**3. A classical culture method of detecting** *Salmonella*

Detection of the presence of *Salmonella* pursuant to Commission Regulation (EC) No 2073/2005 (microbiological criteria for foodstuff) as amended, is carried out according to the ISO 6579 standard - Microbiology of food and animal feeding stuffs - Horizontal method for detection of *Salmonella spp*.(ISO, 2002). Pursuant to the above regulation, detection of *Salmonella* in food should be carried out for such products as raw meat, meat products intended for consumption in the raw state, gelatine, cheese, butter, cream, unpasteurized milk, powdered milk, eggs and products containing raw eggs, crustaceans, molluscs, fruit and vegetables, unpasteurized juice, powdered infant formulas and dietary food for special medical purposes.

Standard ISO 6579 2003 (Microbiology of food and animal feeding stuffs - Horizontal method for detection of *Salmonella* spp.)includes four stages of the detection process and depending on the need to obtain confirmations, it lasts from 5 to 7 days:


During the first stage, in order to proliferate and regenerate damaged cells, the culture is performed on liquid peptone water at 37°C for 18±2 hours. **Buffered peptone water** is applied for non-selective enrichment of *Salmonella* sp. For such products as cocoa or chocolate products, peptone water is applied with an addition of casein or skimmed milk

394 Salmonella – A Dangerous Foodborne Pathogen

truckload) by examining only one 25-gram sample. The answer to the question concerning the required number of single samples is extremely difficult. In view of the high costs of microbiological tests, the number of samples is generally limited. In a microbiological laboratory, samples are taken with the use of sterile tools, e.g. spoons, scalpels, knives, spatulas and pipettes. Frozen products should be first thawed at below 5°C (for not longer than 12 hours). In the case of deeply frozen samples, sterile drills are used for sampling. Determination of *Salmonella* sp. in food products always consists in detecting the presence of those bacteria in a specified amount of the product (generally 25g/ml, very rarely 10g/ml), but the number of those microorganisms in food is not determined. Both in the classical method and in its modifications, the first stage of detection is non-selective enrichment. This is crucial, since food production involves its technological treatment, e.g. heating, which can cause the death of most cells or cause sub-lethal injured. Omission of the stage of preenrichment of the sample and inoculating the material directly on the solid medium can give false negative results. If the examined material includes a very low number of living cells, or the cells have been sub-lethally damaged during the technological processes, we may not receive macroscopically-visible colonies on the solid medium. In such a case there is a risk of releasing the product to market although it does not satisfy safety criteria. During the storage of such a product, damaged cells can be repaired and bacteria can proliferate to a

There are many methods to determine *Salmonella sp.* in food and, for this reason, the present study focuses on the classical culture method – the application of a Vidas device – as the only fully automated one. Additionally, the PCR method (a commonly-applied alternative to the plate method) and the FISH method (which is still not popular, although work on its

Detection of the presence of *Salmonella* pursuant to Commission Regulation (EC) No 2073/2005 (microbiological criteria for foodstuff) as amended, is carried out according to the ISO 6579 standard - Microbiology of food and animal feeding stuffs - Horizontal method for detection of *Salmonella spp*.(ISO, 2002). Pursuant to the above regulation, detection of *Salmonella* in food should be carried out for such products as raw meat, meat products intended for consumption in the raw state, gelatine, cheese, butter, cream, unpasteurized milk, powdered milk, eggs and products containing raw eggs, crustaceans, molluscs, fruit and vegetables, unpasteurized juice, powdered infant formulas and dietary food for special

Standard ISO 6579 2003 (Microbiology of food and animal feeding stuffs - Horizontal method for detection of *Salmonella* spp.)includes four stages of the detection process and

During the first stage, in order to proliferate and regenerate damaged cells, the culture is performed on liquid peptone water at 37°C for 18±2 hours. **Buffered peptone water** is applied for non-selective enrichment of *Salmonella* sp. For such products as cocoa or chocolate products, peptone water is applied with an addition of casein or skimmed milk

level that would be hazardous for the consumers.

