MICROBIOLOGY AND BIOTECHNOLOGY OF FOOD › fileadmin › users › 1 › knjiznice › Vaje_MBH_ANG_20… · MICROBIOLOGY AND BIOTECHNOLOGY OF FOOD. INSTRUCTIONS AND WORKBOOK FOR

Barbara Jeršek

2017

MICROBIOLOGY AND BIOTECHNOLOGY OF FOOD

INSTRUCTIONS AND WORKBOOK FOR II. PART OF LABORATORY EXERCISES

MICROBIOLOGY AND BIOTECHNOLOGY OF FOOD. INSTRUCTIONS AND WORKBOOK FOR II. PART OF LAB. EXERCISES

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Barbara Jeršek Microbiology and biotechnology of food Instructions and workbook for II. part of laboratory exercises Publisher: University of Ljubljana

Biotechnical Faculty Department of Food Science

CI - Kat Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID=290219008 ISBN 978-961-6908-11-5 (pdf) Access: (URL): http/www.bf.uni-lj.si/knjiznice-odd-za-zivilstvo/ucbeniki-v-elektronski-obliki Ljubljana, April 2017

All rights reserved. No part of this publication may be reproduced or used in any other way (graphic, electronic or mechanical, including photocopying, recording or transfer in the database) without the written permission of the copyright owner.


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TABLE OF CONTENTS

1 EXERCISE: MYCOTOXIGENIC MOULDS IN FOOD ................................................................... 4

1.1 PURPOSE ........................................................................................................................................... 4

1.2 COURSE OF EXPERIMENTAL WORK .............................................................................................. 4

1.3 INSTRUCTIONS .................................................................................................................................. 4

1.3.1 ISOLATION OF MOULDS FROM FOOD WITH CLASSICAL MICROBIOLOGICAL METHODS .................... 4 1.3.1.1 HOMOGENISATION ............................................................................................................................. 4

1.3.1.2 WEIGHING OF FOOD AND PREPARATION OF BASIC SOLUTION ................................................... 4

1.3.1.3 PREPARATION OF FOOD DILUTIONS ............................................................................................... 5

1.3.1.4 ISOLATION AND QUATIFICATION OF MOULDS ................................................................................ 5

1.3.2 MOULDS IDENTIFICATION ........................................................................................................................... 6

1.3.3 DETERMINATION OF POTENTIALLY MICOTOXIGENIC MOULDS ............................................................. 7

1.3.4 MYCOTOXYIN DETERMINATION WITH THIN LAYER CHROMATOGRAPHY (tlc) ..................................... 7

1.3.4.1 DETERMINATION OF OTA FORMATION WITH TLC .......................................................................... 8

1.3.4.2 DETERMINATION OF AFB1 FORMATION WITH TLC ......................................................................... 8

1.4 WORKBOOK ....................................................................................................................................... 9

1.4.1 PERFORMANCE ............................................................................................................................................ 9

1.4.2 MATERIAL, WORKFLOW AND RESULTS ................................................................................................... 10

2 EXERCISE: DESINFECTANT ACTIVITY ................................................................................... 14

2.1 PURPOSE ......................................................................................................................................... 14

2.2 COURSE OF EXPERIMENTAL WORK ............................................................................................ 14

2.3 INSTRUCTIONS ................................................................................................................................ 14

2.3.1 PREPARATION OF DESINFICINET............................................................................................................. 14

2.3.2 PREPARATION OF BACTERIAL CULTURES ............................................................................................. 14

2.3.3 BACTERIAL INOCULATION AND DETERMINATION OF SURVIVAL BACTERIA ...................................... 14

2.3.4 BACTERICIDAL ACTIVITY ........................................................................................................................... 15

2.3.5 COMMERCIAL DATA ABOUT DISINFECTANT ........................................................................................... 15

2.4 WORKBOOK ..................................................................................................................................... 16

2.4.1 PERFORMANCE .......................................................................................................................................... 16

2.4.2 MATERIAL, WORKFLOW AND RESULTS ................................................................................................... 17

