy (MS), high pressure gas chromatography (HPLC), thin-layer chromatography (TLC)).



43

Pesticides are plant protection products that are used: • for the control of pathogens of plant diseases, weeds (herbicides), fungi

(fungicides) of harmful insects (insecticides), rodents (rodenticides), snails (limacids) and regulate the growth of plants,

• for the control of pests and fungi that attack the stored crops, • to control insects and other organisms that transmit infectious diseases to

humans and animals.

Major groups of pesticides: • Chlorinated hydrocarbons (DDT, lindane, alpha HCN), • Organophosphates, • Carbamates • Inorganic pesticides based on Cu, Pb, As, Hg, • Plant pesticides (pyrethrin, nicotine).

Veterinary medicines: Veterinary medicinal products are substances or combinations of substances that are prepared and intended for treatment or prevention of diseases in animals; as a medicinal product, any substance that is prepared in order to diagnose or restore, improve or modify physiological functions of animals is considered. Monitoring and control of use of veterinary medicinal products and monitoring of residues in foods is performed by UVHVVR. Major groups of veterinary medicines:

• Antibiotics: (aminoglycosides (streptomycin, neomycin), penicillins, tetracyclines, polyenes (nystantin), polypeptides (bacitracin), macrolides (erythromycin), novobiocin, chloramphenicol,

• Chemotherapeutics: sulfonamides, cabadoxes, nitrofurans, dimethridazole, Antibiotics and chemotherapeutics in foods of animal origin must not be present (meat and meat products may be marketed if they do not contain antibiotics in quantities which can be demonstrated by the prescribed methods), chloramphenicol and dimethridazole are prohibited; for sulphonamides the limit value is 0.1 mg/kg.

• Hormones, thyrostatics, tranquilizers, beta-blockers and beta-antagonists - in foods of animal origin must not be present

DISC DIFFUSION METHOD FOR DETERMINATION OF

ANTIBIOTICS IN FOODS When we suspect that in food (for example, fresh meat, milk) are antibiotic residues, an disc diffusion method may be used as a relatively quick and simple screening method.



44

The method is more commonly known as the method for the determination of antibiotic activity - antibiogram. The principle of disc diffusion method (Four-plate agar diffusion test) is determination of growth inhibition of tested bacterial strains in the presence of tested food sample. As bacterial strains Bacillus subtilis and Micrococcus luteus should be used. In sterile and cooled agar media (48-50o C) that were prepared with different pH (6.0; 7.2; 8.0), pure bacterial cultures are added at concentration (in media) of 104 cfu/ml. Bacillus subtilis is added to media with pH 6.0; 7.2; 8.0 and Micrococcus luteus in medium with pH 8. If we are examined a meat sample, a cylinder with a diameter of 8 mm is aseptically cut from the centre of the sample. Then, we cut 8 discs with thickness of 2 mm and transfer them (two) to four already prepared media (three media with three different pH values and Bacillus subtilis and one with Micrococcus luteus). If we are examined liquid sample (milk, serum), we use sterile filter papers/discs with diameter of 6 mm and impregnate them with a test sample. Then two discs are aseptically with tweezers transferred to the four already prepared media. Agar media with samples and Bacillus subtilis are incubated for 18-24 h at 30o C, agar media with samples and Micrococcus luteus for 18-24 h at 37o C.

By each food analysis with agar diffusion method we must make controls. Controls are filter papers/discs to which standard antibiotic solutions (10 µl of antibiotic solution with a specified concentration, Table 4) are added. The agar plates with control discs are incubated under the same conditions as the plates with the examined samples. After incubation, the size of circular inhibition zone is measured around food samples or around disks (Figure 7). A positive result means that bacterial culture does not grow around two filter papers in one or more medium and the inhibition zone is 2 mm or greater.

Fig. 7: Circular inhibition zone at disc diffusion method

Testni disk / košček mesa

Krožna inhibicijska cona Circular inhibition zone

Food sample or disc



45

The correctness of usage the disc diffusion method is confirmed by the results of control samples - filter papers or discs with standard antibiotic solutions. The results are shown in Table 4.

Table 4: Results of control samples at disc diffusion method for determination of antibiotics in foods

Agar medium with bacterial culture / pH

Antibiotic concentration on disc (µg/ml)

Minimal circular inhibition zone (mm)

Bacillus subtilis / pH 6 penicillin 0.625 µg / ml 6 mm Bacillus subtilis / pH 7.2 sulphamydin 0.5 µg / ml 6 mm Bacillus subtilis / pH 8 streptomycin 0.5 µg / ml 6 mm Micrococcus luteus / pH 8 streptomycin 0.5 µg / ml 6 mm

If antibiotic(s) is(are) in food, it will diffuse from the disc to the solid medium during incubation and create a concentration gradient. Bactericidal effect on bacteria can be seen as an inhibitory zone around the disk, where there is no bacterial growth. As by disc diffusion method the degree of antibiotic diffusion into a bacterial culture medium is very important and the composition itself (for example, volatile components), in the case of positive results, it is essential to carry out additional chemical analyzes to confirm the presence of the antibiotic(s) in the sample.

