COMPARISON OF DIFFERENT BACTERIAL GENOMIC DNA EXTRACTION PROTOCOLS FOR SELECTED GRAM

POSITIVE BACTERIA

Amelia Augustine Te-eng

Bachelor of Science with Honours QR (Resource Biotechnology) 201

2006G76 A498 2006

· M'.lknJ(nat AKaaenu. l'usal KhldmattAi.AY SlA SARAWAJ UNIVERSlTl ~ ~ --_han

94~OO KOla ;"aIDID " ..

Comparison of different bacterial genomic DNA extraction protocols for selected Gram positive bacteria

AMELIA AUGUSTINE TE-ENG

A Thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours

(Resource Biotechnology)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARA W AK

2006

,....

ACKNOWLEDGEMENT

First of all, I thank: God for giving me the wisdom and strength in completing this

thesis. I would like to express my sincere thanks and gratitude to my supervisor, Mr. Micky

for his advice, guidance, support and patience throughout my thesis preparation.

I would also like to thank: the postgraduate students from the Microbiology Lab,

Adnawani, Chong Yee Ling, Evayanti, Lai Lee San and Ng Lee Tze for their advice, support,

encouragement and cooperation during the project. I would also like to express my gratitude

to the lab technicians, Mr. Azis and Mr. Amin for their technical assistance throughout this

project.

Finally, I would like to thank: my family and friends for their support and

encouragement in completing this thesis.

Comparison of different bacterial genomic DNA extraction protocols for selected Gram positive bacterial species.

Amelia Augustine Te-eng

Resource Biotechnology Program Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

In this study, four different DNA extraction protocols were perfonned on three different Gram positive bacterial species to determine the best protocols based on the DNA quantity and purity of the extracted DNA. The evaluation on the DNA quantity and purity were determined by using spectrophotometer and two PCR methods which were Specific PCR and ERIC-PCR. Two species used were from the genus Listeria and one species was from the genus Staphylococcus. The organisms were L. monocytogenes (ATCC 7644), L. innocua (ATCC 33090) and S. aureus (ATCC 25923). For the extraction process, three conventional methods were perfonned on the selected bacterial species namely cell boiling, PCI and CTAB and Promega Wizard® Genomic Purification System (Promega, USA). Serial dilution was done for up to 10-5 dilution. The sensitivity of the methods was also compared between the different number of bacterial count. The overall results of the study indicated that the commercial extraction kit provided satisfying results in the PCR analysis. The most promising method based on DNA quantity and purity differ for different bacterial species.

Keywords: Gram positive bacteria; DNA amplification; DNA quantity; DNA purity.

ABSTRAK

Dalam kajian ini, empat jenis protokol pengekstrakkan DNA telah dijalankan ke atas tiga spesies bacteria untuk mengena/pasti protokol yang terbaik dari segi kuantiti dan ketulenan DNA yang telah diekstrakkan. Penilaian kuantiti dan ketulenan DNA diukur dengan menggunakan spektroJotometer dan dua kaedah PCR iaitu Specific PCR dan ERIC PCR. Dua spesies daripada genera Listeria dan satu spesies daripada genera Staphylococcus. Spesies yang diggunakan ialah L.. monocytogenes (ATCC 7644), L.. innocua (ATCC 33090) dan s.. aureus (ATCC 25923). Bagi proses pengekstrakkan, tiga kaedah lama telah dilakukan ke atas spesies bacteria tersebut iaitu pendidihan, PCL CTAB dan Prom ega WizarJIY Genomic Purification System (Promega, USA). Pencairan bersiri lelah dijalankan ke atas kultur bakteria dan perbandingan dilakukan ke atas kultur sehingga pencairan 1 (J5.

Kuantiti dan ketulenan DNA juga dibandingkan di antara pencairan yang berbeza. Keputusan keseluruhan daripada kajian ini menunjukkan set pengekstakkan DNA komersial memberi keputusan yang baik dalam ana/isis PCR berbanding kaedah lain. Kaedah yang terbaik dari segi kuantiti dan ketulenan DNA berbeza untuk kaedah yang berlainan.

Kata kunci: Bakteria Gram positif; amplifikasi DNA; kuantiti DNA; ketulenan DNA.