3 EXERCISE: ANTIMICROBIAL EFFECTIVENESS ..................................................................... 19

3.1 PURPOSE ......................................................................................................................................... 19

3.2 COURSE OF EXPERIMENTAL WORK ............................................................................................ 19

3.3 INSTRUCTIONS ................................................................................................................................ 19

3.3.1 PREPARATION OF BACTERIAL AND MOULD CULTURES ....................................................................... 19

3.3.2 PREPARATION OF BASIC AND WORKING SOLUTION OF ANTIMICROBIAL AGENT ............................ 19

3.3.3 MICRODILUTION METHOD IN MICROTITRE PLATE FOR BACTERIA ..................................................... 20


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3.3.3.1 PLAN OF MICRODILUTION METHOD ............................................................................................... 20

3.3.3.2 PERFORMANCE ................................................................................................................................. 20

3.3.3.3 EVALUATION OF MIC ........................................................................................................................ 21

3.3.3.4 DETERMINATION OF MBC ................................................................................................................ 22

3.3.4 MICRODILUTION METHOD IN MICROTITRE PLATE FOR MOULD .......................................................... 22

3.4 WORKBOOK ..................................................................................................................................... 23

3.4.1 PERFORMANCE OF MICRODILUTION METHOD FOR BACTERIA ........................................................... 23

3.4.2 MATERIAL, WORKFLOW AND RESULTS FOR BACTERIA ....................................................................... 24

3.4.3 PERFORMANCE OF MICRODILUTION METHOD FOR MOULD ................................................................ 26

3.4.4 MATERIAL, WORKFLOW AND RESULTS FOR MOULDS .......................................................................... 27

4 SAFETY REQUIREMENTS ........................................................................................................ 29

5 REFERENCES ............................................................................................................................ 30

TABLE OF FIGURES

FIGURE 1: SCHEME OF MEDIA AND TEMPERATURES FOR INCUBATION OF PLATES FOR MOULDS IDENTIFICATION (1, 2: DESIGNATIONS FOR TWO MOULDS STRAINS) ....................... 7 FIGURE 2: SCHEME OF TLC .............................................................................................................. 8 FIGURE 3: SCHEME OF MICROTITRE PLATE FOR MICRODILUTION METHOD; (A) DESIGNATIONS, (B) CONCENTRATIONS ....................................................................................... 20 FIGURE 4: EXAMPLE OF READING MIC ........................................................................................ 22


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1 EXERCISE: MYCOTOXIGENIC MOULDS IN FOOD

1.1 PURPOSE

In food sample determine if it contains moulds that produce ochratoxin A or aflatoxin B1.

1.2 COURSE OF EXPERIMENTAL WORK

• Determination of mould in/on food

• Identification of selected mould

• Determination of potential mycotoxigenic mould

• Determination of ochratoxin A and aflatoxin B1

• Evaluation of results

1.3 INSTRUCTIONS

1.3.1 ISOLATION OF MOULDS FROM FOOD WITH CLASSICAL MICROBIOLOGICAL METHODS

1.3.1.1 HOMOGENISATION In order to ensure the most uniform distribution of microorganisms food sample is homogenised. For the determination of moulds on food aseptically cut food on Al-foil into smaller parts. For the determination of moulds in food disinfected it with 70% ethanol (for example, walnut in shell, immerse in ethanol), then cut food aseptically. Homogenize the entire food sample, or at least 100 g and 50 g depending on the type of food and the type of microbiological tests. For homogenization electric mixer with blades or stomacher are used with aseptic technique.

1.3.1.2 WEIGHING OF FOOD AND PREPARATION OF BASIC SOLUTION Depending on the type of microbiological tests aseptically weigh a certain weight of homogenized sample with an accuracy of ± 0.1 g

For quantitative microbiological examinations basic solution is prepared. It is prepared in order to provide the most even distribution of microorganisms in a test quantity of the product. Weigh investigated mass or volume of food into a sterile beaker or sterile plastic bag, add nine times the amount of solvent and homogenise the suspension (ISO 6887-1: 1999 (E)). Stock solution is 10-1 dilution of the food. In the absence of specific rules, the minimum quantity of examined food is 10 g (ml) or 20 g (ml). To 10 g (ml) of the sample, aseptically add 90 ml of the solvent (or 20 g (ml) and 180 ml of solvent). This gives the primary dilution (R 10-1). The sample is homogenized by electric mixer with blades or stomacher. In some cases, 50 g (ml) of food add 450 ml of the solvent.

