Standard bacterial strain of C.sakazakii (MTCC 2958) was procured from MTCC (microbial type culture collection) Chandigarh, India. 3.1 CREATING ASEPTIC ENVIRONMENT FOR THE BACTERIA All glasswares were washed carefully with lab soap solution to create aseptic environment for the bacteria. After washing glasswares were packed with the help of cotton, alumunium foil, paper and thread. Packed glasswares were than autoclaved at a pressure of 15 atm and a temperature of 121 C for 15-20 minutes. Every transfer was done inside the laminar hood chamber wiped with spirit for sterilization. 3.2 REVIVING THE STANDARD CULTURE OF Cronobacter sakazakii (MTCC 2958) Standard strain was revived in 3 ml of EE hi veg mossel broth and incubated at 37 C for 24 hrs. EE broth was sterilized by steaming for 20-25 minutes and then incubating it at 37 C for 24 hrs. 3.2.1 Composition of EE broth EE broth 43.5g 1000ml 3.3 COLLECTION OF SAMPLES Sampling of camel milk was done from different locations of Bikaner. Raw camel milk was collected in sterilized vials directly from the udder of the animal. Collected milk was marked and sealed properly and kept under refrigerated condition and used within 24 hrs. 3.4 ISOLATION AND IDENTIFICATION ON VRBGA (VIOLET RED BILE GLUCOSE AGAR) A total of 16 samples of camel milk from 16 animals were used. 0.5 ml of raw camel milk from each sample was added in 4.5 ml of EE broth and allowed to incubate at 37 C for 24 hrs. After 24 hrs, inoculated EE broth was streaked on VRBGA (Violet Red Bile Glucose Agar) plate and incubated overnight at 37 C to check the presence of C.sakazakii in raw 17
camel milk. Presence of pink mucoid colonies in few samples of raw milk confirms the preliminary presence of C.sakazakii (plate 12). Two colonies from each sample in which they appeared were picked with the help of loop and incubated in EE broth for 24 hrs at 37 C and presence of C.sakazakii was confirmed by performing various biochemical tests and PCR. 3.4.1 Composition of VRBGA VRBGA dehydrated 40.62g 1000ml Agar 22g VRBGA was sterlized by boiling it for 15 to 20 minutes and sterility checked by incubating it at 37 C for overnight. 3.5 BIOCHEMICAL CHARACTERIZATION 3.5.1 METHYL RED TEST Principle Bacteria are able to ferment glucose to produce sufficient amount of acidity which gives red color with the help of methyl red indicator. Reagents used Glucose phosphate broth MR-VP medium 17gm 1000ml This was autoclaved at 15 atm, 121 C for 15 20 minutes. Methyl red indicator Methyl red 0.1gm 95% alcohol 300ml 200ml 18
Procedure In 1 ml of sterile glucose phosphate broth, a colony of test organism was inoculated. It was incubated overnight at 37 C. After incubation few drops of methyl red indicator were added and color change was observed.(plate 1) Interpretation Red color Positive test Yellow or orange color Negative test 3.5.2 VOGES PROSKAUER (VP) TEST Principle The test is based on the ability of certain organism to produce acetoin when cultured in glucose broth. In presence of KOH and on exposure to air the acetoin produce from fermentation of glucose is oxidized to diacetyl. This reaction is catalyzed by α naphthol. Diacetyl reacts with the guanidine group associated with molecules contributed by peptone in the medium, to form pinkish-red-colored product. Reagents used Glucose phosphate broth MR-VP medium 17gm 1000ml This was autoclaved at 15 atm, 121 C for 15 20 minutes. Naphthol solution α naphthol Absolute ethylate 5 gm 100 ml 19
Potassium hydroxide (40 %) KOH 40 gm 100 ml Procedure In 1 ml of sterile glucose phosphate broth, a colony of test organism was inoculated. It was incubated overnight at 37 C. After incubation 0.6 ml α naphthol and 0.2 ml KOH was added and vortexed after removing the cotton plug. The test was left for 1 hour at room temperature. The development of red pink color awaited. (plate 2) Interpretation Pink red color- Positive test No pink red color- Negative test 3.5.3 INDOLE TEST Principle Certain bacteria have ability to split amino acid tryptophan into indole and pyruvic acid. Indole can be detected with Kovac s reagent, which reacts with indole and produce a dark pink colored compound. Reagents used Tryptone broth Tryptone 10 gm 1000 ml This was autoclaved at 15 atm, 121 C for 15 20 minutes. 20