**3. A classical culture method of detecting** *Salmonella*

depending on the need to obtain confirmations, it lasts from 5 to 7 days:

Serological and biochemical identification of suspected colonies

Pre-enrichment in non-selective liquid medium

Selective enrichment in liquid media

Plating on selective media

optimization is ongoing) are also described.

medical purposes.

and brilliant green in order to inhibit the growth of Gram-positive bacteria. In the case of acid and soured food products, peptone water should be used with double concentration of components, while for meat and food of high fat content, pre-enrichment should be performed in lactose broth with the addition of Triton X-100.


Fig. 1. Flow diagram for detection of *Salmonella*.

After the non-selective pre-enrichment stage, a 0.1cm3 sample is taken from the culture and inoculated on 10cm3 of selective medium, Rappaport-Vassiliadis with soya, and on **Muller-Kauffmann** medium in the amount of 1 cm3. **Rappaport-Vassiliadis (RVS)** medium is solid, strongly selective and contains malachite green and sodium chloride (inhibiting the growth of accompanying microflora). Soya peptone, pH 5.2, and increased temperature of incubation (41.5°C) favour the growth of *Salmonella* sp. strains. The medium is dark blue and clear. *Salmonella* sp. strains grow on this medium in the form of milky residue, while the colour of the medium itself does not change. The other selective medium, **Muller-Kauffmann broth (MKTTn)**, contains sodium thiosulphate and potassium iodide, which react to form a compound known as sodium tetrathionate, inhibiting the growth of the coliforms. *Salmonella* sp. are able to reduce this compound. The broth also contains brilliant green, which, in turn, inhibits the growth of Gram-positive bacteria.

Detection of *Salmonella* spp. Presence in Food 397

TSIa H2S production No black colour Black colour UREA BROTH Urease Yellow Rose pink – deep

INDOLE Indole production Yellow ring Red / pink ring

S production; +: Black colour; -: No black colour

Table 1. Interpretation table. aRegarding TSI: Read the colour of the butt and of the surface of the medium; ALK: A red colour corresponding to no acid production; NC: No change in the colour of the medium ; A: A yellow colour corresponding to acid production; G: Gas

After 48 h incubation at 37°C, a preliminary identification is made on the basis of the appearance of colonies grown on selective media. Five characteristic colonies are selected from each plate and are plating on the nutrient agar medium, followed by biochemical examinations. In order to perform these examinations, biochemical tests are carried out on

lactose and/or sucrose Surface red Surface yellow

colour

RESULTS

NEGATIVE POSITIVE

Butt red Butt yellow

butt Air bubbles in butt

colourless Yellow

colourless A pink/red colour

cerise

A purple colour (and a yellow/brown colour in the LDC control medium if used)

MEDIUM REACTIONS/ENZYMES

TSIa Acid production from

Acid production (if the butt is yellow, and the slope is red, acid production is only from glucose)

TSIa Gas production No air bubbles in

LCD TEST Lysine decarboxylase A yellow/brown

ONPG β-Galactosidase Remain

PROSKAUER Acetoin production Remain

TSIa

VOGES

production in the butt; H2

the following media:

TSI medium (Triple-sugar iron agar)

Clark medium (V-P reaction)

 Christensen medium with urea (urease production) peptone medium with tryptophan (indole production)

medium with lysine (lysine decarboxylation)

ONPG medium (β-galactosidase detection)

After incubation at 37°C for 48±3 hours, cultures are inoculated on two selective media, so as to receive individual colonies. The first of them is XLD (xylose lysine deoxycholate) agar. The other can be chosen by the laboratory, and it can be BGA (brilliant green agar), Hektoen or Wilson-Blair agar for example.

**XLD agar** contains lactose, saccharose, L-lysine, sodium thiosulphate, sodium deoxycholate, ferric ammonium citrate (III) and phenol red. Differential agents of the agar include: lactose, saccharose, xylose, lysine and sodium thiosulphate, from which hydrogen sulfide is released, forming in reaction with iron salts (III) black residue of iron sulfide in the centre of the colony. The pH indicator is phenol red. The agar makes it possible to determine the sugar fermentation ability. Incubation is carried out at 37ºC for 24±3 hours. Typical colonies can be colourless, very light, slightly shiny and transparent (colour of the medium) with a dark tinted centre, surrounded by a light red area and yellow edge, or of pink to red colour, with a black centre or without a black centre. H2S (–) colonies are colourless or light pink with darker centres, and lactose (+) colonies are yellow or without the characteristic blackening.