Food additives: Food additive is any substance that is normally not used or consumed as a food and does not constitute an ordinary, typical food ingredient, whether it has a nutritional value or not, but it is intended to be added to food for technological or sensory reasons in the manufacture, packaging, transport, storage and has direct or indirect effects on the food and becomes a constituent of the food. Only food additives that are legally permitted can be used in food production. Additives are added to foods in the smallest possible amount, which still achieves the desired technological effect. Additives must have adequate purity, as they are expected to be consumed permanently. The products of their degradation in the organism must also be known. For individual additives and foods, the maximum permitted quantities are prescribed. Examples of the main categories of additives are sweeteners, colorants, preservatives, antioxidants, emulsifiers, stabilizers, and packaging gases. Chemicals fro packaging materials: vinyl monomers, oligomers Chemicals from technological processes: nitrosamines, polycyclic aromatic hydrocarbons (PAH) Environmental contaminants: dioxins, polychlorinated biphenyls (PCB), metals and non-metals (lead, cadmium, mercury, arsenic, antimony, zinc, copper)



46

PRACTICAL WORK

Purpose:

1. By using disc diffusion method determine if food samples contain residua of

antibiotics! Materials and practical work: 1. Describe food samples, list materials and describe practical work! Food samples:

Control samples:

Materials and practical work:

Draw a plan of the experimental work!



47

Results 1. Measure circular diameter zones for control samples and food samples and

asses presence of antibiotic(s) in food samples!

Control sample Bacterial culture, pH Circular diameter zone (mm)

Bacillus subtilis, pH 6.0

Food sample Circular diameter zone (mm) Estimation





Micrococcus luteus, pH 8.0




48

7. EXERCISE: CLEANING: DETERMINATION OF WORKING

SURFACE CLEANILINESS

INTRODUCTION

anitation in food industry is a procedures for achieving that surfaces, equipment and places are in such hygienic conditions to prevent contamination that could lead to hygienically unsuitable foods. The main purpose of sanitation is to prevent contamination of foods with pathogenic microorganisms and to reduce the

potential for multiplication of spoilage microorganisms. Sanitation includes various methods with which we perform cleaning, disinfection, destroying certain insects (disinfection), rodent destruction, destroying poisons (detoxification) and other methods.

CLEANING Cleaning, in the strict sense of the word, means the removal of impurities from certain surfaces and places. In addition to constant regular cleaning and disinfection during work or final cleaning after the work has finished, general cleaning and disinfection (daily, weekly, three months, annually) must be organized. Cleaning and disinfection is difficult to separate, because the effectiveness of disinfection is highly questionable without pre-cleaning. The cleaning itself includes the following phases:

• mechanical removal of surface contamination (scrubbing, scraping, water jet with high pressure),

• dispersion of impurities in the cleaning agent, • preventing the re-settling of dispersed impurities in a cleaning agent on already

cleaned surfaces - washing, • drying.

Cleaning products are used for cleaning or combined cleaning and disinfection agents. When choosing the means and method of cleaning, it is important to take into account the following parameters: the nature and properties of the impurities, type of material, method of cleaning, properties of the cleaning agent, composition of the water and the degradability of the cleaning agent.

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Nature of impurities Impurities in food industry are of organic and inorganic origin and, by chemical composition, are very heterogeneous. For inorganic impurities, low pH cleaning agents are more suitable, and for organic with alkaline pH. Impurities are very complex in nature and often organic impurities are "protected" by inorganic deposits and vice versa (for example, milk film). Therefore, it is very important to correctly determine the type of impurities and the correct choice of a cleaning agent or a combination of various cleaning agents (two-stage cleaning) in order to remove the impurities effectively. The main cleaning agents used in the food industry can be divided into three main groups: basic cleaning agents (strong bases, weak bases), acidic cleaning agents and surfactants. Nature of material Various types of materials are used in food industry: stainless steel sheet, galvanized sheet, glass, plastic materials, ceramics, concrete, rubber, wood. Depending on the structure and roughness of the surface, the corrosion resistance of the material and the mechanical and thermal resistance of the material, appropriate cleaning technology and suitable cleaning agent are selected. For example, stainless steel is the most corrosion-resistant material with a smooth surface, resistant to high temperatures. On the other hand, cleaning of wood is a very problematic because it is permeable to moisture, grease and oil, it is not suitable for high pressure cleaning, it is not resistant to alkaline cleaning agents Type and method of cleaning The method of cleaning depends on the surfaces, equipment, the rooms being cleaned, and the available cleaning agents and equipment. The basic methods of cleaning are manual and machine cleaning. Manual cleaning covers several stages: rough cleaning, main cleaning with detergent and flushing with water. But more and more, machine cleaning is underway with high pressure water cleaning machines, pressurizers for foamy application of cleaning solutions, CIP - cleaning in place and other devices. All cleansing and disinfection equipment should be regularly cleaned and disinfected. Properties of cleaning agent The ideal cleaning agent should have a good water softening ability and emulsification of fats and oils, good washing and dissolving ability, preventing the deposition of already dispersed particles, should be well washed from surfaces, having good solubility, not to be corrosive, easy to use and ecologically suitable at an affordable price.

The efficiency of cleaning depends on many factors, most important of which are: operating time, temperature, mechanical effect, and the type and concentration of the cleaning agent.



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CONTROL OF CLEANING EFFICIENCY

Control of cleaning can be visual, but also need to periodically carry out laboratory control of microbiological examination of swabs of surface or washes. In addition to the conventional microbiological methods, the so-called "fast tests" are increasingly used, which, with rapid results, contribute to effective action (for example: measurements of ATP-bioluminescence, measurements of NAD and NADP).