11

TABLE OF CONTENTS

Page ACKNOWLEDGEMENT

ABSTRACT 11

11ABSTRAK

IIITABLE OF CONTENTS

LIST OF TABLES v

LIST OF PLATE V11

LIST OF ABBREVIATIONS Vlll

LIST OF FIGURES vi

CHAPTER! INTRODUCTION

1.0 Introduction 1

1.1' Objectives 3

CHAPTER 2 LITERATURE REVIEW

2.0 DNA extraction protocols 4 2.0.1 Boiling cell method 5 2.0.2 Phenol/Chloroform/Isoamyl alcohol (PCI) method 5 2.0.3 Cetyltrimethylammoniumbromide (CTAB) method 6 2.0.4 Promega DNA Extraction Kit 7

2.1 Description of the bacteria 8 2.1.1 ' . Listeria monocytogenes 8 2.1.2 Listeria innocua 9 2.1.3 Staphylococcus aureus 10

2.2 DNA quantity and purity 11

2.3 Polymerase Chain Reaction (PCR) 12 2.3.1 Specific PCR 13 2.3.2 Enterobacterial Repetitive Intergenic Consensus PCR 13

iii

CHAPTER 3 MATERIALS AND METHODS

3.0 Materials 14 3.0.1 Bacterial strain preparation 14 3.0.2 Dilution of culture 14 3.0.3 DNA extraction and purification 15

1. Boiling cell method 15 11. PhenollchloroformJisoamyl alcohol (PCI) method 15 iii. Cetyltrimethylammoniumbromide (CTAB) method 16 iv. Promega DNA extraction Kit 16

3.0.4 PCRmethod 17 1. Specific PCR 17 11. Enterobacterial Repetitive Intergenic Consensus PCR 18

3.1 Methods 19 3.1.1 Bacterial preparation 19 3.1.2 Culture dilution and colony counting 19 3.1.3 DNA extraction and purification 20

1. Boiling cell method 20 11. PhenollchloroformJisoamyl alcohol (PCI) method 20 iii. Cetyltrimethylammoniumbromide (CTAB) method 21 iv. Promega DNA Extraction Kit 22

3.1.4 Determination of DNA quantity and purity 23 3.1.5 DNA amplification and visualization 24

1. Specific PCR 24 ii. Enterobacterial Repetitive Intergenic Consensus PCR 26

CBAPTER4 RESULTS

4.0 Culture dilution and colony counting 27

4.1 DNA quantity and purity 28

4.2 PCR analysis 32 4.2.1 Specific PCR 32 4.2.2 Enterobacterial Repetitive Intergenic Consensus PCR 35

CHAPTERS DISCUSSION 39

CHAPTER 6 CONCLUSION 44

REFERENCES 45

APPENDIX 48

iv

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

LIST OF TABLES

Specific PCR primer sequences for L. monocytogenes, L. innocua and S. aureus.

Page

24

Sequences of the primer for ERIC-PCR. 26

The number of colonies for the selected bacterial species at different dilutions

27

DNA quantity and purity for L. monocytogenes different dilutions and DNA extraction methods

obtained from 29

DNA quantity and purity of L. innocua dilutions and DNA extraction methods.

obtained from different 30

DNA quantity and purity of S. aureus dilutions and DNA extraction methods.

obtained from different 31

PCR analysis for different number of colonies obtained from different DNA extraction methods.

38

v

,.... I

LIST OF FIGURES

Page

Figure 1 Promega Wizard® Genomic Purification System 48

Vl

LIST OF PLATES

,.. i

Page

Plate 1 The Specific PCR products from four different extraction methods at 32 three different dilutions for the L. monocytogenes strain . .

Pate 2 The Specific PCR products from four different extraction methods at 33 three different dilutions for the L. innocua strain.

Plate 3 The Specific PCR products from four different extraction methods at 34 three different dilutions for the S aureus strain.

tplate4 The ERIC-PCR products from four different extraction methods at three 35 different dilutions for the L. monocytogenes strain.

!plate 5 The ERIC-PCR products from four different extraction methods at three 36 different dilutions for the L. innocua strain.

IPlate 6 The ERIC-PCR products from four different extraction methods at three 37 different dilutions for the S. aureus strain.