**BGA**. Differential factors of this agar are sugars: saccharose and lactose. Brilliant green is a selective agent. Typical colonies are transparent, colourless or light pink, and the colour around colonies changes from pink to light red.

**Hektoen agar**. Selective agents include bile salts, inhibiting the growth of Gram (+) bacteria Differential factors are three sugars: lactose, saccharose and salicin. Increased lactose content ensures that bacteria fermenting this sugar with a delay are not omitted. Bacteria colonies producing hydrogen sulfide had a dark centre as a result of the reaction between hydrogen sulfide and iron (III). Typical colonies of *Salmonella* sp. are green, with or without a black centre.

**Wilson-Blair agar**. This is a strongly selective and differential medium for *Salmonella*, including *S.* Typhi isolated from food. *Salmonella spp*., depending on the strain, grow in the form of black colonies surrounded with an area of black medium or dark brown and brown without this area. A characteristic feature of *Salmonella spp*. colonies is a metallic, shining surface as a result of produced hydrogen sulfide, forming a metallically-black residue in reaction with iron ions. The growth of Gram-positive bacteria and other *Enterobacteriaceae*, including *Shigella spp*., is strongly inhibited by brilliant green and bismuth sulfite present in the medium.

**Rambach-agar chromogenic medium** – with sodium deoxycholate, proplylene glycol and chromogenic mix. Colonies of *Salmonella* sp. are red as a result of glycol fermentation, lactose positive bacteria from the coli group, due to the activity of galactosidase, destroy a bound between the components of chromogenic mix and released chromophore gives those colonies a blue-violet or blue-green colouring. *Salmonella* Typhi and *Salmonella* Paratyphi form colourless or yellowish colonies on this medium.

New selective media have been developed based on biochemical characteristic of *Salmonella*  such as α-galactosidase activity in the absence of β-galactosidase activity, C8-esterase activity, catabolism of glucuronate, glycerol and propylene glycol, hydrolysis of X-5-Gal, and H2S production. e.g. SMID agar (BioNerieux, France), Rainbow *Salmonella* agar (Biolog, USA), CHROMagar *Salmonella* (CHROM agar, France), chromogenic *Salmonella* esterase agar (PPR Diagnostics Ltd, UK), Compass *Salmonella* agar (Biokar diagnostics, France), and chromogenic ABC medium (Lab M. Ltd., UK) (Maciorowski et al., 2006; Manafi, 2000; Perry et al., 2007; Schonenbrucher et al., 2008)

After incubation at 37°C for 48±3 hours, cultures are inoculated on two selective media, so as to receive individual colonies. The first of them is XLD (xylose lysine deoxycholate) agar. The other can be chosen by the laboratory, and it can be BGA (brilliant green agar), Hektoen

**XLD agar** contains lactose, saccharose, L-lysine, sodium thiosulphate, sodium deoxycholate, ferric ammonium citrate (III) and phenol red. Differential agents of the agar include: lactose, saccharose, xylose, lysine and sodium thiosulphate, from which hydrogen sulfide is released, forming in reaction with iron salts (III) black residue of iron sulfide in the centre of the colony. The pH indicator is phenol red. The agar makes it possible to determine the sugar fermentation ability. Incubation is carried out at 37ºC for 24±3 hours. Typical colonies can be colourless, very light, slightly shiny and transparent (colour of the medium) with a dark tinted centre, surrounded by a light red area and yellow edge, or of pink to red colour, with a black centre or without a black centre. H2S (–) colonies are colourless or light pink with darker centres, and lactose (+) colonies are yellow or without

**BGA**. Differential factors of this agar are sugars: saccharose and lactose. Brilliant green is a selective agent. Typical colonies are transparent, colourless or light pink, and the colour

**Hektoen agar**. Selective agents include bile salts, inhibiting the growth of Gram (+) bacteria Differential factors are three sugars: lactose, saccharose and salicin. Increased lactose content ensures that bacteria fermenting this sugar with a delay are not omitted. Bacteria colonies producing hydrogen sulfide had a dark centre as a result of the reaction between hydrogen sulfide and iron (III). Typical colonies of *Salmonella* sp. are green, with or