S A M P L I N G W I T H S W A B A N D M I C R O B I O L O G I C A L I N V E S T I G A T I O N

Sampling with swabs is used to determine the hygienic condition in production facilities, catering establishments, to determine effectiveness of cleaning and disinfection. We sample the working surfaces, equipment, cooking utensils and kitchen utensils, containers, hands, inlets, pipes, etc. For sampling with swabs, sterile tubes containing a certain volume of sterile saline (5 ml or 10 ml) are used. On the inside of the cap of tube is a stick which is wrapped with wadding. When sampling surfaces with a brush, the surface of a certain size (20 cm2)is wiped in such a way that the swab is rotated during sampling and that the direction of surface is changed at least twice. Then swab is added to the test tube with a saline solution and mixed well. In saline solution from swab different microbiological criteria are established, for example:

• total number of bacteria • Escherichia coli, coagulase-positive staphylococci, streptococci, Proteus, Pseudomonas

aeruginosa The number of microorganisms in swab on the surface is calculated as:

VRofcolonynoN ××= . (cfu/20 cm2) R sample dilution V volume of physiological saline in swab Example of evaluation: surface is clean when

• Total number of bacteria is up to 100 cfu/20 cm2 (cutlery, glasses, cups, plates) or up to 200 cfu/20 cm2 (working surface)

• Swab does not contain Escherichia coli, coagulase-positive staphylococci, streptococci, Proteus, Pseudomonas aeruginosa.



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S A M P L I N G W I T H W A S H E S A N D M I C R O B I O L O G I C A L I N V E S T I G A T I O N

Sampling with washes is used for determination of hygienic conditions of difficult to reach places, for example Vzorčenje z izpirki uporabljamo za ugotavljanje higienskega stanja težko dostopnih mest, for example packaging. Washes of bottle: place 10 ml of sterile saline in bottle, cover the bottle with original cap and shake it so that the physiological solution spills over the entire inner surface of the bottle and the inside of the cap. The microbiological examination is carried out in the same way as in the case of swabs.

M E A S U R E M E N T O F A T P - B I O L U M I N I S C E N C E

Adenosine triphosphate (ATP) is a universal energy carrier in metabolic reactions in all living cells. Determination of cleaning with ATP-bioluminescence measurements is based on measurement of light, which is released as a by-product during the enzyme conversion of luciferin to oxyluciferin (Fig. 8).

Fig. 8: Principle of ATP-bio-luminescence measurement

Light emission shall be measured with a luminometer at 562 nm in relative light units RLU (Relative Light Unit). The measured light is proportional to amount of ATP in sample under standardized reaction conditions. Since the amount of ATP in the metabolic active microbial and other cells is relatively constant (in dead cells ATP is rapidly decomposed), the proportion of the measured light can be proportional to the number of living cells in the sample.

Relation between the amount of ATP and the number of microorganisms determined on agar plates is good in cases of laboratory samples of cultures. In practice, the amount of ATP in samples is in correlation with all living cells (microbial, plant, animal), and not only with ATP of microbial origin. It is generally believed that there are a large number of microorganisms on the dirty surface and a large amount of ATP, while there are few microorganisms on the clean surface and a small amount of ATP. ATP measurements

ATP

O2

Luciferin

Luciferase

Mg2+ AMP

CO2 + PPi

Oxyluciferin

Bio-luminescence



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also detect residues of organic origin (residues of foods on working surfaces), which may be the ideal medium for microorganisms, and therefore the decision on the cleanliness or dirtiness of the surface is based on the actual presence of residual food and microorganisms. The method of measuring ATP-bioluminescence is very fast (a few minutes) and allows appropriate rapid action. As a limitation, the following parameters are often mentioned in the literature: relatively low sensitivity (104 to 106 cfu/ml), microbial cells in the stationary growth phase and under stress have smaller amounts of ATP, the measurement is dependent on the enzyme reaction and this from the environmental situation (for example a disinfectant means decrease the activity of luciferase).

M E A S U R M E N T O F N A D A N D N A D P

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are coenzymes that participate in cell metabolism. NAD is primarily involved in catabolic reactions, and NADP in anabolic reactions. Biological oxidation - dehydrogenation means that organic molecule is oxidized, loses two hydrogen atoms, while the NAD molecule is reduced. Coenzyme NAD receives two electrons and one proton, and NADH is formed, another proton goes into the environment (Fig. 9). Reduced NADH coenzyme has more energy than NAD and energy is used in subsequent reactions of ATP formation. Determination of cleanliness by measurements of NAD and NADP is based on the oxidation-reduction enzyme reaction between organic molecules (residues of foods and/or microorganisms) on investigated surface, added substrate and enzyme. If there are residues of foods and/or microorganisms on the surface, NAD is reduced and the result is a change in colour. He method is fast (5 min) and easy to perform. No special apparatus is required for measurements. HY-RiSETM (Merck) includes:

• sterile tape for sampling of surface, • a bottle with a surface wetting solution (A), • a bottle with substrate solution (B), • a bottle with enzyme solution (C).