As solvent peptone water, buffered peptone water (ISO 6887-1: (1999)), phosphate buffer, or a saline solution can be used. For foods with a high fat content, such as the raw butter, cream, cheeses, ice cream, use instead of saline solution 2% solution of sodium citrate, which is heated to 45 °C. For acidic foods, such as fruit juice, syrup, refreshments, first measure pH and then neutralize 100 ml of the sample with 0.1 N KOH solution. The basic solution is prepared from neutralized sample.


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1.3.1.3 PREPARATION OF FOOD DILUTIONS Next a 10-fold dilution is obtained, such that one volume of food basic solution (1 ml) is added in a nine-fold volume of the solvent (9 ml). The dilution is repeated so many time to obtain the appropriate dilution of the food by inoculating in/on the medium. By diluting the food reduces the amount of food being investigated and the number of microorganisms in unit volume. For the preparation of food dilutions use the same solvent as for preparing basic solution. The time from preparation of basic solution to microbiological testing should be no longer than 45 minutes. Dilutions should be prepared in the first 30 minutes.

1.3.1.4 ISOLATION AND QUATIFICATION OF MOULDS Appropriate food dilutions are inoculated on/in an appropriate solid or liquid medium. To isolate the mould DRBC (Dichloran Rose-Bengal chloramphenicol; medium with a dye dicloran red and Bengal and chloramphenicol), OGY (Oxytetracycline Glucose Yeast Agar, the medium with glucose, yeast extract and oxytetracycline), MEA (Malt Extract agar medium containing malt extract) and DG-18 (medium with dicloran and glycerol) can be used. DRBC and OGY are media used for the isolation and quantification of moulds from foods that contain a higher concentration of bacteria, because they contain an antibiotic that inhibits their growth. MEA is used for isolation and quantification of moulds and yeasts from the food. It contains malt extract and peptone; it also has the lactic acid, which inhibits the growth of bacteria (pH of 3.5). The medium DG-18 is used for isolation and quantification xerophilic moulds from dried and semi-dried foods (dried fruits, candied fruits, spices, cereals, meat and fish products). DG-18 contains peptone as the nitrogen source, glucose as a source of energy, and vitamins and minerals required for the growth of fungi. The medium has also dicloran, which inhibits the spread of colonies, chloramphenicol and chlor-tetracycline to inhibit bacterial growth. The medium has 18% (w/w) glycerol, which reduces aw (water activity) from 0.999 to 0.95. Plates of MEA and DG-18 are incubated at 25 °C for 7 days.

After incubation of the medium at a particular temperature and after a certain time the results were quantified. Examples:

• When for isolation solid medium is prepared in advance in Petri dish (2r = 90 mm) we need to inoculate on medium 0.1 ml of appropriate food dilution and spread the sample with sterile glas rod on whole medium surface.

• When for isolation solid medium is not prepared in advance we need to inoculate 1 ml of appropriate food dilution in sterile Petri dish. Solid medium should be dissolved, cooled to 45 oC and added (15 ml) in Petri dish with already inoculated food sample. The sample should be mixed with media immediately as "3x up-down, 3x left-right".

After incubation of solid isolation medium, characteristic colonies are counted in these Petri dishes where countable numbers of colonies are grown (in general, countable plate for bacteria is 15 – 300 colonies, countable plate for fungi is 15 – 150 colonies). To calculate the number of microorganisms in 1 g or 1 ml of food sample (cfu/g or cfu/ml), average number of colonies and the dilution of food sample should be considered.

