Protocol for inoculating bottled mineral water with bacteria to receive artificial contaminated water samples

Summary Here, we present a protocol for inoculating drinking water samples with a variety of pathogens or facultative pathogen bacteria. We describe steps for preparing bacterial solutions, inoculating mineral water bottles and other drinking water samples, filtration and incubation of the agar plates, and counting colony-forming unit per mL. We also detail procedures for determining selected chemical properties, such as anions and cations, which can also affect the bacterial growth. For complete details on the use and execution of this protocol, please refer to Schalli et al.1


SUMMARY
Here, we present a protocol for inoculating drinking water samples with a variety of pathogens or facultative pathogen bacteria.We describe steps for preparing bacterial solutions, inoculating mineral water bottles and other drinking water samples, filtration and incubation of the agar plates, and counting colony-forming unit per mL.We also detail procedures for determining selected chemical properties, such as anions and cations, which can also affect the bacterial growth.For complete details on the use and execution of this protocol, please refer to Schalli et al. 1

BEFORE YOU BEGIN
The protocol below describes a method of inoculating drinking water samples with a variety of pathogens or facultative pathogen bacteria.We describe steps for preparing bacterial solutions, inoculating mineral water bottles and other drinking water samples, filtration and incubation of the agar plates, and counting CFU/mL.We also detail procedures for determining selected chemical properties, such as anions and cations, which can also affect the bacterial growth.

Institutional permissions
All experimental procedures require compliance to the safety guidelines of the institution or laboratory.

Preparation one: Contaminant solution and incubators
Timing: 30-60 min 1.First, prepare the contamination solution to inoculate the water samples.
a. Autoclave a sterile glass bottle containing 1000 mL of distilled water at 121 C for 20 min for each strain.b.Dilute one ceramic bead in a bottle of distilled water (1000 mL): c.Gently mix the solution by inverting the bottle.d.After 10 min the contaminate solution is ready.e.To determine the CFU/mL in the contaminated starting solution, prepare a serial dilution and plate it on tryptic soy agar.Afterwards count your plates and note the CFU/mL you started with.2. For the incubation step of the artificial contaminated water at the period of 31 days set the incubator to 25 C 3. The incubators should be set to 37 C to provide an optimal growth rate for counting the CFU/mL of the strains.
Note: This protocol requires bacterial strains to inoculate the water samples.The strains we used were obtained from commercial sources (E. coli DSM 1103 and P. aeruginosa DSM 50071, Leibnitz-Institute, DSMZ-German Collection of Microorganisms und Cell Cultures GmbH, Braunschweig, Germany), which were shipped in glass vials and contain freeze-dried material on ceramic beads.More important, we started the protocol using the beads covered in a cryopreservative solution with around 10 6 CFU/mL.The CFU/mL of the beads covered in the cryopreservative solution can be found in the datasheet of the respective strains that are listed in the table REAGENT or RESOURCE.
Preparation two: Equipment for the filtration Timing: 10-15 min 4.For this protocol, take a cylindrical funnel with a volume of 100 mL and filtrate an amount by 10 mL-100 mL of the artificial contaminated water samples.2,3 5. To verify Enterobacteriaceae filtrate the 100 mL of the artificial contaminated water and put the filter on Endo agar plates.
Note: For the filtration of the water samples through a porous membrane, filtration equipment is necessary.
CRITICAL: The handling of the filtration equipment can adversely affect the efficiency of this technique.Furthermore, the right handling avoids cross contaminations to receive reliable results.

Note:
The membrane disc must have the correct pore size of 0.45 mm to ensure the right strains are filtered. 3te: If you store the plates at 4 C in the fridge, be sure to preheat to 25 C before it's usage.
Preparation three: Eluent for ion chromatography and reagents for photometry Timing: 45 min 6.To prepare the anion eluent add 42.4 g Na 2 CO 3 and 4.2 g NaHCO 3 to 500 mL deionized water to reach an eluent concentrate of 800 mM Na 2 CO 3 and 100 mM NaHCO 3 .a. Freshly prepare the eluent using 20 mL of the solution prepared in 1. a. and dilute in 1980 mL deionized water.

Note:
We use the stock solution to prepare the eluent for the ion chromatography ICS 1100.

