Brain-heart infusion broth. Himedia /India. Brilliant green agar (BGA). Himedia/India. Simmon's citrate agar. Himedia/ India bismuth sulphate agar himedia/ India.
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
Materials and Methods
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
[26]
Chapter three
Materials and Methods
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
[27]
Chapter three
Materials and Methods
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.
[28]
Chapter three
Materials and Methods
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
[29]
Chapter three
Materials and Methods
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
Materials and Methods
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
[31]
Chapter three
Materials and Methods
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.
[32]
Chapter three
Materials and Methods
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
Materials and Methods
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
Materials and Methods
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) .
[35]
Chapter three
Materials and Methods
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
Materials and Methods
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).
[37]
Chapter three
Materials and Methods
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.
[38]
Chapter three
Materials and Methods
: 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.
[39]
Chapter three
Materials and Methods
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: beta- galactosidase ;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.
[40]
Chapter three
Materials and Methods
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
Materials and Methods
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)
[42]
Chapter three
Materials and Methods
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
[43]
Chapter three
Materials and Methods
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
Materials and Methods
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..
[45]
Chapter three
Materials and Methods
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]