Examples:

• If we have countable plates at many dilutions of food, the number of microorganisms is calculated according to the following formula:


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• If we have countable plates at a single dilution of food, the number of microorganisms is calculated according to the following formula:

• If we have no countable plates the results is expressed as estimation of the number of microorganisms which is calculated according to the following formula:

1.3.2 MOULDS IDENTIFICATION After MEA (or OGY or DRBC) and DG-18 plates incubation, describe the results (qualitative and quantitative) and choose single colony for mould identification For mould identification prepare spore suspension in 0.5 % agar with Tween (AT) – with inoculating loop aseptically transfer spores into AT (in laminar flow cabinet).

Mould spores are then inoculated on following media: CYA (Czapek yeast extract agar), MEA, G25N (25 % Glycerol nitrate agar) (Pitt in Hocking, 1997) according to the scheme in Figure 1.

N number of microorganisms in food (cfu/ml, cfu/g) ∑c sum of all colonies on countable plates n1 number of plates at the first dilution of food sample n2 number of plates at the second dilution of food sample R the first dilution of food sample by which colonies are counted

N number of microorganisms in food (cfu/ml, cfu/g) np average number of colonies R dilution of food sample by which colonies are counted

N* < 1/R N* estimation of number of microorganisms (cfu/ml, cfu/g) R the lowest dilution with uncountable plates


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FIGURE 1: SCHEME OF MEDIA AND TEMPERATURES FOR INCUBATION OF PLATES FOR MOULDS IDENTIFICATION (1, 2: DESIGNATIONS FOR TWO MOULDS STRAINS)

After 7-day incubation of media shown in Fig. 1 describe macro-morphological properties for each mould strain (2r of colony, colour and structure of substrate mycelium, colour and structure of aerial mycelium, formation of exudate) and micro-morphological properties seen under stereo magnifier (40x-80x) (formation of cleistotecium,and ascus) and on native microscopic slide under microscope (100x-1000x) (septate or non-septate mycelium, formation of sporangiospores or conidia, kindof conidiogenic cells, properties of conidia or aporangiospores, apex, metula, phialide,…). Native microscopic slide is prepared in a drop of water with added lacto-fuchsine. Detailed description, identification keys and species descriptions are in Pitt and Hocking (1997) and Samson et al. (2000). After microscopy decontaminate microscopic slide in NaOCl solution (18%) and clean microscopis objectives.

1.3.3 DETERMINATION OF POTENTIALLY MICOTOXIGENIC MOULDS Depending on identified mould predict from literature if the isolate can form mycotoxins and which one, ochratoxin A and/or aflatoxin B1. These isolates are re-inoculated on YES (Yeast Succrose Agar) and MEA or CYA or DG-18 as described in 1.3.2. Plates are incubated at 25 oC for 7 to 28 days.

1.3.4 MYCOTOXYIN DETERMINATION WITH THIN LAYER CHROMATOGRAPHY (TLC) When working with mycotoxins is the mandatory use of protective equipment! All material that comes into contact with mycotoxin throw in a prepared solution of NaOCl! All solvents must be treated as hazardous substances and work must be carried out in a fume hood.

1. Depending on type of mycotoxin that will be analysed with TLC, first prepare mixture of solvents – mobile phase (200 ml for bigger chamber, 20 ml for smaller chamber).

2. As stationary phase use TLC-plate (TLC Silica gel 60 F294), which is consisted of the support (aluminium plate or glass) with a thin layer of stationary phase (silica gel and aluminium oxide).

3. On TLC plate label with a pencil (very gently) the line (2.5 cm from the bottom edge), and mark the samples at intervals of 1.5 cm (Figure 2).

MEA, 25 oC CYA, 25 oC G25N, 25 oC

CYA, 37 oCCYA, 5 oC

1

1

2

2

1

1

2

2

1

1

2

2

1 1

CYA, 37 oCCYA, 5 oC

2 2


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4. On the first place is always standard solution of investigated mycotoxin (S), pipette standard solution (5 µl) very carefully so that diameter of spot is smaller than 3 mm.

5. Afterword add on places marked as V1, V2, V3, … moulds samples like plugs obtained from YES medium. Each mould is sampled 3 times and each plug is added to plate 3 times.

