Chapter three Materials and Methods

Chapter three

Materials and Methods

3:Materials and Methods: 3.1: Materials: 3.1.1: Instruments and Equipments: The instruments and equipments that were used in this work are listed in table (3) below: Table (3): Instruments and equipments with their remarks Instrument / equipment

Manufacturer / state

Autoclave

Sturdy (Taiwan)

Compound light microscope

Olympus (Japan)

Digital camera

Sony (Japan)

oven

Memmert

Hot plate with magnetic stirrer

Heidolph (Germany)

Incubator

Memmert (Germany)

Api20E System

Biomeriux (France)

PCR system: -Thermo cycler apparatus

MWG Biotch / Germany

-Ultraviolet transilluminator

(European)ECX-15.m.

-Gel electrophoresis

(UK)Shandod Scientific

-Eppendorf centrifuge

Co.Hettich EBA 20(Germany)

-Micropipettes (different volumes)

Eppendrof Oxford (USA)

Eppendorf tubes

Sigma(England)

Sensitive balance

Sartorius (Germany)

Sterilized cotton swabs

Sterile EO. / China

Water bath

Kottermann (Germany)

Water distillator

LapTech(Korea) Sterellin Ltd. / UK

Tips

[25]

Biomeriu

Chapter three


3.1.2: Chemical Materials: The chemical and biological materials used in this work are listed in Table( 4) below: Table (4): Chemical materials with their remarks Material

Manufacturers name

Boric acid

BDH/UK

Bromophenol blue

BDH

Casein

Fluka (Switzerland)

Crystal violet

Oxoid

DNA marker

Promega/USA

Ethanol 96%

BDH

Ethidium bromide

BDH

Ethylene diamine tetra-acetic acid (EDTA)

BDH

Glucose (C6H12O6)

BDH

HCl

BDH

Hydrogen Peroxide (H2O2)

BDH

Isoamylalcohol

BDH

KH2PO4

BDH

Malachite green

BDH

Methyl red

BDH

NaOH

BDH

Peptone

Difco / USA

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3.1.3: Culture Media: Culture media used in this work are listed in Table (5).They were prepared according

to the manufacturer’ instructions on their containers and sterilized

according to the suitable method. Table (5): Culture media used with their remarks Medium

Manufacturer (State)

Agar –Agar

Biolife (Italy)

Agarose

Promega(USA)

Brain-heart infusion broth

Himedia /India

Brilliant green agar (BGA)

Himedia/India

Simmon’s citrate agar

Himedia/ India

urahaphsuhplusihtuisib

usaatmai/ India

Nutrient agar

Himedia/ India

Nutrient broth

Himedia/ India

Chromoagar salmonella

uhcarios/ France

Triple Sugar Iron agar (TSI)

Himedia/ India

Urea agar base

Himedia/ India

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3.1.4. Kits 3.1.4.1 API20 E System It was provided by Biomeriux, France, which include :  Culture of bacterial species.  0.85% NaCl solutions (5ml)  Sterile Pasteur pipettes +bulbs  Sterile mineral oil  API 20E test strip (for oxidase- gram negative rods)  API test strip incubation chamber.  FeCl3, Barrett's reagents A and B.  Kovac's reagent.

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3.1.4.2 Genomic DNA Extraction Kit:-

Table (6): The kits used in this study with their companies and countries of origins: No. 1

Kit Genomic DNA Extraction Kit

Company

Country

Geneaid

USA

Bioneer

Korea

Cell lysis buffer Protein removel buffer Isopropamol Ethanol70% Elution buffer TE buffer Collection tube 2ml 2

AccuPowerTM PCR PreMix Taq DNA polymerase dNTPs Tris-HCl pH 9.0 KCl MgCl2 Stabilizer and Tracking dye

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3.1.5 : Solutions and Reagents: 3.1.5.1: Reagents: A : Oxidase Reagent: This

reagent

was

prepared

by

dissolving

1g

phenylenediaminedihydro chloride in distilled water

of

tetramethyl-P-

then the volume was

completed in to 100 ml( final concentration 1%), keeping in dark

flask for few

days. This reagent used for oxidase test(Collee ,et al., 1996). B: Catalase Reagent: Hydrogen peroxide (3% H2O2) was used for detection the ability of bacteria to produce catalase enzyme (Cloeckaert, and Chaslus-Dancla,2001). C: Kovac´s Reagent: This

reagent was prepared

by dissolving 10 g

P-dimethyl-aminobenz

aldehydein 150ml isoamylalcoholand slo wly add 50 ml of concentrated hydro chloricacid ( HCl),prepared in small quantities and stored in the refrigerator, shacked gently before its use. This reagent is used for detection of indole

production

(Collee et al., 1996). E: Methyl Red Reagent: This reagent was prepared by dissolving0.1g of methyl red in 300 ml of ethanol, then adding 200 ml of distilled water . This reagent is used for detection of glucose fermentation by bacteria (Collee et al.,1996). F : Voges-Proskauer(VP) Reagent: Solution I:This solution was prepared by dissolving 40 g of distilled water then the volume was completed into 100 ml