Note:
The photometric determination of the ammonium values in the samples requires two reagents. 4 Add 100 mL Orthophosphoric acid to 500 mL deionized water, to prepare the reagent to measure the nitrite.5 a. Add 40 g sulfanilamide to the orthophosphoric acid solution and mix it well.b. Ctinue to get the reagent by adding 2 g naphthylethylenediamine dihydrochloride.
Note: We use the Griess test to measure the nitrite ions in the water samples.

MATERIALS AND EQUIPMENT
Note: The eluent concentrate can be stored for 2 months at 25 C. Further chemicals that we used in this protocol, e.g., acidifying samples, are listed in the key resources table.
Note: If you keep reagent 1 in the fridge at 4 C, the solution can be stored for 2 weeks.Contrariwise, freshly prepare reagent 2 every day.

Note:
The reagent for Griess test is stable for 1 month, since it's stored in the fridge at 4 C.If you want to store the solution up to one year, keep the reagent at À25 C.

STEP-BY-STEP METHOD DETAILS
Step one: Analyses on the chemical properties Follow this procedure to determine the chemical properties of mineral water ( Note: We use the Metrohm Tritator connected to the sample changer 730 to move the samples.For this reason, we measured the pH and conductivity values of the samples consecutively and the raw data was processed using the Tiamo software. 3. Add 50 mL sulfuric acid (2M) with additional 80 g/L sodium peroxidsulfate to the reaction tube and put the sample into the TOC analyzer. 8Troubleshooting 1.
Note: You have to ensure that the argon gas flow is set between 130 and 150 mL/min.4. To assay the dissolved metal ions, take 100 mL of the water samples, filter it through a 0.45 mm membrane and add 1 mL of HNO 3 conc.to acidify the sample for the determination of Fe, Mn, Ca, Mg, Na and K. To determine the anions (Nitrate, chloride and sulfate) using the ion chromatography run the correct program on the Dionex ICS-1100. 10Before you start with the samples rinse the system with the eluent solution to receive a linear baseline.Troubleshooting 2.

Note:
We measured the ammonium and nitrite ions using a spectrophotometer. 4Take a reaction tube and fill in 5 mL of the water sample to measure the ammonium ions.a. Add 0.4 mL of the previously prepared reagent 1 to the water sample and mix it well.b.Additionally, add 0.4 mL of freshly prepared reagent 2 and mix the reaction tube.The pH of the samples should now reach 12.6.The values in the units [mg/L] for the chemical components and the hardness is calculated in German hardness degree [ dH].
c. Measure the extinction at a wavelength of 665 nm to get the ammonium values.7. To measure the value of the nitrite ions add one drop of the Griess reagent to the samples (5 mL). 5 a. Wait for 20 min until the reaction is finished.Step two: Inoculation of the water samples

Timing: 30-45 min
To prepare the contaminated water samples, following steps are mandatory.
8. Set up 3 water bottles for each condition and pathogen, e.g., Figure 1.a. Open a fresh water bottle or your sample and inoculate with 1 mL of the contaminant solution, you have prepared previously.b.Immediately close the bottles again and then gently mix your water samples by orbital movements of the bottle.Troubleshooting 3. 9. Put the artificial contaminated water samples to incubation at RT for 1-31 days (dark place).
CRITICAL: Due to the pathogenicity of the used strains (key resources table), please be careful during the preparation procedure.
Note: Once you have inoculated all your samples, prepare non-artificial contaminated samples to determine your background-flora.
Optional: If you want to do any investigations on the background-flora in artificial contaminated water, prepare the samples for the same period.
Step three: Filtration and cultivation over a period of 31 days Timing: 1 h After incubating the water bottles for 1 day (24 h), 2 days, 7 days, 2 weeks, 3 weeks and 31 days proceed with the filtration to get the CFU/mL over this period. 3,11,12After Filtration (Figure 2) and incubation of the agar plates count all the colonies on the plates to calculate the CFU/mL.10.First, make sure that the equipment, which you have prepared before, is ready to use and open the bottle at the respective day (e.g., Figure 3).a. Place the filter on the cylindrical funnel and check that the funnel top is not leaking out, otherwise you will lose the feed or get no vacuum to filtrate sufficient (Figure 3).b.Fill in 100 mL of the inoculated water samples and filter it through the cellulose ester filter using vacuum (Figure 4). 2,311.Place the filter on an Endo Agar plate for cultivating the pathogens.Troubleshooting 4.
a. Incubate the plates for 24 h at 37 C. b.Afterwards count the colonies (e.g., Figure 5) and calculate the CFU/mL for both strains.
Optional: If you work under different conditions or if you want to test new strains, vary your collection time points to gain significant results.
Note: Further down the period do duplicates using 10 mL (1:10) of the samples as higher colony counts may occur.The purpose of this step is to confirm the respected colonies of the artificial contaminated water samples. 3,11. Select different single colonies on the plate for the identification and transfer them on TSA agar to get pure cultures.a. First, confirm your respected strains checking the morphological pattern.b.Take an inoculation loop to test whether your single colony is oxidase positive or negative.c.Pick the same colonies from pure cultures and place them on the well of the MS-chip.
Note: For the determination of the respected strains, MALDI TOF analyses can be performed.