Before sampling, apply a drop of solution A to the tape (if we sample wet or damp surfaces, this level is not necessary). Smooth surfaces are sampled by slowly pulling the strip over the surface (3 cm x 10 cm), the rough surfaces are sampled with a 10-inch surface impression. Then add 1 drop of solution B and 1 drop of solution C. Then, put the tape back into the overcoat so that the tape is in the dark. After 4-5 minutes, depending on the colour reaction, read the result. Yellow colour means pure, pink, red, and blue purple colour means a dirty surface.



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Fig. 9: Principle of NAD+ measurement

The sensitivity of the HY-RiSE method is in the range of pmol of NAD. The determination of cleanliness by NAD measurements is equivalent to the measurement of ATP-bioluminescence. The method is better than visual control, since at the same time it determines residues of foods and microorganisms on the investigated surface. It is intended for determining cleanliness of various surfaces, hands and liquids in small and medium-sized food establishments, canteens, restaurants, shops, butchers, bakeries, hotels, hospitals. Most often is used for sampling surfaces containing food residues, for example, work desks, cutting, cutting boards, equipment, interior of refrigerators, microwaves, various containers, kitchen utensils, hooks, handles, drains, scales, storage shelves, conveyor belts). The method is rapid and has very good sensitivity. Therefore, it is suitable for inclusion in the HACCP system, with its application we improve the general hygiene level and identify the dangerous areas with food and microbial residues.

Coenzyme NAD+ (carrier of electrons)

NADH + H+ (reduced carrier of electrons

Organic molecule with 2 atoms of H

z

Oxidated organic molecule

oxdation

reduction



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PRACTICAL WORK

Purpose: 1. Determine cleanliness of selected surface! Use a classic microbiological surface swab,

and the method of measuring NAD and the method of measuring ATP! 2. Clean the surface! 3. Determine the effectiveness of the cleaning by (i) classic microbiological examination

of the surface swab and by (ii) alternative methods for controlling cleanliness! Materials and practical work: 1. Describe selected surface and practical work!

2. Visually determine cleanliness of selected surface!



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Results: 1. Indicate the results of classic microbiological swab tests and the results obtained by

alternative methods for controlling purity cleanness!

Selected surface

Results of classic microbiological swab tests

Results of NAD measurements

Results of ATP measurements

N

(cfu/swab)before cleaning

N (cfu/

swab) after

cleaning

Before cleaning

After cleaning

Before cleaning

After cleaning

2. Comment results!



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8. EXERCISE: DISINFECTION

INTRODUCTION

isinfection in the wider sense of the word, means destruction of microorganisms that may have a negative effect on foods. Disinfection is a complex area of the food industry, where it is important to ensure minimal risk of contamination with microorganisms mainly in processing,

manufacturing, storage and transport of food.

PROCEDURES FOR DISINFECTION 1. Physical procedures Different procedures for disinfection are of great importance, both in limiting the transmission of pathogenic microorganisms as well as in the restriction of the transmission of widespread microorganisms (ubiquitous microorganisms). Because of its simplicity, efficiency and few side effects, physical disinfection methods are commonly used. In addition to mechanical cleaning and high temperatures, the most common procedures are ventilation, filtration, and various types of radiation and low temperatures. 2. Chemicals used for disinection Chemicals that inhibit the growth of microorganisms are called bacteriostatic agents (inhibiting bacterial growth) or fungistatic agent (inhibiting fungal growth), and chemical agents that destroy microorganisms are called bactericides or fungicides. Disinfectants have different mechanisms of action on microbial cells: they destroy outer membrane of bacterial cell wall, destroy bacterial cell wall, destroy cytoplasmic membrane, destroy energy metabolism of the microbes by acting on ATP, destroy cytoplasm or core of the cell, or destroy structure of the bacterial endospores. Factors that affect the effectiveness of disinfectants:

• Kind and number of microorganisms, and their sensitivity to disinfectants • Disinfectant concentration • Time of disinfectant action

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• Temperature of disinfectant • Type of surface material • Cleanness of surface.

Properties of the ideal disinfectant:

• It should be active against broad spectrum of micro-organisms • It should be effective at a lower concentration • It should operate as rapidly as possible in wider pH range and independent of the

hardness of the water • It should also operate in the presence of organic residues, cleaning agents • It should not be toxic, non-corrosive • It should have an unlimited expiration date and is cheap • It should be easy to use • It should not have an unpleasant smell and should not change the smell and taste

of foods • It should be rapidly degradable and environmentally friendly

Since there is no ideal detergent and ideal disinfectant, all parameters must be taken into account when selecting medium or combination that has the least negative properties.

DILUTION-NEUTRALIZATION METHOD By dilution-neutralization method (SIST EN 1040, 2001) the effect of different concentrations of disinfectant in different contact times on the investigated bacterial culture is determined. According to SIST EN 1040 (2001) standard disinfectant has bactericidal activity when the number of tested bacteria (Pseudomonas aeruginosa, Staphylococcus aureus) is decreased in defined contact time at least for 105 cfu/ml. The method is standardised and as investigated bacteria are prescribed Pseudomonas aeruginosa and Staphylococcus aureus. A simplified method comprises of following steps: Preparation of disinfectant Prepare three different concentrations of disinfectant; and two concentrations should be within the range specified by the manufacturer. For the solution use freshly distilled and sterilized water (not deionized) and sterile glassware. The disinfectant solution is poured (9 ml) into sterile tubes. Preparation of bacterial cultures, their inoculation and determination of survival bacteria