KOH in

to obtain final

concentration (40%).This is presented (VP1). Solution II: This solution was prepared by dissolving 5 g of α , naphthol in 100 ml of absolute ethanol (99%). This is represented (VP2). They are used for detection the ability of bacteria to produce acetoin (Collee ,et al., 1996) [30]

Chapter three


3.1.6 . Primers These primers are specific for designed in this study by using NCBI Gene-Bank and Primer online and provided by (Bioneer company, Korea) as following table:

Table( 7) Specific Primers Sequence Used for PCR Amplification.

Sequence

Orientation

Forward

CGG,ACG,GGT,GAG,TAA,TGT,CT

GTT,AGC,CGG,TGC,TTC,TTC,TG

Reverse

ATG,CCC,GGT,AAA,CAG,ATG,ATG ,AG

Forward

Position

Size of PCR product(bp)

16s rRNA

406

avni 558

CTC,GCC,TTT,GTC,GGT,TTT,AG

Reverse

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3.2 Methods :3.2.1 Collection of Samples : This study was carried out during a period extended from( December 2011 to June 2012 ) . A total of 200 samples from imported chicken meat and beef in the different markets of the city of al- Diwaniyah divided into a one hundred samples of chicken and a one hundred samples of beef were selected five origins of chicken and beef available in the local market, the origins of chicken meat (al-murad , alkafeel, thighs U.S.A, Turkish Chicken, Jordan Chicken) the beef origins (Australian meat, Emirates meat , al-murad , India , meat AL-kafeel) 25 g were taken from each sample from wing and thighs muscles and in 5 ml of tetrathionate broth and transported to the laboratory in portable container, then incubated for 18-24 hrs at 42 C˚.

3.2.2: Isolation and Identification of Salmonella spp.: A loopful of u hthtbihsacviht broth culture was streaked on surface of , bismuth sulphate agar and the positive growth than cultured on chromogenic salmonella agar plates then incubated at 37C˚ for 24 hrs . The biochemical characters of non – lactose fermenting bacteria was determined by using TSI agar and urease test and other biochemical tests. Colonies that showed biochemical characteristics similar to that of Salmonella spp. were tested by API 20- E system and the confirmation was identified by PCR with 16s r RNA genes primers for detection of Salmonella spp. and S.typhimurium

was detected by PCR with invA

gene primers . Figure (1) summarizes the steps of isolation and identification of Salmonella spp.

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Samples 100 chiken meat

100 Beef

Tetrathionate broth

Chicken meat

Beef

Bismuth sulphate agar

Chicken meat

Beef

Chromogenic salmonella agar

Beef

Chicken meat

Biochemical test & Api 20-E

PCR (16 s rRNA gene )

PCR (inv A gene) Figure (1) Summarizes the steps of isolation and identification of Salmonella spp. [33]

Chapter three


3.2.3: Sterilization methods A: Sterilization by autoclave : all media except chromogenic agar were sterilized by autoclave at 121C˚ and pressure 15 pounds/inch for 15-20 minutes. 2

B: Sterilization by filtration :the solutions which were affected by high temperature were sterilized by filtration through Millipore filter (0.2µm) C: Dry heat sterilization: The glasses were sterilized by dry heat in an electric oven at 180C˚ for 2 hrs. 

3.2.4.: Laboratory Prepared Media:  



A : Urea Agar Medium: This medium was prepared by dissolving 24g of urea agar base in 1000 ml of distilled water, the pH was adjusted to 6.8and sterilized by autoclaving, cooled to 50C˚,then aseptically adding50ml of 40% sterile urea solution, mixed thoroughly, distributed into sterile test tubes and allowed tubes to solidify in a slanted position (Collee, et al., 1996).  

 B: Peptone Water Medium: 



This medium was prepared by dissolving 10 g of peptone and 5g of NaClin1000 ml distilled, deionized water , mixed thoroughly, the pH was adjusted to 7.2, distributed in 5ml

sterile test tubes, then sterilized by

autoclaving and store at 4C˚ until use (Collazo,et al . 1997). 