6. When finishing application of plugs, evaporate the solvent in a fume hood. 7. TLC-plate is then carefully placed in chamber with mobile phase, cover the chamber and wait

until mobile phase is approximately 0.5 cm from the top of TLC-plate. 8. TLC-plate carefully take from the chamber and live it in the fume hood until mobile phase is

evaporated. 9. Illuminate the TLC-plate with UV light (366 nm) so that spots become visible spots and mark

them. Measure the distances travelled by each compound and mobile phase (d). 10. Calculate retention factors. The ratio between the distance travelled by each compound and

the one travelled by mobile phase is called the retention factor (Rf):

Rf = d sample / d mobile phase

FIGURE 2: SCHEME OF TLC

TLC can be used as quick screening method for assessment of production of mycotoxin and when appropriately worked it can be semi-quantitative method.

1.3.4.1 DETERMINATION OF OTA FORMATION WITH TLC For OTA determination with TLC prepare mobile phase as mixture of solvents – TEF in ratio 5:4:1 (toluene, ethyl acetate, formic acid) in the fume hood. Continue analysis according to the description in 1.3.4.

1.3.4.2 DETERMINATION OF AFB1 FORMATION WITH TLC For AFB1 determination with TLC prepare mobile phase as mixture of solvents – KAC in ratio 9:1 (chloroform: acetone) in the fume hood. Continue analysis according to the description in 1.3.4.

S V1 V2

TLC plate Chamber with mobile phase

d V1

d Moble phase

S V1 V2 S V1 V2

d V2

Results evaluation


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1.4 WORKBOOK

1.4.1 PERFORMANCE Draw a plan of the experimental work!


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1.4.2 MATERIAL, WORKFLOW AND RESULTS

1. Description of food on/in which moulds are determined

2. Description of moulds isolation from food

3. Determination of moulds in food by conventional microbiological methods - qualitative and quantitative description of isolated moulds on the isolation medium; selection and designation of isolates for identification


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4. Moulds identification

In table below write macro-morphological properties of mould with designation …………..

Medium Macro-morphological properties

MEA 25 oC

2r:

SM:

ZM:

EK:

CYA 25 oC

2r:

SM:

ZM:

EK:

CYA 37 oC

2r:

SM:

ZM:

EK:

CYA

5 oC

2r:

SM:

ZM:

EK:

G25N 25 oC

2r:

SM:

ZM:

EK:

Legend: 2r: colony diameter; SM: substrate mycelium; ZM: aerial mycelium; EK: exudate


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Micro-morphological properties of mould with designation ………………………….are:

Isolate is identified as mould species: …………………….……………………………………..

In table below write all identified moulds and add data from literature about possible mycotoxin(s) formation.

Mould Mycotoxin(s)


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5. Determination of OTA with TLC – draw TLC-plate with results, measure distances and calculate retention factors! Describe the results according to food

6. Determination of AFB1 with TLC – draw TLC-plate with results, measure distances and calculate retention factors! Describe the results according to food!


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2 EXERCISE: DESINFECTANT ACTIVITY

2.1 PURPOSE

By dilution-neutralization method determine whether the examined solution which is based on quaternary ammonium compounds has the effect of disinfectants on tested bacteria of Staphylococcus aureus and Pseudomonas aeruginosa!


• Check the commercial information of disinfectant

• Prepare the appropriate concentrations of the disinfectant and bacteria of Staphylococcus aureus and Pseudomonas aeruginosa

• Determine the bactericidal action of disinfectant in planned contact times

• Evaluate the results

2.3 INSTRUCTIONS

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 the 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 in Staphylococcus aureus. A simplified method comprises of following steps:

2.3.1 PREPARATION OF DESINFICINET Prepare three different concentrations of disinfectants; 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 (9ml) into sterile tubes.

2.3.2 PREPARATION OF BACTERIAL CULTURES Determine the number of bacteria in overnight cultures (for P. aeruginosa and S. aureus) by colony count method.

2.3.3 BACTERIAL INOCULATION AND DETERMINATION OF SURVIVAL BACTERIA In tubes with disinfectant (2.3.1) add 1 mlob overnight bacterial culture (18-h culture with 108 cfu/ml), mix, and measure the 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).