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Determine the number of bacteria in overnight cultures of P. aeruginosa and S. aureus by colony count method. In tubes with disinfectant add 1 ml of overnight bacterial culture (18-h culture with approximately 108 cfu/ml), mix, and measure contact time (i.e. 1 min, 5 min, 15 min, 30 min, 45 min, 60 min). After a certain contact time, the content of tube is mixed and 1 ml of the suspension is transferred into 9 ml of neutralizer. Tubes with disinfectant and culture suspensions are further incubated at 20 ° C and sampling is repeated at each contact time. In each sample (suspension of disinfectant and neutralizer) determine the number of surviving bacteria with colony count method (homogenization and transfer 2 x 1 ml suspension in sterile empty Petri dish). Add in Petri dishes then solid medium that is previously dissolved and cooled to 45o C (Triptic Soy Agar with yeast extract) and gently mix the suspension in medium. After 24-h incubation count the colonies and calculate number of bacteria (N (cfu/ml)). Bactericidal activity Bactericidal activity of disinfectant is determined as the concentration of disinfectant, which in a certain contact time, reduce the number of test bacteria for at least 105 cfu/ml.



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PRACTICAL WORK

Purpose: 1. By dilution-neutralization method determine whether the examined solution which is

based on quaternary ammonium compounds has the effect of disinfectant on tested bacteria of Staphylococcus aureus and Pseudomonas aeruginosa!

Materials and practical work: 1. Commercial data about disinfectant: Name: Neutral disinfectant based on quaternary ammonium compounds. Application: Disinfectant with active cationic agents suitable for disinfection by

spraying, pouring and immersion after thorough pre-cleaning. Appearance: Colourless clear liquid. Smell: An almost odourless. Density: 1g cm-3 (1 kg = 1 l) pH (1 %): Neutral. Use: It is used from 1 to 3% solution, contact time of 15 min. Aggressiveness: At the prescribed concentration, disinfectant will not damage

normal materials. Special notes: Surfaces that come into contact with food, after using disinfectant

thoroughly rinse with plenty of water. Safety precautions: Irritant. Irritating to eyes and skin. After contact with skin, wash

immediately with plenty of water. If swallowed, seek medical advice immediately and show the container and label of disinfectant.

Packaging: Plastic bottles, 20 kg. Expiration date: 18 months 2. In table below write data! Prepared concentrations of disinfectant

Bacterial culture

Contact times

Neutraliser

Solid medium

Incubation conditions



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3. Draw a plan of the experimental work!



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Results: 1. The number of bacteria in time 0 Bacteria: N (cfu/ml)

2. Write the number of bacteria (N, cfu/ml) in disinfectant at different contact times

and different concentrations of disinfectant!

Contact time (min)

The number of survival S. aureus (N (cfu/ml)) at different concentrations of disinfectant (%)

Contact time (min)

The number of P. aeruginosa (N (cfu/ml)) at different concentrations of disinfectant (%)

3. Comment the results!



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9. EXERCISE: ANTIMICROBIAL ACTIVITY OF DISINFECTANT

INTRODUCTION

n practice, the effect of disinfectants is lower than in ideal conditions. The effect of the disinfectant is influenced by the purity of the surfaces, presence of dirt and organic residues, time and operating temperature, and the nature and concentration of microorganisms. Of course, the concentration of the disinfectant is also

influenced by the manner of preparation, packaging, use and storage. For disinfectants in use, we must carry out control of the mode of use and periodic verification of contamination with microorganisms, in particular less efficient or to determine their effectiveness.

DETERMINATION OF DISINFECTANT EFFECTIVENESS

n order to determine the effectiveness of disinfectant, we use microorganisms according to individual pathogens or determine the effect of disinfectant on ubiquitous microbes (commonly spread microorganisms), coliform bacteria and staphylococci. The effect of disinfectant is determined by surviving microorganisms

per unit area. Some definitions of a good disinfectant are: For a good disinfectant there should be less than 100 microorganisms on disinfected surface of 100 cm2 (Pintarič, Dobeic, 2001); good disinfectant destroys from 7.5 x 107 to 1.25 x 108 Escherichia coli and Staphylococcus aureus after 30 s at 20 o C (Marriott, 1995). According to the SIST standard EN 1040 (2001), the disinfectant has bactericidal activity when it decreases the number of test bacteria (Pseudomonas aeruginosa, Staphylococcus aureus) at a specific contact time for at least 105 cfu/ml.

Determination of the effectiveness of disinfectant is a very large topic and includes many methods that can be divided into diffusion (agar diffusion method) and dilution method (broth dilution method, agar dilution method and broth micro-dilution method). Based on obtained results, the minimum inhibitory concentrations (MIC) and microbicidal concentrations (MBC) are determined. In some cases, direct surface sweep testing and further microbiological or chemical testing can be used to determine the effectiveness of the disinfectant

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A G A R D I F U S S I O N M E T H O D

The effect of the disinfectant can be easily determined by the agar diffusion method so that different concentrations of disinfectant are applied to the sterile disks placed on the medium with the investigated microbial culture. After incubation, the inhibition zones are measured. A detailed description of the procedure is described in the 6th exercise. B R O T H D I L U T I O N M E T H O D