**Wilson-Blair agar**. This is a strongly selective and differential medium for *Salmonella*, including *S.* Typhi isolated from food. *Salmonella spp*., depending on the strain, grow in the form of black colonies surrounded with an area of black medium or dark brown and brown without this area. A characteristic feature of *Salmonella spp*. colonies is a metallic, shining surface as a result of produced hydrogen sulfide, forming a metallically-black residue in reaction with iron ions. The growth of Gram-positive bacteria and other *Enterobacteriaceae*, including *Shigella spp*., is strongly inhibited by brilliant green and bismuth sulfite present in

**Rambach-agar chromogenic medium** – with sodium deoxycholate, proplylene glycol and chromogenic mix. Colonies of *Salmonella* sp. are red as a result of glycol fermentation, lactose positive bacteria from the coli group, due to the activity of galactosidase, destroy a bound between the components of chromogenic mix and released chromophore gives those colonies a blue-violet or blue-green colouring. *Salmonella* Typhi and *Salmonella* Paratyphi

New selective media have been developed based on biochemical characteristic of *Salmonella*  such as α-galactosidase activity in the absence of β-galactosidase activity, C8-esterase activity, catabolism of glucuronate, glycerol and propylene glycol, hydrolysis of X-5-Gal, and H2S production. e.g. SMID agar (BioNerieux, France), Rainbow *Salmonella* agar (Biolog, USA), CHROMagar *Salmonella* (CHROM agar, France), chromogenic *Salmonella* esterase agar (PPR Diagnostics Ltd, UK), Compass *Salmonella* agar (Biokar diagnostics, France), and chromogenic ABC medium (Lab M. Ltd., UK) (Maciorowski et al., 2006; Manafi, 2000; Perry

or Wilson-Blair agar for example.

the characteristic blackening.

without a black centre.

the medium.

around colonies changes from pink to light red.

form colourless or yellowish colonies on this medium.

et al., 2007; Schonenbrucher et al., 2008)


Table 1. Interpretation table. aRegarding TSI: Read the colour of the butt and of the surface of the medium; ALK: A red colour corresponding to no acid production; NC: No change in the colour of the medium ; A: A yellow colour corresponding to acid production; G: Gas production in the butt; H2 S production; +: Black colour; -: No black colour

After 48 h incubation at 37°C, a preliminary identification is made on the basis of the appearance of colonies grown on selective media. Five characteristic colonies are selected from each plate and are plating on the nutrient agar medium, followed by biochemical examinations. In order to perform these examinations, biochemical tests are carried out on the following media:


Detection of *Salmonella* spp. Presence in Food 399

compound cadaverin and CO2 . A paraffin oil layer is added after inoculation to keep the pH alkaline. Often glucose is metabolised in the beginning of the incubation period and a yellow colour develops in the media after some hours of incubation, but later the media turns purple if the lysin decarboxylase is present because of formation of the alkaline compound cadaverin. As other compounds in the media could be broken down to alkaline compounds, the LDC control media without lysine is also inoculated, a layer of paraffin oil added and it is incubated at the same time. If both the LDC media and the LDC control media turn purple, it cannot be shown that lysine decarboxylase is present and the test is

**Medium VP.** This is a test for acetoin production from glucose. The acetoin produced is oxidised to diacetyl, which produces a red colour with α-naphtol at alkaline pH. A positive

**ONPG medium.** This medium shows the presence of β-galactosidase producing bacteria. βgalactosidase liberates o-nitrophenol, which is yellow at alkaline pH, from ONPG. The

**API.** Determination of biochemical features of the examined bacteria can also involve the application of API 20E tests (Biomerieux), aimed at identification of bacteria from the family *Enterobacteriaceae*. The API 20E system facilitates the 24-hour identification of *Enterobacteriaceae* as well as 24 or 48-hour identification of other Gram negative bacteria. The API 20E strip consists of microtubes containing dehydrated substrates for the demonstration of enzymatic activity and carbohydrate (CHO) fermentation. The substrates are reconstituted by adding a bacterial suspension. After incubation, the metabolic end products are detected by indicator systems or the addition of reagents. CHO fermentation is detected

**Serological tests**. These tests are carried out for strains of bacteria which have been classified into the *Salmonella* genus on the basis of their biochemical features, in order to detect the presence of somatic O, capsular Vi and flagellar H antigens. The examinations are carried out by slide agglutination on the basis of Kauffmann-White antigenic schema. Polyvalent and monovalent serums should be used to determine somatic antigens, and anti-Vi and anti – H serums to detect the presence of Vi and H antigen. Determination of flagellar antigens makes it possible to determine the serological type of the examined