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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)).

2.3.4 BACTERICIDAL ACTIVITY Bactericidal activity of disinfectants 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.

2.3.5 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.

Smel: An almost odorless.

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


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2.4 WORKBOOK

2.4.1 PERFORMANCE Draw a plan of the experimental work!


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2.4.2 MATERIAL, WORKFLOW AND RESULTS

1. In table below write data!

Prepared concentrations of disinfectant

Bacterial culture

Contact times

Nevtraliser

Solid medium

Incubation conditions

2. The number of bacteria in time 0:

Staphylococcus aureus

Pseudomonas aeruginosa

3. Write the number of Staphylococcus aureus (N, cfu/ml) in disinfectant at different contact times and different concentrations of disinfectant!

Contact time (min)

The number of survival bacteria N (cfu/ml) at different concentrations of disinfectant (%)


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4. Write the number of Pseudomonas aeruginosa (N, cfu/ml) in disinfectant at different contact times and different concentrations of disinfectant!

Contact time (min)

The number of survival bacteria N (cfu/ml) at different concentrations of disinfectant (%)

5. Comment the results


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3 EXERCISE: ANTIMICROBIAL EFFECTIVENESS

3.1 PURPOSE

Determine antimicrobial (antibacterial and antifungal) effectiveness of plant extracts.


• Prepare overnight cultures of Gram-positive and Gram-negative bacteria and mould suspension,

you have isolated in the first exercise

• Prepare a plan of microdilution method in microtitre plate

• Prepare basic solution of antimicrobial agent

• Prepare working solution of antimicrobial agent

• Determine MIC and MBC of antimicrobial agent for bacteria

• Determine MIC and MBC of antimicrobial agent for mould

• Evaluate the results

3.3 INSTRUCTIONS

With microdilution method in microtitre plate MIC (minimal inhibitory concentration) and MBC (microbicidal concentration) of antimicrobial agent are determined.

3.3.1 PREPARATION OF BACTERIAL AND MOULD CULTURES From the overnight bacterial cultures (24 hours, 37 °C) 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.

From mould colony on medium (MEA or DG18, saved from exercise 1) prepare a suspension of mould spores so that add 3-5 splitting loop of spores in 4 ml RPMI medium. The concentration of spores in the medium should be from 106 to 107 cfu/ml. The exact number is determined by the colony count method on MEA or DG18 medium.

3.3.2 PREPARATION OF BASIC AND WORKING SOLUTION OF ANTIMICROBIAL AGENT Basic solution of antimicrobial agent is prepared by weighting antimicrobial agent and dissolving it in 1 ml of ethanol (99.9%), for example 500 mg of antimicrobial agent in 2 ml-tube add 1 ml ethanol.

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


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3.3.3 MICRODILUTION METHOD IN MICROTITRE PLATE FOR BACTERIA

3.3.3.1 PLAN OF 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 Figure 3.

FIGURE 3: SCHEME OF MICROTITRE PLATE FOR MICRODILUTION METHOD; (A) DESIGNATIONS, (B) CONCENTRATIONS

(A) DESIGNATIONS:

B1: bacterial culture 1 in TSB medium with different concentrations of antimicrobial agent

B2: bacterial culture 2 in TSB medium with different concentrations of antimicrobial agent

PK1: positive control for bacterial culture 1 in TSB medium with different concentrations of ethanol

PK2: positive control for bacterial culture 2 in TSB medium with different concentrations of ethanol

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

(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

3.3.3.2 PERFORMANCE Aseptically take the following steps:

1. In the first row, in wells in which bacteria will be tested (A1, A2, B4, B5), add 100 ml 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.