By using broth dilution method, we determine how different concentrations of disinfectant work on the investigated microbial culture. Pre-prepared disinfection solutions with different concentrations and investigated 24-hour microbial culture are added in a series of tubes with a suitable liquid medium (Mueller Hinton broth, Triptic Soy broth, Brain Heart Infusion broth). We need to include in the experiment a control sample that is prepared in the same way as other samples, except that instead of the disinfectant, the same volume of sterile distilled water is added. All the suspensions are then incubated at a predetermined temperature. After incubation, the number of surviving micro-organisms is determined in each tube. The broth dilution method may also be performed in microtiter plates where the principle of the preparation is the same, except that the volume of the test suspension is smaller. The difference is also in the evaluation of the results, since after incubation, the optical density or change in the colour of the suspension is determined. If a change in the colour of the suspension is determined, a chromogenic agent should be included in the preparation of the test suspension (for example TTC - 2,3,5-triphenyl tetrazolium chloride). The actively growing bacteria reduce colourless TTC, resulting in precipitation of formazane, which is visually seen as a red colour. The most common measure of the effectiveness of the disinfectant is the determination of minimal inhibitory concentration (MIC) and microbicidal concentration (MBC). The lowest concentration of the disinfectant that inhibits growth of culture is called the minimal inhibitory concentration (MIC), and the lowest concentration of disinfectant in which the microorganisms do not survive is called the microbicidal concentration (MBC). The definitions of the MIC are somewhat different between the authors and, therefore, the results of various surveys are sometimes difficult to compare with each other. Some examples: MIC is the smallest concentration of a substance that completely prevents the reproduction of the selected microorganism by at least 48 hours (Cannilac and Mourey, 2001). MIC is the smallest concentration of a substance that prevents the visible growth of the selected micro-organism (Delaquis et al., 2002; Hammer et al., 1999).



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MIC is the smallest concentration of a substance that causes more than 90% reduction of viable selected microorganisms (Cosentino et al., 1999). Similarly, the MBC is the concentration of the selected substance that kills 99.9% or more of the microorganism (Cosentino et al., 1999), or MBC is the concentration of a substance in which there is no growth of microorganisms, and they do not grow after inoculating them into the fresh medium. D I R E C T T E S T I N G W I T H S U R F A C E S W A B

The effect of disinfectant can be determined by the number of surviving microorganisms on a certain surface after a certain disinfection time. A description of the sampling procedure with the swab and further swab analysis are described in the 7th exercise. In similar ways, the antimicrobial effects of preservatives and natural antimicrobial substances can also be determined.



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PRACTICAL WORK

Purpose: 1. Determine MIC for disinfectant (Chlorhexidine diacetate) with broth micro-dilution

method for Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Materials and practical work: Commercial data about disinfectant Name: Chlorhexidine diacetate Molecular formula: C22H30Cl2N10 . 2CH3COOH . H2O Usage: Disinfectant with active cation agents from bi-guanide group Solubility: Water-soluble; 15 mg/ml Preparation of bacteria, medium and disinfectant working suspensions

• From the overnight bacterial cultures (24 hours, 37 °C) of Staphylococcus aureus and Escherichia coli bacterial inocula are prepared so that 0.15 ml of the overnight culture is transferred into 10 ml of TSB. The concentration of bacterial cells in the medium should be from 106 to 107 cfu/ml. The exact number of bacteria is determined by colony count method on TSA.

• Basic solution of disinfectant is prepared by weighting or dissolving it in 1 ml of water, for example 15 mg of antimicrobial agent in 2 ml-tube add 1 ml water.

• Working solution of disinfectant is prepared by diluting basic solution with liquid medium (TSB), for example 250 µl of basic solution add 750 µl TSB

Microdilution method

• For each experiment first prepare a plan of microdilution method. With plan follow concentrations of antimicrobial substances in samples and in particular in control samples. Example is on Fig. 10.

• Aseptically take the following steps: 1. In the first row, in wells in which bacteria will be tested (A1, A2, B4, B5), add 100 µl of working solution antimicrobial substance. 2. In all other rows (form B to H) in columns 1, 2, 4 in 5 add 50 µl of TSB. 3. 2-times dilution of antimicrobial substance is carried out in each column so that from the first well (A1) 50 µl of working solution of antimicrobial substance is transferred to the second well (B1) and gently mix the contents (with tip). Then from the second well (B1) take 50 µl of suspension and transfer it to the third well (C1) and gently mix the contents. Dilute antimicrobial substance on the same way to the last well (H1) and from this well, after mixing 50 µl of the suspension is discarded. The same process is repeated in column 2, then the columns 4 and 5.



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4. In column 7 in the first well (A7) add 100 µl of working suspension of ethanol (it is prepared by mixing 250 µl of ethanol and 750 µl TSB medium). Repeat the same in column 8 (A8). 5. In all other rows (B-H) in columns 7 and 8 add 50 µl of TSB medium. 6. 2-times dilution of working solution of ethanol is carried out so that from the first well (A7) 50 µl of working solution of ethanol is transferred in the second well (B7) and gently mix the contents (with tip). Then from the second well (B7) take 50 µl of suspension and transfer it to the third well (C7) and gently mix the contents. Dilute working solution of ethanol on the same way to the last well H7 and from this well, after mixing 50 µl of the suspension is discarded. The same process is repeated in column 8. 7. In column 10 in all wells (A-H) add 50 µl TSB medium. 8. In columns 1, 2 and 7 in all wells add 50 µl of bacterial culture 1, in column 10 add bacterial culture 1 ONLY in the first four wells (A10, B10, C10, D10). 9. In columns 4, 5 in 8 in all wells add 50 µl of bacterial culture 2, in column 10 add bacterial culture 2 ONLY in the last four wells (E10, F10, G10, H10). 10. In column 12 in wells A12, B12, C12, D12 add 50 µl TSB and add 50 µl of working solution of ethanol, in wells E12, F12, G12 and H12 add 100 µl of TSB. 11. Microtitre plate mix on microtitre plate-mixer, incubate microtitre plate at 37 oC for 24h.