Culture methods are labor intensive and time consuming when handling many samples. In addition, detection can be prevented by the presence of other competing microorganisms during cultural enrichment, and the selective agar media have a very poor specificity creating an abundance of false positives (such as *Citrobacter* or *Proteus*) (Manafi, 2000). Therefore, there is a need for *Salmonella* detection methods that provide results more rapidly

Due to its high sensitivity, specificity, and rapid results, PCR is an efficient alternative to conventional microbiological culture methods to detect specific types of microorganisms in foods, water, and environmental samples (Moganedi et al., 2007; Glynn et al., 2006; Piknova´et al., 2002). The International Standardization Organization (ISO) recently published standards which address the PCR methodology for the detection of food-borne

with sensitivity similar to or greater than, the conventional methods.

evaluated as negative.

reaction is seen as a very pale red colour.

by colour change in the pH indicator.

**4. Polymerase chain reaction** 

pathogens (Tomás et al., 2009).

bacteria.

reaction is positive if a yellow colour develops.


1) These percentages indicate only that not all strains of *Salmonella* show the reactions marked + or -. These percentages may vary from country to country and from food product to food product.

2) *Salmonella* Typhi is anaerogenic.

3) The *Salmonella* subspecies III (Arizona) gives positive or negative lactose reactions but is always βgalactosidase positive. The *Salmonella* subspecies II gives a negative lactose reaction, but gives a positive β-galactosidase reaction. For the study of strains, it may be useful to carry out complementary biochemical tests.

<sup>4</sup> *S*. Paratyphi A is negative.

Table 2. Biochemical results for *Salmonella.*

**Triple-sugar iron agar** is used for differentiation of *Enterobactericeae* according to their ability to ferment lactose, sucrose and glucose. The colour of the slope and the butt and gas production are noted. Acid production from fermentation of one or more of the sugars results in a yellow colour because the phenol red indicator turns yellow at low pH. Very little glucose is present in the medium, so if a bacteria, like *Salmonella*, only ferments glucose then only a little acid will be formed. On the slope, the acid will be oxidised by the air and by the breakdown of protein in the medium and the colour will remain red while the butt is yellow. H2 S production from thiosulphate will be seen as black areas in the medium due to

FeS production. Gas production from fermentation of sugars will be seen as gas bubbles in the medium. The medium is only lightly inoculated.

**Christensen medium with urea.** Urea medium tests for high urea activity. It is the most common method to detect urease production by Enterobacteriaceae (1):

$$\text{(NH}\_2\text{)}\_2\text{CO} + \text{H}\_2\text{O} \rightarrow 2\text{NH}\_3 + \text{CO}\_2 \tag{1}$$

The phenol red turns red at alkaline pH so a positive reaction is shown as the development of a red-pink colour.

**Tryptone/tryptophane medium for indole reaction.** The media is used for testing the liberation of indole from tryptophane. When Kovacs reagent containing amyl alcohol and pdimethylaminobenzaldehyde is added, indole can be extracted into the amyl alcohol layer by shaking a little. Indole and p-dimethylaminobenzaldehyde produces a red or pink colour.

**L-Lysine decarboxylation medium for the LDC test.** The LDC broth is used for the test of production of lysine decarboxylase. This enzyme decarboxylates lysine to yield the alkaline

reaction

+ + - - + - + - - -

1) These percentages indicate only that not all strains of *Salmonella* show the reactions marked + or -. These percentages may vary from country to country and from food product to food product.

3) The *Salmonella* subspecies III (Arizona) gives positive or negative lactose reactions but is always βgalactosidase positive. The *Salmonella* subspecies II gives a negative lactose reaction, but gives a positive

**Triple-sugar iron agar** is used for differentiation of *Enterobactericeae* according to their ability to ferment lactose, sucrose and glucose. The colour of the slope and the butt and gas production are noted. Acid production from fermentation of one or more of the sugars results in a yellow colour because the phenol red indicator turns yellow at low pH. Very little glucose is present in the medium, so if a bacteria, like *Salmonella*, only ferments glucose then only a little acid will be formed. On the slope, the acid will be oxidised by the air and by the breakdown of protein in the medium and the colour will remain red while the butt is