B1 B1 B2 B2 PK2PK1 PK3 NK1








1 2 43 5 6 87 9 10 1211

A

B

C

D

E

F

G

H

250 250 250 250 2525 PK3 NK1

125 125 125 125 12.512.5 PK3 NK1

62.5 62.5 62.5 62.5 6.36.3 PK3 NK1

31.3 31.3 31.3 31.3 3.13.1 PK3 NK1

15.6 15.6 15.6 15.6 1-61.6 PK4 NK2

7.8 7.8 7.8 7.8 0.80.8 PK4 NK2

3.9 3.9 3.9 3.9 0.40.4 PK4 NK2

1.9 1.9 1.9 1.9 0.20.2 PK4 NK2

1 2 43 5 6 87 9 10 1211

A

B

C

D

E

F

G

H

(A) (B)


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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.

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.

3.3.3.3 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 (Figure 4) in a column means MIC for a particular combination of bacteria and antimicrobial substance. In parallel to check positive and negative control samples.


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FIGURE 4: EXAMPLE OF READING MIC

3.3.3.4 DETERMINATION OF MBC After the incubation of microtitre plate MBC is determined so those in wells with no visible colour change evaluate the survival bacteria with colony count method on agar plates. For example for bacterial culture 1 (Figure 4) 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.

3.3.4 MICRODILUTION METHOD IN MICROTITRE PLATE FOR MOULD Plan and performance are the same as at bacterial cultures (3.3.3), just instead of TSB use RPMI medium and incubations are longer (7-10 days) at 25 oC.

250 250 250 250 2525 PK3 NK1

125 125 125 125 12.512.5 PK3 NK1

62.5 62.5 62.5 62.5 6.36.3 PK3 NK1

31.3 31.3 31.3 31.3 3.13.1 PK3 NK1

15.6 15.6 15.6 15.6 1-61.6 PK4 NK2

7.8 7.8 7.8 7.8 0.80.8 PK4 NK2

3.9 3.9 3.9 3.9 0.40.4 PK4 NK2

1.9 1.9 1.9 1.9 0.20.2 PK4 NK2

1 2 43 5 6 87 9 10 1211

A

B

C

D

E

F

G

H

MIC for bacterium 1

MIC for bacterium 2


23

3.4 WORKBOOK

3.4.1 PERFORMANCE OF MICRODILUTION METHOD FOR BACTERIA Draw a plan of the experimental work!


24

3.4.2 MATERIAL, WORKFLOW AND RESULTS FOR BACTERIA


Antimicrobial substance

Basic solution of antimicrobial substance

Working solution of antimicrobial substance

Bacterial culture 1

Bacterial culture 2

Working solution of ethanol

2. Plan of microtitre plate:

1 2 43 5 6 87 9 10 1211

A

B

C

D

E

F

G

H


25

3. Determination of MIC


Bacterial culture 1

Bacterial culture 2

4. Determination of MBC


Bacterial culture 1

Bacterial culture 2



26

3.4.3 PERFORMANCE OF MICRODILUTION METHOD FOR MOULD Draw a plan of the experimental work!


27

3.4.4 MATERIAL, WORKFLOW AND RESULTS FOR MOULDS



Basic solution of antimicrobial substance

Working solution of antimicrobial substance

Mould culture 1

Mould culture 2

Working solution of ethanol

2. Plan of microtitre plate:

1 2 43 5 6 87 9 10 1211

A

B

C

D

E

F

G

H


28

3. Determination of MIC


Mould culture 1

Mould culture 2

4. Determination of MBC


Mould culture 1

Mould culture 2



29

4 SAFETY REQUIREMENTS

At practical laboratory work strictly follow the basic instructions of safe work and detailed instructions presented in each laboratory exercise:

• In microbiology laboratory always wear protective gowns. • In microbiology laboratory smoking, eating and drinking is not allowed. • On the work-bench is only material that you need to carry out an investigation. • Windows and doors must be closed during the experimental work due to the risk of

contamination. • Always use aseptic technique. • Gas burners at the beginning and at the end of the work open and close assistant or

technician. • When working near the gas burner be careful. • If you come to direct contact with microorganisms, tell the assistant or technician. • To avoid contamination of the environment, place of contamination must first be disinfected,

washed with water and wipe with a paper towel. • Contaminated glassware and plastic tips for automatic pipettes always place in the prepared

basket or container with disinfectants. • Waste should never be placed in trash as it is contaminated with microorganisms. • Microorganisms can not be out of the microbiological laboratory. • Work with microscopes with care; make sure that the microscope and cleaned. • Microscopic slides can not be throwing in the trash, but in prepared dish with disinfectants. • After finishing work clean and disinfect work desk. • After finishing work, disinfect hands, wash them thoroughly with soap and water and dry with

a paper towel. • With dangerous and toxic substances, work in a fume hood.