 C: Methyl Red – Voges- Proskauer Medium (MR-VP Medium): 

This medium was prepared by dissolving 5g of glucose, 5g of peptone and 5 gof phosphate buffer in 1000ml distilled water, distributed in 5mltest tubes, then sterilized by autoclaving and store at 4C˚ until use al.,1996).

[34]

(Collee et

Chapter three


 D: Chromogenic salmonella agar : Salmonella chromogenic agar is a new selective Rombach ,(1990)

used for the

chromogenic medium,

detection and presumptive identification of

Salmonella species from stool samples, foods and Waters .The media used, traditionally,

to

differentiate

species

of

Salmonella

from

the

rest

of

Enterobacteriaceae family, based on their capacity to produce hydrogen sulfide associated with its inability to ferment lactose,( Narquet and Roupas., 1996a) . are not really adequate as there are more than 2000 species of Salmonella, which do not have these characteristics. Casein peptone and Beef extract are the nutrient sources of nitrogen, vitamins, amino acids and minerals; the Chromogenic mixture aids in inhibiting Gram-positive organisms and Proteus and

Coliforms in

conjunction with the Sodium citrate. Bacteriological agar is the solidifying agent. To

identify Salmonella species, this chromogenic agent is based on the

combination of two

chromogenic substrates that ease quick identification. These

two chromogenes are X-gal and

Magenta-caprylate. X-gal is a substrate

incorporated to visualize the ,, ß-D-galactosidaseproducing

organisms as blue

colonies. Magenta colonies are a result of the hydrolysis of Magenta-caprylate by the Lactose negative Salmonella species. Thus, non-Salmonella organisms appear blue or are not stained by any of the chromogenes of the medium. Inoculate with sample and incubate at 35 ± 2°C for 18 – 24 hours. This media was prepared by dissolving 34.9 g in 1000ml distilled water disperse powder slowly in water by rotating the agar than heating and boiling to 100c .  E. Bismuth sulphate agar : This media prepared by dissolve 52 g in 1000 ml distilled water disappears powders slowly by rotating and heating and boiling to 100 c ,then sterilized by autoclaving. (Gurtler, 2009) .

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3.2.5: Biochemical Tests: A: Triple-Sugar Iron Agar (TSI Agar): Heavy inoculums were streaked over the surface of the slant of TSI medium and stab into the button, incubated aerobically at 37C˚ for 24 hrs. Interpretation the results were recovered by the change of color at slant e and buttons, with or without H2S production as follows:

(MacFaddin, 2000).

Reactions seen in the TSI agar slants Slant/ ButtonColor

Result

Alkaline /Acid

Red / Yellow

Acid /Acid

Yellow / Yellow

Alkaline / Alkaline

Red / Red

H2S

Black precipitation

B: Simmons Citrate Utilization Test: Medium was inoculated by streaking from saline suspension of the organism to be tested and Incubate for 24 - 48 hrs at 37C Positive result was indicated by blue color and streak of growth while negative result it is original green color and no growth (Collee et al., 1996).

C : Voges-Proskauer test: Methyl red – VogesProskauer (MR.VP) medium was inoculated with new culture colonies, and incubated at 37C˚for 24-48 hrs, then (1ml) of reagent VP1, and 3ml of reagent VP2 were added . A positive reaction was development of pink color in 2-5 minutes (Collee etal., 1996). [36]

indicated by

the

Chapter three


D: Methyl Red Test: This test was done by inoculating MR-VP medium with new culture colonies, incubated at 37C˚ for 48 hrs . About five drops of the methyl red reagent was added and mixed then read immediately. The positive tests were bright red, while the negative tests were yellow (D’Aoust, et al .,2001)

E: Motility Test: The motility test medium was inoculated with a straight inoculation, making a single state down the center of the tube to about half the depth of the medium . It was incubated under the conditions favoring motility incubation at 37C˚. An examination was done after hours 1, 2 and 6 days (Collee et al., 1996). F: Indole Test: Peptone water medium was inoculated with new culture colonies and at incubated 37C˚. for 24 hrs , then drops from Kovacs reagent were added . A red color in the alcohol layer indicated a positive reaction (Collee et al., 1996). G: Urease Test: The test was done by inoculation of urea agar medium with new culture colonies and incubated aerobically

at 37C˚ for 24 hrs. A positive result was

recorded by changing the color of the media from yellow to pink due to the ability of an organism to split urea , forming two molecules of ammonia by the action of the urease H : Catalase Test: A small amount of the bacterial growth was obtained and suspended in a drop of hydrogen peroxide 3% on a glass slide, and observed for evolution of bubbles as a positive result (MacFaddin, 2000).