Vll

l

LIST OF ABBREVIATIONS

base pairs

double distilled water

deoxynucleotide triphosphates

Deoxyribonucleic Acid

ethylenediamine tetra-acetic acid

base pairs

Colony Fonning Unit

Luria Bertani

minute(s)

molar or molarity

miliMolar

magnesium chloride

sodium chloride

sodium acetate

picomole per milliliter

phenol-chlorofonn-isoamyl alcohol

Polymerase Chain Reaction

revolution per minute

sterile distilled water

Sodium Dodecyl Sulphate

seconds

Thermus aquaticus DNA Polymerase

Tris-Borate EDT A

Tris-EDTA buffer

microliter

Ultraviolet

percentage

degree Celsius

Vlll

.0

CHAPTERl

INTRODUCTION

Introduction

Since the emergence of molecular biology, genomic DNA extraction protocQls have

me one of the most important and basic technique in this field. Genomic DNA extraction

applied in many other fields such as biotechnology and clinical microbiology, which aids in

existence of genetically modified crops and the detection of bacterial based diseases in

uman. In recent years, genomic DNA extraction has been performed on plant cells, animal

lIs and bacterial cells with a variety of different protocols.

There are many protocols introduced by early scientists in order to extract genomic

NA from various types of bacteria. These different extraction protocols are performed or

metimes modified to make the methods compatible to the types of microorganisms and to

crease the efficacy of the extraction process. The compatibility and the efficacy of the

otooois are portrayed in the sensitivity of the protocols towards certain bacteria and the

'ty of the extracted DNA product at the end of the extraction processes. Pure DNA is

sential for downstream processes, for example, in peR applications and sequencing. The

odification of the methods usually arises from the previous studies of early scientists.

ently, there are two types of DNA extraction methods that can be used which are the

nventional methods and the methods utilizing commercial DNA extraction kits. As many

lecular biology applications involving Gram positive bacteria utilize the extraction of

omic DNA method, it is important to establish the best extraction protocol for different

plications. The standard DNA extraction commonly used by scientists may have many

vantages and disadvantages in relation to the downstream processes.

1

In this study, four different protocols were compared in terms of duration of the

il:Xtraction process, DNA quality and DNA quantity. The four DNA extraction methods were

Perfonned on three Gram positive bacteria from two different genera. The chosen species

" ere Listeria monocytogenes and Listeria innocua from the genus Listeria and

~taphylococcus aureus from the genus Staphylococcus. These species were chosen as they are

~own to be associated with diseases found in human (Hugo, 1992). Both conventional and

j=ommercial methods were performed on these bacteria in different culture dilutions namely

~e cell boiling method (Ausubel et aI., 1990), Phenol/ChloroformlIsoamyl alcohol (PCI)

~ethod (Ausubel et al., 1990), Cetyltrimethyammoniumbromide (CT AB) method (Ausubel et

~., 1990) and DNA extraction using the Wizard® Genomic DNA Purification Kit (Prom ega,

PSA). Evaluation of the efficacy of the different extraction protocols were done by using a

~trophotometer, two Polymerase Chain Reaction (PCR) methods which were the Specific

.olymerase Chain Reaction and Enterobacterial Repetitive Intergenic Concensus (ERIC)

.olymerase Chain Reaction and direct visualization using the Agarose Gel Electrophoresis

ystem. The determination of the best protocol will indeed assist greatly in further

Iownstream processes.

2

l

•. 1 Objective

J'he main objectives of this study were:

To compare the DNA purity yielded from the respective genomic DNA extraction

processes.

[10 To compare the DNA quantity obtained from the respective genomic DNA extraction

processes.

fO To detennine the amplification products generated from the respective DNA extraction

methods via Specific PCR and ERIC-PCR.

ro To detennine the advantages of the respective genomic DNA extraction processes by

assessing their rapidness and efficiency.