The results of the observations should be written in the workbook.


30

5 REFERENCES

Downes F. P., Ito K. 2001. Compendium of methods for microbiological examination of foods. 4th edition. Washington, APHA, 659 str.

Harrigan W. F. 1998. Laboratory methods in food microbiology. 3rd edition. London, Academic Press Limited, 532 str.

International standard. ISO 7954. 1987. Microbiology - General guidance for the enumeration of yeast and moulds - Colony count technique at 25oC. 1st ed. Geneve: International Organization for Standardization, 3 str.

International standard. ISO 4833. 1991. Microbiology - General guidance for the enumeration of microorganisms - Colony count technique at 30 oC. 2nd ed. Geneve: International Organization for Standardization, 4 str.

International standard. ISO 7218. 1996. Microbiology of food and animal feeding stuffs - General rules for microbiological examination. - 2nd ed., Geneve: International Organization for Standardization, 43 str.

International standard. ISO 6887-1. 1999. Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination. Part 1, Genearal rules for the preparation of the initial suspension and decimal dilutions. - 1st ed. - Geneve: International Organization for Standardization, 5 str.

Jeršek B. Praktikum mikrobiološke analize: skripta in delovni zvezek za študente IV. letnika živilstva. 2008. Barbara. Ljubljana: Biotehniška fakulteta, Oddelek za živilstvo, Katedra za živilsko mikrobiologijo, 45 str.

Jeršek B. Higiena živil : laboratorijske vaje za študente živilstva in prehrane. 2. dopolnjena izd. Ljubljana: Biotehniška fakulteta, Oddelek za živilstvo, 2009. 79 str. ISBN 978-961-6333-77-1. http://www.bf.uni-lj.si/fileadmin/groups/2752/Higiena_zivil.pdf.

Jeršek B., Poklar Ulrih N., Skrt M., Gavarić N., Božin B., Smole Možina S. 2014. Effects of selected essential oils on the growth and production of ochratoxin A by Penicillium verrucosum. Arhiv za higijenu rada i toksikologiju, 65, 2, 199-208

Jeršek B. 2014. Osnovni principi identifikacije plesni, kvasovk in bakterij v živilih : skripta in delovni zvezek za laboratorijske vaje pri predmetu Živilska mikrobiologija. Ljubljana: Biotehniška fakulteta, Oddelek za živilstvo, 2014. ISBN 978-961-6333-68-9. http://www.bf.uni-lj.si/fileadmin/groups/2752/Skripta_ZM_2014.pdf.

Jeršek B. 2014. Toksikologija in kontaminacija živil : navodila in delovni zvezek za laboratorijske vaje. Ljubljana: Biotehniška fakulteta, Oddelek za živilstvo, 2014. ISBN 978-961-6908-06-1. http://www.bf.uni-lj.si/fileadmin/groups/2752/Skripta_ToKo_2014.pdf.

Klančnik A., Piskernik S., Jeršek B., Smole Možina S. 2010. Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. Journal of microbiological methods, 2, 81, 121-126

Pitt J.I., Hocking, A.D. Fungi and food spoilage. London, Blackie Academic & Professional, cop. 1997, 593 str.

Samson R. A., Hoekstra E.S., Frisvad J.C., Filtenborg O. Introduction to food- and airborne fungi. Utrecht : Centraalbureau voor Schimmelcultures. 389 str.

SIST EN 1040:2001 Kemična razkužila in antiseptiki - Osnovno baktericidno delovanje - Preskusna metoda in zahteve (faza 1). 2001, 31 str.

Zakon o zdravstveni ustreznosti živil in izdelkov ter snovi, ki prihajajo v stik z živili (ZZUIZS). Uradni list RS 52/2000; 42/2002

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