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3.2.6: API20-E system: A: Preparation of bacterial suspension: 1: A large colony (2-3 mm diameter) of the bacterium was inoculated into the 0.85% NaCl solution, making sure that the suspension was homogenous and without clumps of floating bacteria. 2:

A MacFerland barium sulfate standard(0.5) ( growth equivalent to1.5 X108 cell/ml ) was prepared.

B: Inoculation of the API strip: 1: The strip was hold at a slight angle up from the table top then ,

the bacterial

suspension was inoculated into each well with the sterile pipette. 2: The end of the pipette was touched to the side of the cupule, allowing capillary action to draw the fluid into the well as the bulb

was slowly squeezed. This

should eliminate any bubbles forming in the wells. Each well was filled up to the neck (Table 8). 3: Citrate utilization (Cit), acetoin production(VP) and gelatinase (GEL) had boxes around their names. These test wells were filled all the way up to the top of the well. 4: Lysinedecarboxylase (LDC), Ornithine

decarboxylase (ODC), arginine

dihydrolase (ADH),H2S production and Urea hydrolysis (URE) were filled as described in step B, but they were then filled up to the top with sterile mineral oil. C: Incubating the Strip in its Chamber: 1: The bottom of the incubation chamber had small indented wells in the bottom filled with water just enough to fill these indentations. 2: The strip into this bottom was placed . 3:The top of the incubation chamber was placed over the bottom and labeled it. 4: The strip was incubated aerobically at 37C˚ for 18-24 hours.

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Chapter three


: Interpretation: 1. The proper reagents were added to the compartments:  1 drop of Kovac's reagents added to the Indole production ( IND) (reading within a couple of minutes).  1drop of

Barritt's A and B added to acetoin production (VP) ( a + ve

reaction may take up to10 min)

)

 1 drop of FeCl3 to TDA 2. All other tests as described (chart below) were read without reagents. 3. Results on the diagram handed out to you in lab (1, 2 or 4 points for +ve reaction, 0 points for –ve reaction) were recorded. The oxidase test reaction was negative, and was added as the last test results. 4. Three test reactions were added together at a time to give a 7- digit number, which could then be looked up in the codebook.

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Chapter three


Table (8): Readings of the API 20-E system and their interpretations TESTS

SUBSTRATE

ONPG

ONPG

ADH

arginine

LDC

Lysine

ODC

Ornithine

CIT

Citrate

REACTION TESTED betagalactosidase arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilization

H2S URE TDA IND VP GEL

Na thiosulfate Urea Tryptophan Tryptophan Na pyruvate Charcoal gelatin

H2S production Urea hydrolysis deaminase Indole production acetoin production gelatinase

GLU

glucose

MAN

mannitol

INO

inositol

SOR

Sorbitol

RHA

Rhamnose

SAC

Sucrose

MEL

Melibiose

AMY

Amygdalin

ARA

Arabinose

Fermentation/ oxidation Fermentation/ oxidation Fermentation/ oxidation Fermentation/ oxidation Fermentation/ oxidation Fermentation/ oxidation Fermentation/ oxidation Fermentation/ oxidation Fermentation/ oxidation

OX

Oxidase

Oxidase

- RESULTS

+ RESULTS

Colorless

Yellow

Yellow

Red/ orange

Yellow

Red/ orange

Yellow

Red/ orange

Pale green/ yellow Colorless/ gray Yellow Yellow Yellow Colorless No diffusion of black Blue/ bluegreen Blue/ blue-green

blue- green/ blue

Blue/ blue-green

Yellow

Blue/ blue-green

Yellow

Blue/ blue-green

Yellow

Blue/ blue-green

Yellow

Blue/ blue-green

Yellow

Blue/ blue-green

Yellow

Blue/ blue-green

Yellow

Colorless/ yellow

Violet

Black deposit Red/ orange Brown- red Red(2 min) Pink/ red (10min.) Black diffuse Yellow Yellow

1: ONPG: betagalactosidase ;2: ADH: arginine dihydrolase ;3: LDC: Lysine decarboxylase;4: ODC: Ornithine decarboxylase;5: CIT :Citrate utilization;6: H2S: H2S production;7: URE: Urea hydrolysis;8: TDA: Tryptophan deaminase;9: IND :Indole production;10: VP : acetoin production; 11 GEL: gelatinase;12: GLU glucose Fermentation;13: MAN mannitol Fermentation;14: INO: inositol Fermentation;15: SOR: Sorbitol Fermentation;16: RHA Rhamnose Fermentation;17: SAC: Sucrose Fermentation ;18: MEL :Melibiose Fermentation ;19: AMY: Amygdalin Fermentation;20 :ARA: Arabinose Fermentation;21: OX: Oxidase Fermentation.