3

to

CHAPTER 2

LITERATURE REVIEW

o DNA extraction protocols

There have been many genomic DNA extraction protocols emerging SInce the

very of genetics. According to Gerhardt (1994), new methods that emerged became the

solve major problems in science and subsequently creating in-coming ideas and

'ments that may result in the formation of the final solution to the problems. Besides

1, the different methods of genomic DNA extraction that are suitable for different species

genera might be connected to the wide range of diversity of morphological phenotypes

'n the bacteria and the existence of additional surface structure (Ridgway and Stokes,

The choice of method for DNA extraction and the complexity of obtaining the DNA

ds on the source, for example, the types of organisms to be studied. For bacteria, the

ption of the cell wall depends on the nature of the cell wall (Johnson, 1994). The DNA

ction generally involves collecting cells, disrupting the cells with lytic enzymes and

gents, separating the DNA from other biomolecules and cellular debris, and finally

centrating the DNA (Burden and Whitney, 1995)

4

.1 Cell boiling method

According to Angellis et al. (2004), the cell boiling method has been used in cell lysis

also to detect pathogens in plant tissues. It involves the use of high temperature which is

C to disrupt the cell wall. This method also assist in the inactivation of compounds such

Proteinase K., which inhibits Taq Polymerase. Besides that, Cell Boiling method has other

antages as being very efficient, rapid and simple because it involves few steps. This

od is also applicable for high number of samples to be processed. Apart from that, Cell

iling method is also suitable for various downstream PCR applications such as RAPD

. que. It also does not produce hazardous wastes compared to other extraction methods,

providing a safer environment during the extraction process. Furthennore, boiling

od is less expensive because it requires no special chemicals or equipment (Angell is et

.2 Phenol/Chloroform/Isoamyl alcohol (PC I} method

The Phenollchlorofonnlisoamyl alcohol (PCl) is a conventional method used to

DNA and it involves the use of phenol, chlorofonn and isoamyl alcohol. This method

involves the use of lysozymes, Proteinase K and isopropanol. According to Burden and

'tney (1995), the weakening of the bacterial cell wall is contributed by lysozyme which

ts the polymeric compounds that helps in the cell wall rigidity. The protein denaturation

contributed by Proteinase K, an enzyme obtained from a Streptomycete (Penn, 1991).

I and chloroform are organic solvents that denature and remove protein contamination

aqueous solutions. These solvents contribute in the precipitation of proteins which

uently separates the proteins from the aqueous phases. The separation of the phases is

5

er aided by isoamyl alcohol. The DNA precipitate is obtained by adding isopropanol to

solution. The ethanol added to the solution is used to remove the residual salts, phenol and

orofonn. There are disadvantages present in this method whereby the process may result in

ease yield of DNA (Boccuzzi et al., 1997) and it requires the removal of carry-over

01 that can inhibit PCR applications (McOrist et al., 2002).

Cetyltrimethylammoniumbromide (CTAB) method

The Cetyltrimethylammoniumbromide (CTAB) method is another conventional

od used for DNA extraction. It involves the use of CT AB, a detergent that can partially

ove humic compounds and forms insoluble complexes with denatured proteins,

ccbarides and cell debris (Robe et al., 2003). It is also known to disrupt the function of

in cell. This method also utilizes another detergent called sodium dodecyl sulphate

S) which assists in the lysis of the cell membrane by removing lipid molecules but SDS

cause the shearing of DNA (Burden and Whitney, 1995). Besides that Proteinase K,

IIchlorofonnlisoamyl alcohol (PCI) and chloroforml isoamyl alcohol (CIA) are added to

in the extraction and separation process by separating the DNA from the organic solvent

debris. The DNA precipitate is achieved by the addition of isopropanol.

6

Wizarde Genomic DNA Purification Kit (Promega, USA)

Although conventional DNA extraction methods are currently being used widely to

DNA, many of these methods are not only complicated and time consuming, they also

toxic reagents. According to Jungkind and Kessler (2002), molecular diagnostic

IPUlcaDOns should avoid the contamination of DNA in order to reduce false results. One of

best way is by using commercially available kits. The use of kits are often more rapid and

less manipulation steps as compared to conventional methods. One of the most used

DNA extraction kits is the Promega Wizard® Genomic Purification System

rrorD~:a. USA) which is considerably faster and safer, especially in isolating high molecular

genomic DNA. It does not involve hazardous chemicals such as phenol and can be

to extract high quality genomic DNA. This method basically consists of four steps. The

step of the extraction process requires the lysis of cells and the nuclei. The second step is

obtain the DNA from the bacteria while at the same time removing the RNA with RNase.

third step is to purify the DNA from impurities such as proteins by precipitating the

in salt solution. Finally, the DNA is concentrated and cleansed by isopropanol

~J:mation in order to obtain purified DNA (Micka et al., 1996).