Kovac s reagent Para-dimethylaminobanzaldehyde 5 gm Isoamyl alcohol 75 ml HCl was added till pale yellow color develops. Procedure In 1 ml of sterile Tryptone broth, a colony of test organism was inoculated. It was incubated overnight at 37 C. After incubation few drops of Kovac s reagent were added. The development of pink color ring at the surface layer of broth within 10 minutes was awaited. (plate 4) Interpretation Pink surface layer Positive test No Pink surface layer Negative test 3.5.4 NITRATE TEST Principle Certain bacteria have the ability to convert nitrate into nitrite. Sulphanilic acid reagent is used to detect presence of nitrite. If nitrite is present then reagent is diazotized and forms dark red compound with α-naphthylamine. Reagents used Nitrate broth Potassium nitrate Beef extract Peptone NaCl 1gm 10 gm 10gm 05gm This was autoclaved at 15 atm, 121 C for 15 20 minutes. 21
Sulphanilic acid reagent 0.8%Sulphanilic acid in 5N acetic acid Acetic acid 0.04gm 5ml α-naphthylamine reagent 0.5%Sulphanilic acid in 5N acetic acid Acetic acid 0.025gm 5ml Procedure In 1 ml of sterile nitrate broth, a colony of test organism was inoculated. It was incubated overnight at 37 C. Both the reagents were mixed in 1:1 ratio. And a few drops were added in the tubes. The color change was observed (plate 3). Interpretation Red color Positive test No red color Negative test 3.5.5 CATALASE TEST Principle Catalase is an enzyme, which converts hydrogen peroxide into water and oxygen. Catalase producing organism when brought into contact with hydrogen peroxide produce oxygen bubbles. 22
Reagents used Hydrogen peroxide Procedure Using a sterile loop the test cells was immersed in 2-3ml of hydrogen peroxide. Immediate formation of bubbles was looked for. (plate 7) Interpretation Formation of bubbles Positive test No formation of bubbles Negative test 3.5.6 OXIDASE TEST Principle The oxidase test identifies organisms that produce the enzyme cytochrome oxidase. If the organism is capable of producing oxidase it can be oxidized by phenylenediamine to produce deep purple color. Reagents used N,N, N, N,-tetramethyl-pphenylenediamine 0.1gm 10 ml Procedure Autoclaved whatman filter paper was soaked with 2-3 drops of freshly prepared Oxidase reagent. Colony of test organism was placed on filterpaper by taking a swab from the streaked TSA plates using a sterile toothpick. Development of blue color was checked within a few seconds.(plate8) 23
Interpretation Blue color Positive test No blue color Negative test 3.5.7 TRIPLE SUGAR IRON (TSI) AGAR TEST Principle This test is used to differentiate Enterobacteria by performing multiple tests on a single medium. Acid from sugars (glucose, lactose and sucrose) and H 2 S production are observed. Reagents used Peptone Yeast extracts Beef extract Lactose Sucrose Dextrose NaCl Ferric citrate Sodium thiosulphate Phenol red Agar 20gm 3gm 3gm 10gm 10gm 1gm 5gm 0.3gm 0.3gm 0.024gm 15gm 1000ml This was autoclaved at 15 atm, 121 C for 15 20 minutes, sugars were added and poured in test tubes and steamed for 10 to 15 minutes. Procedure Slants of TSI agar were prepared in tubes. With the help of sterile loop the slant was streaked and then butt was stabbed. This was incubated at 37 C for 24-48 hrs.(plate 6) 24
Interpretation Reaction Slant Butt Interpretation Yellow color Yellow color + + Acid from lactose/ sucrose - - Acid from glucose Red color/no color change + + No acid from any sugar Blackening Anywhere Anywhere Hydrogen sulphide production Gas bubbles/ Anywhere Anywhere Gas from sugars lifting of medium 3.5.8 Citrate utilization test Principle When the cell culture is grown in a medium containing sodium citrate and ammonium salt and bromophenol blue indicator, the color changes from green to blue if the growth of the organism causes alkalinity in broth. Reagents used Simmon s citrate agar dehydrated 22.28 gm Agar 22gm 1000ml This was boiled for 10 minutes, poured in test tubes and autoclaved at 15 atm, 121 C for 15 20 minutes. 25