Fig. 10: Scheme of microtitre plate for microdilution method; (A) designations; (B) disinfectant concentrations

(A) DESIGNATIONS: B1: bacterial culture 1 in TSB medium with different disinfectant concentrations B2: bacterial culture 2 in TSB medium with different disinfectant concentrations PK1: positive control for bacterial culture 1 in TSB medium (only when ethanol is used instead of water for antimicrobial agent solution) PK2: positive control for bacterial culture 2 in TSB medium (only when ethanol is used instead of water for antimicrobial agent solution) PK3: positive control for bacterial culture 1 in TSB medium PK4: positive control for bacterial culture 2 in TSB medium NK1: negative control of TSB medium (and ethanol) NK2: negative control of TSB medium



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(B) CONCENTRATIONS Columns 1, 2, 4 and 5: concentration of antimicrobial agent (mg/ml) in TSB medium Columns 7 and 8: concentration of ethanol (%) in TSB medium Evaluation of MIC After incubation add aseptically in all wells 10 µl of INT (p-iodo-nitro-tetrazolium violet, 2 mg/ml) reagent. INT reagent is indicator of metabolic activity, which can accept electrons from dehydrogenase and is reduced in red coloured formazan. After addition of INT microtitre plate is incubated (0.5 to 2 h at 37 °C). The results are read visually according to the colour change. The first uncoloured well (Fig. 11) in a column means MIC for a particular combination of bacteria and antimicrobial substance. In parallel to check positive and negative control samples.

Fig. 11: Example of reading MIC

Determination of MBC After the incubation of microtitre plate MBC is determined so that in wells with no visible colour change survival bacteria are evaluated with colony count method on agar plates. For example for bacterial culture 1 (Fig. 11) samples are taken from wells C1, B1 and A1, plated on solid media, incubated and after incubation of agar plates determine MBC as concentration at that plate that has no colony.



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Results 1. Write data! Bacteria: Preparation of basic and working solutions: Composition of control samples: Tested concentrations of disinfectant:

2. Plan of microtitre plate:

1 2 43 5 6 87 9 10 1211

A

B

C

D

E

F

G

H



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3. Determination of MIC

Antimicrobial substance

Bacterial culture 1

Bacterial culture 2

4. Determination of MBC

Antimicrobial substance

Bacterial culture 1

Bacterial culture 2

5. Comment the results



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10. EXERCISE: PERSONAL HYGIENE

INTRODUCTION

ersonal hygiene includes everything that an individual needs and can do to maintain and strengthen his/her health. In spite of modern technologies and the increasing degree of automation in food industry, people, or people involved in food production and trade, are still the main source or cause of food

contamination with bacteria. Human hands, hair, breathing air, sweating, coughing, sneezing are just some of factors that are important as microbial carriers. The appropriate personal hygiene of all employees in food industry is therefore one of the most important factors contributing to the production of safe foods. For a better understanding of the importance of personal hygiene, the following parts of the human organism are listed below, which may be a potential source of contamination. Microorganisms that inhabit the skin and mucous membranes of healthy people can be classified into two groups. Permanent microbial flora is those microorganisms that are found in the same age range in individuals at the same parts. Transient microbial flora is non-pathogenic and conditionally pathogenic microorganisms temporarily inhabiting the skin or mucous membranes. If there is a disturbance in the composition of a normal microbial flora, transient flora microbes may colonize a region, multiply and cause infection.

NORMAL MICROBIAL POPULATION

SKIN The skin has different important functions in human body: it protects tissues and organs beneath it, feels and participates in the regulation of body temperature and excretion. The outer part (corneum) of the external layer of skin (epidermis) contains 20 to 30 cells thick layer that is impermeable to microorganisms. This tissue is regenerated with new growing cells every 4 to 5 days. The size of the dead cells is about 30 x 0.6 µm and they spread easily into the air. The inner layer of the skin (dermis) consists of connective tissue, elastic fibers, blood and lymph vessels, nervous tissue, muscle tissue, various glands and water. The sweat glands eliminate sweat and the sebaceous glands fat. The skin acts as an organ that permanently secretes sweat and fat and deposits dead cells. When these materials mix with substances from the environment, for example with dust, dirt, grease, this is the ideal medium for bacterial growth. Thus, the skin becomes a potential source of bacterial contamination. Inadequate hand washing and maintenance of body hygiene thus mean an

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increased number of bacteria on the skin, and these can also spread with dead skin cells into the environment. With appropriate personal hygiene, however, the skin acts as a natural barrier to passage of microorganisms. Most microorganisms are inactivated on the skin (except commensals - microbes of normal flora), since fatty acids and some other secretions are potent antimicrobial agents (due to fatty acids pH of skin is about 5.5). Commensals on skin: Two dominant bacteria of Staphylococcus aureus and S. epidermis are normally on the skin. These bacteria are also found in hair follicles and ducts of the sweat glands. Deep in the skin pores, Micrococcus luteus and S. epidermis are often present.