7

·

are

Description of the bacteria

Bacteria are single-celled organisms that reproduce by simple division for example by

fusion. Most bacteria are free living and contain the genetic information and energy

.,aucmg and biosynthetic systems that are essential for growth and reproduction. For

growth, the nutrients needed differ from Gram positive to Gram negative bacteria.

positive bacteria require yeast extract to grow as the extract provides nitrogen, sugars,

both organic and inorganic nutrients for the bacteria. Gram positive bacteria also differ

Gram negative bacteria in terms of the composition of the cell wall. Gram positive

....~'''. contain thick peptidoglycan as part of the cell wall as compared to Gram negative

~.._.w. According to Wheat (1992), the thick cell wall layer overlying the plasma membrane

_ eru,ltnre to lysozyme and most of Gram positive bacteria have polysaccharide covalently

to the peptidoglycan which contributes 10% to 50% of the mass of the cell wall.

Listeria monocytogenes

Listeria is Gram positive non-sporing bacilli or rod-shaped organism (Ridgway and

1987). According to Bille and Doyle (1991), this genus is divided into seven species

categorized under two groups in terms of genomic characteristics. L. murrayi and L. grayi

known to be non-pathogenic. Listeria is also known to be hemolytic and non-hemolytic.

species are L. monocytogenes, L. seeligeri, and L. ivanovii whereas non-hemolytic

L. innocua and L. weshimeri. One of the species, L. monocytogenes, is a

" It8tive anaerobe, motile and catalase-positive (Brooks et al., 2001). Besides that, L.

"OI!)'lo~~enles is known to be associated with diseases in human and animal (Bemer et al.,

which was isolated by Murray et at. in 1926 (Cossart and Lecuit, 2001). L.

8

rmocvtc'l!e.nes is a food-borne pathogen that has the ability to cause diseases through

usumD1tion of contaminated foods such as dairy products and raw vegetables (Ridgway and

1987). This species is also known to be able to survive and multiply at low

imM:nture (Hugo, 1992). In medical microbiology, this species has been used as a model

for bacterial intracellular parasitism study in tenus of genetic and for the study of

cell biology (Lecuit and Cossart, 2001). Based on previous studies, L. monocytogenes

be differentiated from other Listeria species through applying Polymerase Chain Reaction

IPIllllca1tIon of the hlyA gene that encodes listeriolysin 0 (Karpiskova et al., 2000).

__,n",.,," 0, the sulfhydryl-activated hemolysin is an important virulence factor for the

PUlliN.. (Bessesen et al., 1990).

Listeria innocua

Listeria innocua is a gram-positive, nonhemolytic, nonpathogenic rod that can be

_lted from soil, vegetation and both human and animal feces. It can also be isolated from

and dairy products. From the studies done by Johnson et al. (2004), certain isolates ofL.

contains all the members of the PrfA-regulated virulence gene cluster. This gene has

used in the study of the virulence factor of this species by Polymerase Chain Reaction

prutlca~no:n. L. innocua is non-hemolytic bacteria and categorized under the genus Listeria

9

IU&lJLUU.<U.

Stilphylococcus aureus

Staphylococcus is another Gram positive bacteria that has a morphology of grape-like

• It was first named by Sir James Ogston in 1881 when he discovered the bacteria in

ICCSISeS (Cookson, 1997). According to Novick (1993), Staphylococcus is non-motile,

nltllm~ aerobic and glucose fermenting bacteria. It can be differentiated by growth as

cluster and by pentaglycine cross-bridges in its peptidoglycans. This genus contains

r.CWlbDlct species, among which is Staphylococcus aureus. This species can be found on

skin and mucous membrane. The growth of this species can be observed as

yellow pigment. The optimum growth for S. aureus is between 30°C to 37°C and

well in nutrient agar. Most of the strains of S. aureus are relatively heat resistant and

them requires a temperature of at least 60°C (Willet, 1992). The peptidoglycan of the

wall which contains lysine as the diamino acid and pentaglycine residues are the

wmanc target of the lytic enzyme lysostaphin (Cossart and Lecuit, 2001) but are resistant

action of lysozyme (Penn, 1991). The characteristic of coagulase-positive from this

is the cause of skin infection in human (Bille and Doyle, 1991). It is thought to be an

virulence factor ofS. aureus. Besides that, coa gene has been implicated in binding

pmlDoJ~en inS. aureus (Dickinson et al., 1995). The coa gene has been used in Polymerase

Reaction amplification for the detection of this species (Motta et ai., 2001).