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Chapter three


3.2.7 : Polymereas chain reaction PCR 3.2.7.1. Genomic DNA extraction

Genomic DNA of salmonella spp.isolates were extracted by using Genomic DNA Kit, as following steps: 1-One ml of

incubated cultured bacterial cells was transferred to a 1.5 ml

microcentrifuge tube. 2- Then centrifuged in high speed centrifuge at 15000 rpm for 1 minute then the supernatant discarded. 3- Cell lysis buffer ( 300 μl) were added to the tube and re-suspended the cell pellet by shaking vigorously by vortex, . 4- Then incubated at 60 °c for 10 minutes, until the sample lysate and the tubes inverted every 3 minutes through incubation periods. 5- Protin Removel Buffer (100μl ) were added to each sample lysate and mixed by shaking vigorously for 10 seconds. 6- Then the tubes were incubated at ice 5 minutes and then centrifuge at 15000x g for 3 minutes . 7- Transfer the supernatant to clean 1.5 ml microcentrefuge tube. 8- Add 300 μl of Isopropanol and mix by inversion and centrifuge at 15000x g for 5 minutes . 8 - Discard the supernatant and add 300ml of 70 % ethanol to wash the pellet and then centrifuge at 15000x g for 3 minutes [41]

Chapter three


9- Discard the supernatant and air –dry the pellet for 10 minutes . 10-

Add 50-100 ml of TE Buffer and incubate at 60c for 30 _60 minutes to

dissolve the DNA pellet .During incubation ,tap the bottom of the tube to promote DNA rehydration. 3.2.7.2: DNA Amplification: The results of amplification was performed on the DNA extracted from all the studied isolates was

confirmed by electrophoresis analysis. By this analysis, the

strands of DNA resulted from the successful binding between specific primers and isolates extracted DNA. These successful bindings appeared as single bands under the U.V light using ethidium bromide as a specific DNA stain. The electrophoresis was also used to estimate DNA M.W. depending on DNA marker 2000 bp DNA ladder).

3.2.7. 3. Preparation of PCR master mix for Single plex PCR Detection 16s r RNA Gene And invA Gene : Polymerase chain reaction master mix reaction was prepared by using (Accu Power PCR PreMix Kit) and this master mix done according to company instructions as showing in Table:(9)

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Chapter three


Table (9): Components of PCR master mix reaction

PCR Master mix reaction components

PCR PreMix* (Lyophilized)

Volume / µL

5 5

DNA template

F. primer

1.5

R. primer

1.5

Primers

PCR water

7

Total volume

20

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Chapter three


3.2.7.4. PCR Thermo cycler Conditions: PCR thermo cycler conditions were done by using conventional PCR thermo cycler as following table: Table( 10) PCR Thermo cycler Conditions:

PCR cycle

Temp.

repeat

Initial

ᴄ˚ 1

denaturation

Denaturation

30

Annealing

Extension

Final extension

Hold

1

-

[44]

Time

95

5min

95

5sec.

55

30sec

72

45sec

72

7min

4

Forever

Chapter three


3.2.7.5. Gel electrophoresis PCR products of 16s rRNA gene and invA was analyzed by loading in 1.5% Agarose as following steps: 1- 1.5% Agarose gel was prepared in using 1X TBE and dissolving in water bath at 100 °C for 15 minutes, after that, left to cool 50°C. 2- Then 3µ of ethidium bromide stain were added into agarose gel solution. 3- Agarose gel solution was poured in tray after fixed the comb in proper position after that, left to solidified for 15 minutes at room temperature, then the comb was removed gently from the tray and 10µl of PCR product were added to each comb well and 5ul of (2000 bp Ladder) in one well. 4- The gel tray was fixed in electrophoresis chamber and fill by 1X TBE buffer. Then electric current was performed at 80 volt and 60 AM for 1.5 hour. 5- PCR products (406 bp) as specific for 16 r RNAs gene and PCR products (558 bp) as specific for invA gene were visualized by using gel documentary system..

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3.3 : Statistical Analysis :the statistical analysis of the data performed by f test analysis (One-Way ANOVA )then the least significant differences (LSD) were conducted to find significant differences between the percentage of isolation .some data were analyzed using the statistically analysis system (SAS) and the significant differences were determined at (p< 0.05) (Steel and Torrie 1960) .

[46]