HANDS, NAILS Special attention should be paid to hand hygiene, because hands are those with which a person touches different objects (dirty equipment, other body parts, clothes) and can directly transfer microorganisms to foods. Nail hygiene is also very important, as under the nail dirt, microbes and eggs of parasites might be present. HAIRS Also on the hair are microorganisms, which are predominantly staphylococci. For this reason it is essential that employed in food industry after combing hair wash their hands. It is also essential for employees in food industry to wear protective headgear. EYES There are no bacteria in the eye. But even smaller infections mean an increased number of bacteria. Bacteria are on eyelashes and in the eye corner. Therefore, it is also in such cases need to know that touching with hands mean spreading bacteria into environment. RESPIRATORY TRACT There is normally less bacteria in nose and throat than in the mouth. Nasal cavity, throat, trachea, bronchi and alveoli are usually sterile. Coagulase-negative staphylococci, corinebacteria and najseria are the most dominant in the nasal cavity, while in the throat there are several alpha-hemolytic streptococci. Thirty percent of healthy people have in the nasal cavity Staphylococcus aureus, 25-80% of people have Haemophilus influenzae in the upper part of the respiratory tract. Bacteria of Streptococcus pyogenes and Enterococcus faecalis are also important in the upper part of the respiratory tract. Occasionally, microorganisms can pass mucous membranes and settle into the throat and respiratory tract. Most often these are staphylococci, streptococci and enterococci. A common cold is the most common infectious disease. In the initial phase, this is a viral infection, usually followed by a secondary infection, which may also be bacterial. Therefore, even in mild cold, special attention is needed in hygiene of the nose and hands.



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GASTROINTESTINAL TRACT In the gastrointestinal tract, microbes are inhabited in the mouth, esophagus, stomach, small and large intestine, while the liver and gallbladder generally do not contain microorganisms. There are many bacteria in the oral cavity (Streptococcus spp., Corynebacterium spp., Actinomyces spp., Propionibacterium spp., Lactobacillus spp.). In the mouth can also be pathogenic bacteria and viruses, especially if a person is ill. Digestive tract secretions are the primary source of bacterial contamination. Approximately 30 to 35% of the dry weight of intestinal contents is bacterial cells. In the upper part of the small intestine, Enterococcus faecalis and staphylococci are predominant, and their number is increasing in the course of the small intestine. In the colon, the microbial flora is very heterogeneous and numerous and contains parasites and fungi in addition to bacteria. In the large intestine, more than 100 different types of microorganisms (Pseudomonas aeruginosa, Staphylococcus, Streptococcus spp., Enterococcus spp., Corynebacterium spp., Lactobacillus spp., Bacteroides spp., Bifidobacterium spp., Clostridium perfringens, Salmonella, Shigella, Vibrio cholerae, Escherichia coli and many others). If personal hygiene is inadequate, bacteria are transferred to the environment. When a person gets sick, the microbial population greatly increases, and this also greatly increases the potential for contamination of the environment. Streptococci are normal around the acne, acne, torso, infected cuts, eyes and ears. Inflammation of sinuses, throat inflammation, stinging coughing and other symptoms of colds also mean an increase in the number of microbial populations. The same applies to various gastrointestinal disorders such as diarrhoea and gastric disorders. Diseases or bacteria that are transmitted from a sick person to a foodstuff are diseases of the respiratory tract (e.g. sore throat, various inflammations in the oral and nasal cavity, pneumonia, tuberculosis) and gastrointestinal disorders (for example, abdominal pain, vomiting, spasms, diarrhoea). In many diseases, bacteria that have caused the disease can remain in the body after a disease. These microorganisms can cause re-contamination when personal hygiene is poor. For example, Salmonella may remain in humans for many months or years after illness. Also, viruses in the gastrointestinal tract remain many years after hepatitis has been reported. Washing / disinfection of hands:

• wet and rinse hands with running hot water, • apply soap to hands and wash hands for a certain amount of time (soap

concentration and washing time prescribed by the manufacturer), • rinse hands well under warm running water, • dry hands completely with a paper towel.



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PRACTICAL WORK

Purpose: 1. Determine microorganisms on hair! 2. Determine microorganisms on lip! 3. Determine microorganisms under the nail! 4. Determine microorganisms on palm before washing hands, after washing hands with

water, after washing hands with liquid soap and after washing hands with liquid soap and disinfection! Determine the effectiveness of hand washing after different kind of washing hands.

Materials and practical work:



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Results: 1. Microorganisms on hair

2. Microorganisms on lip

3. Microorganisms under nail



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4. Microorganisms on palm before hand washing, po umivanju z vodo, after washing hands with water, after washing hands with liquid soap and after washing hands with liquid soap and disinfection

No. of microorganisms N (cfu/palm)

Water Liquid soap

Liquid soap and disinfection

Before 1-min washing

After 1-min washing


After 3-min washing


After 6-min washing

5. Effectiveness of hand washing

Effectiveness of hand washing (%)

Time Water Liquid soap Liquid soap and

disinfection

1-min washing

3-min washing

6-min washing



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NOTES



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