10

1IIl'01J)D<)res,ls system.

DNA Quantity and Purity

Quantity of the DNA is often associated with the concentration of the DNA and purity

DNA is detennined by the level of contaminants that are present after the extraction

Detennination of DNA concentration and purity can be estimated either

by using a spectrophotometer or qualitatively by using agarose gel

DNA, RNA and protein can strongly absorb ultraviolet light in the range of 260 run to

mn. A pme DNA shows between the values of 1.8 to 2.0 in the ratio of 260 nml280 run.

value of less than 1.8 indicates the presence of protein contaminants while value of more

shows the presence of RNA. The presence of protein can give a false value of DNA

cen~ion since protein also absorb light within the ratio of 260 nml280 run (Burden and

1995). Absorption spectrophotometer which is widely used can detect 1 to 50 ).lg of

per m1 (Cassin et aI., 2000). Another mean to determine the quantity of DNA

IIDIID.VIElIV is by using agarose gel electrophoresis. This method is sensitive towards a small

of DNA. The DNA fluorescence is proportional to its concentration and it has been

that a band of DNA less than 5 ng is not detectable by human eyes (Burden and

11

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is one of the most important discovery in the field

lDO~r=cu.IaI biology. According to Hoorfar and Lubeck (2003), the first publication on

f,lD4DIC Chain Reaction (PCR) in science work is from R.K. Saiki and colleagues in 1985

Mullins and F.A. Faloona in 1987. The main advantage of PCR is in having the

10 amplify small amounts of genetic materials, which cannot be detected or analyzed

by other methods.

PCR involves a primer mediated enzymatic amplification of DNA sequences. Birch

Komoldin (2002) stated that this method can be applied in the identification and

ditBtion of environmental and food pathogen at high sensitivity in complex matrices with

sample preparation techniques. The technique requires a repetitive series ofthree steps

PCR cycle which are the double stranded DNA template denaturation, annealing of

(QligollucJleo1jde primers to the single stranded template and enzymatic extension of the

to produce copies that works as template in subsequent cycles. Every cycle doubles

inmhf!r of the target copies and this means an amplification of 25 cycles could generate

copies. The denaturation process occurs rapidly at 94°C to 96°C and the primer

depends on the melting temperature of the primer-template hybrids but the best

temperature is determined by optimization (Birch and Komoldin 2002).

12

pedficPCR

pecific PCR is widely used in Polymerase Chain Reaction based study. This type of

involves the oligonucleotide DNA strands which controlled the specificity of the

In an exponential reaction, the original target sequence is amplified into many

within a few hours. This technique also amplify the specific target sequence of

ERIC-PCR

Enterobacterial Repetitive Intergenic Concensus-Polymerase Chain Reaction (ERIC­

a DNA-based typing technique that generates strain-specific fingerprinting using

directed to specific nucleotide sequences. Based on Ventura et al. (2003), this

involves the use of oligonucleotides targeting short repetitive sequences dispersed

various bacterial genomes. Their various locations in bacterial genomes allows

_Dation at the genus, species and strain levels based on their electrophoretic pattern of

IIlcltiCm products which also involves the generation of species-specific patterns of

bacterial speCies. A 35 PCR cycles of 103 CFU/ml bacterial sample can still be easily

whereas at a dilution corresponding to 100 bacterial cells, onl y very few ERI C-PCR

could be detennined (Ventura et al., 2003).

13

TERIALS

CHAPTER 3

MATERIALS AND METHODS

Sources of bacterial strains and preparation

1. L. monocytogenes (ATCC 7644) (Microbiologics, USA)

2. L. innocua (ATCC 33090) (Microbiologics, USA)

3. S. auTeus (ATCC 25923) (Makmal Makanan, Miri)

• Luria Bertani broth with 0.6% yeast extract

.1 DUution of culture

1. Bacterial culture (L. monocytogenes, L. innocua and S. aureus)

~ Peptone (Merck, Gennany)

• Total Plate Count(TPC) Agar (Oxoid, UK)

14