Procedure Slants of Simmon s citrate agar were prepared in test tubes. With the help of sterile loop the slant was streaked with the test organism. This was incubated at 37 C for 24-48 hrs and the change was observed.(plate 5) Interpretation Bright blue color Positive test No color change Negative 3.6 MOIECULAR ANALYSIS OF THE ISOLATES BY PCR 3.6.1 Preparation of DNA template Extraction of the template DNA was done as per the method of Nair and Venkitanarayanan, (2006). Pure culture of the isolates and the standard were grown overnight in their respective broth. 1 ml of 24 hour pure culture of test organism was taken in an eppendorf tube and boiled at 100 C for 10 minutes, and centrifuged at 1500 g for 30 seconds. Supernatant obtained was used as DNA template and mixed with Master Mix. 3.6.2 Preparation of Master Mix Ready to use DNA amplification kit (Fermantas) was used to prepare the Master Mix. All the components were mixed serially as given in the table below, in a fixed ratio except the DNA template. 26
3.6.3 Calculation for the preparation of Master Mix SERI AL NO. CHEMICALS FINAL CONCENTRATION 1. 10 X PCR buffer 1X 5 2. 25mM MgCl 2 2mM 4 3. 10mM dntp mix 0.2mM 1 4. 100 µm primer 1µM 0.5 ESSF 5. 100 µm primer 1µM 0.5 ESSR 6. Taq DNA 2.5U 0.833 polymerase(3u/ml) 7. Template 5 VOLUME FOR 1 SAMPLE (in µl) 8. DNase and RNase free water 33.167 The total PCR Master Mix required for the reaction was prepared in a single tube to minimize pipetting error. To 45 µl of PCR mixture 5 µl of template DNA was added. The whole procedure was carried out at a constant temperature of 4ºC. The prepared mixture was then subjected to amplification by using the thermal cycler maintaining the cycling conditions as given in the table below 3.6.4 PCR cycle SERIAL NO. STEP TEMPERATURE TIME (MIN) 1. Initial incubation 94ºC 2 2. Denaturation 94ºC 0.15 3. Annealing 60ºC 0.15 4. Extension 72ºC 0.30 5. Final extension 72ºC 5 No. of cycles = 30 cycles Storage - At 4ºC till removal. 27
3.7 AGAROSE GEL ELECTROPHORESIS 3.7.1 Preparation of Agarose gel Agarose gel electrophoresis efficiently separates and analyzes the DNA. The purpose of the gel might be to look at the DNA, to quantify it or to isolate a particular band. Bands were visualized in the gel by the addition of ethidium bromide. Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log 10 of their molecular weight. Several actors have important effects on the mobility of DNA fragments in agarose gels, and can be used to optimize separation of DNA fragments. Chief among these factors are agarose concentration, voltage, electrophoresis buffer and effects of ethidium bromide as it retards the migration by causing a decrease in the negative charge of DNA. 3.7.2 Apparatus The apparatus consists of a horizontal submarine unit having reservoirs at the two ends, which serve as the buffer tank. The gel tray sealed with cello tape at both ends containing agarose is placed in between the two reservoirs, which are then connected to the respective electrodes and power was applied. 3.7.3 Preparation of agarose gel 30 ml agarose suspension (1.5%) was prepared by mixing molecular biology grade agarose (SRL 0140M) in 0.5X TBE buffer and heated in microwave oven for 30 seconds, then swirled to dissolve the agarose. It was heated again for 30 seconds in the microwave and the process was repeated till the agarose dissolved completely. 5X tris borate EDTA (TBE) buffer (stock solution) Tris base 54g Boric acid 27.5 0.5M EDTA (ph 8.0) 20ml 980ml 28