FeS production. Gas production from fermentation of sugars will be seen as gas bubbles in

**Christensen medium with urea.** Urea medium tests for high urea activity. It is the most

The phenol red turns red at alkaline pH so a positive reaction is shown as the development

**Tryptone/tryptophane medium for indole reaction.** The media is used for testing the liberation of indole from tryptophane. When Kovacs reagent containing amyl alcohol and pdimethylaminobenzaldehyde is added, indole can be extracted into the amyl alcohol layer by shaking a little. Indole and p-dimethylaminobenzaldehyde produces a red or pink colour. **L-Lysine decarboxylation medium for the LDC test.** The LDC broth is used for the test of production of lysine decarboxylase. This enzyme decarboxylates lysine to yield the alkaline

S production from thiosulphate will be seen as black areas in the medium due to

2 2 32 <sup>2</sup> NH CO H O 2NH CO (1)

β-galactosidase reaction. For the study of strains, it may be useful to carry out complementary

Percentage of *Salmonella* inoculations showing the reaction1)

> 100 91.92) 99.23) 99.5 91.6 99 94.64) 98.43) 100 98.9

Test Positive or negative

TSI glucose (acid formation) TSI glucose (gas formation)

TSI hydrogen sulfide Urea splitting

Indole reaction

biochemical tests.

yellow. H2

of a red-pink colour.

<sup>4</sup> *S*. Paratyphi A is negative.

Lysine decarboxylation β-Galactosidase reaction Voges-Proskauer reaction

2) *Salmonella* Typhi is anaerogenic.

Table 2. Biochemical results for *Salmonella.*

the medium. The medium is only lightly inoculated.

common method to detect urease production by Enterobacteriaceae (1):

TSI lactose TSI sucrose compound cadaverin and CO2 . A paraffin oil layer is added after inoculation to keep the pH alkaline. Often glucose is metabolised in the beginning of the incubation period and a yellow colour develops in the media after some hours of incubation, but later the media turns purple if the lysin decarboxylase is present because of formation of the alkaline compound cadaverin. As other compounds in the media could be broken down to alkaline compounds, the LDC control media without lysine is also inoculated, a layer of paraffin oil added and it is incubated at the same time. If both the LDC media and the LDC control media turn purple, it cannot be shown that lysine decarboxylase is present and the test is evaluated as negative.

**Medium VP.** This is a test for acetoin production from glucose. The acetoin produced is oxidised to diacetyl, which produces a red colour with α-naphtol at alkaline pH. A positive reaction is seen as a very pale red colour.

**ONPG medium.** This medium shows the presence of β-galactosidase producing bacteria. βgalactosidase liberates o-nitrophenol, which is yellow at alkaline pH, from ONPG. The reaction is positive if a yellow colour develops.

**API.** Determination of biochemical features of the examined bacteria can also involve the application of API 20E tests (Biomerieux), aimed at identification of bacteria from the family *Enterobacteriaceae*. The API 20E system facilitates the 24-hour identification of *Enterobacteriaceae* as well as 24 or 48-hour identification of other Gram negative bacteria. The API 20E strip consists of microtubes containing dehydrated substrates for the demonstration of enzymatic activity and carbohydrate (CHO) fermentation. The substrates are reconstituted by adding a bacterial suspension. After incubation, the metabolic end products are detected by indicator systems or the addition of reagents. CHO fermentation is detected by colour change in the pH indicator.

**Serological tests**. These tests are carried out for strains of bacteria which have been classified into the *Salmonella* genus on the basis of their biochemical features, in order to detect the presence of somatic O, capsular Vi and flagellar H antigens. The examinations are carried out by slide agglutination on the basis of Kauffmann-White antigenic schema. Polyvalent and monovalent serums should be used to determine somatic antigens, and anti-Vi and anti – H serums to detect the presence of Vi and H antigen. Determination of flagellar antigens makes it possible to determine the serological type of the examined bacteria.

Culture methods are labor intensive and time consuming when handling many samples. In addition, detection can be prevented by the presence of other competing microorganisms during cultural enrichment, and the selective agar media have a very poor specificity creating an abundance of false positives (such as *Citrobacter* or *Proteus*) (Manafi, 2000). Therefore, there is a need for *Salmonella* detection methods that provide results more rapidly with sensitivity similar to or greater than, the conventional methods.