0.5X working solution of electrophoresis buffer was prepared by diluting 5X stock solution in the ratio 1:10. 0.5M EDTA ph 8.0 186.1 g of dehydrated EDTA was mixed in 800 ml of distilled water and stirred vigorously on a magnetic stirrer. Disodium salt of EDTA will not go into solution until the ph of the solution is adjusted to 8.0 by adding NaOH, if required. EDTA 186.1g Sodium hydroxide 20g 800ml Molten agarose was cooled to 50ºC and to it 1.5µl of ethidium bromide was added to make the final concentration of.5µg/ml from the prepared stock of 10 mg/ml, and it was mixed slowly. Ethidium bromide (stock solution) EtBr 1g 100ml The gel tray was placed on a horizontal, even surface, sealed with adhesive tape and molten agarose was poured in it.the comb was placed with its teeth 1mm above the base. The gel tray was left undisturbed for an hour to allow the gel polymerization. After which the comb was taken out and the adhesive tape removed. The gel tray was then placed in the electrophoresis tank (biotech, 003017) with the wells towards the cathode. Electrophoresis buffer (TBE 0.5X) was filled in both the reservoirs to immerse the gel. 3.7.4 Sample Preparation and loading The PCR product (8µl) was mixed with 2µl of 6X bromophenol blue loading dye (provided with the amplification kit) to increase the density of the samples and loaded into well along with the DNA marker ladder in the adjoining lane. The electrophoresis tank was covered with a lid and 29
connected to the power supply unit (Alliance ENPS 300). Electrophoresis was carried out at 5 volts/cm until the dye reaches two thirds of the gel (an hour and half). 6X loading dye/buffer Glycerol 3000µl 10% bromophenol blue 25 µl 675 µl The solution was stored at 4ºC. DNA ladder (100 kb) Ladder.8µl 6X loading dye 3µl DNase and RNase free water 12µl 3.7.5 Gel documentation The resolution of the bands on the gel was visualized by a UV transilluminator. 3.8 CONFIRMATION OF THE SELECTED ISOLATES 3.8.1 UREASE TEST Principle: The test organism is cultured in a medium, which contains urea and the indicator phenol red. When the strain is urease producing, the enzyme will break down the urea to librate ammonia and carbon dioxide. With the release of ammonia, the medium becomes alkaline as shown by a change in color of broth. 30
Reagents used Special peptone D (+) glucose NaCl Potassium di-hydrogen phosphate Phenol red 1gm 1gm 5gm 2gm.012gm 1000ml These is Autoclaved and after cooling to 45-50ºC 100 ml of 20% aqueous solution of filter sterilized urea was added. Procedure: The test organism was heavily inoculated in a test tube containing 1ml sterile urea broth. It is Incubated at 35-37ºC for 3-12 hours. A pink color in the medium is looked for. Interpretation: Pink color Positive urease test No pink color Negative urease test 3.9 STUDY OF GROWTH CONTROL OF THE SELECTED ISOLATES 3.9.1 Checking antibiotic susceptibility Antibiotic susceptibilities were determined by using disk diffusion method. Susceptibility of presumptive Enterobacter cloacae after reidentification was done. Susceptibility was checked against the following antibiotics: gentamicin, streptomycin, chloramphenicol, spectinomycin, ciprofloxacin and sulphadiazine. 31
Procedure MHA plates were prepared and 100 µl of 24 hour grown culture was spread on MHA agar and antibiotic disks were placed on it. The zone of inhibition was measured after incubation of 24 hours at 37 C. 3.9.2 Checking antimicrobial activity of plant products Plant extracts of Amla (Emblica officinalis), Clove (Syzygium aromaticum), Triphala and Harad were procured from the lab. Procedure The screening of the alcoholic extracts of the plant products for antibacterial activity was performed using well diffusion method. MHA was prepared and 100µl of test culture was inoculated. Wells of 6mm in diameter were punched in the agar and filled with 40µl (Such as 2 10 6 µg/ml) of the extract. After holding the plates at room temperature for 2 hours to allow diffusion of the extract into the agar, the plates were incubated at 37 C for 24 hours. After incubation the zone of inhibition was measured. 3.9.3 Checking antibacterial activity of lactic acid bacteria Antibacterial activity of lactic acid bacteria was studied by using well diffusion method. MHA was prepared and 100µl of presumptive Enterobacter cloacae was inoculated. When media was solidified wells were made and in each well 40 µl of lactic acid bacteria culture was added. Inhibition zones were measured after 24 hours of incubation at 37ºC. 32