EXPECTED OUTCOMES
After the bacterial strains adapted to the minimal nutrient conditions of the bottled water they started growing in mineral water.At day one, we found a reduced CFU/mL in sparkling mineral water (E.coli).Moreover, E. coli showed a decreased lifespan in mineral water without CO 2 due to the fact of low nutrition in the water.In contrast, the CFU/mL for P. aeruginosa in mineral water without CO 2 escalates after an incubation of 31 days.However, the low pH values due to the carbonation of the water and the damaging effects of CO 2 lead to decreased CFU/mL (Table 2) of both strains in sparkling water.

LIMITATIONS
Our method of artificially contaminated water is not investigating the interaction of different pathogens in a consortium.Therefore, the CFU of the pathogenic strains or any other bacterial strains in the contaminant solution has to be adjusted.Our protocol determines the CFU/mL of the single pathogen with no data of interaction or biofilm formation.

TROUBLESHOOTING Problem 1
If the sample is containing high amounts of chloride ions (R3 g/L) or a carbonate hardness R30 dH (German Hardness degree), see the solution below.

Problem 2
Cross-sensitivities or insufficient resolutions are rarely observed, but can occur at large concentration differences between the anions in step 5.

Potential solution
Proceed with the instruction of EN ISO 10304-1:2009.

Problem 3
If the water sample touches the screw cap it can consequently lead to cross-contaminations, especially in Step 8.b.

Potential solution
Mix the inoculated water carefully to avoid any contaminations.

Problem 4
Transferring the membrane to the agar plate is fussy, referring to step 11.The transfer membrane disc should have no cracks or air inclusions among.Moreover, any of those defects will lead to loss of CFU and consequently a wrong outcome (Figure 6).

Potential solution
It is important to take forceps with no bended tips or any damaged parts.Since the forceps are broken, you will easily damage the membrane.
b. Set the wavelength to 540 nm and measure the extinction.Note: The calculation of the values was done after the procedure mentioned in O ¨NORM ISO 7150-1:1987 12 01 (DIN 38406-5:1983 (DEV-E5))

Figure 1 .
Figure 1.Inoculating mineral water bottles with and without carbon dioxide An example of commercial sparkling mineral water bottles (green capped) and ones without carbonization (red cap) that we used in this investigation.

Figure 2 .Figure 3 .
Figure 2. Make sure, that the sterilized filtration equipment is ready

Figure 4 .Figure 5 .
Figure 4. Fill the respective amount of sample into the prepared apparatus

Figure 6 .
Figure 6.Avoid air inclusions and folding in the membrane In the case of air inclusions, you may lose CFU, since the nutrition of the agar cannot diffuse through the membrane.

TABLE REAGENT
(Continued on next page)

Table 1
7or analyzing the pH fill a 150 mL beaker with 100 mL of the respective water sample and run the program to measure the pH value at RT.62.To get the conductivity score use the same beaker filled with 100 mL of the water sample and do the measurements with the conductometer.7 ) without carbon dioxide and with additional CO 2 .
For mineral water samples, please prepare a 1 to 10 dilution and set it in the schedule.Put the vial to the sample holder and run the program.
9 a.Put the samples to the ICAP 7000 sampler module and start the program.b.Calculate the hardness by the values of Calcium and Magnesium.DIN 38409:1986:01 DEV H6.Note: We use the samples changer ASX-280 for the ICP-OES to analyze the water samples in serial, applying the Qtegra software version 2.8.2944.202 to calculate the results and values for each ion.5.

Table 1 .
The chemical properties of mineral water with and without CO 2 Chemical properties of bottled water without additional CO 2

Table 2 .
Mean values for CFU/mL of E. coli and P. aeruginosa in mineral water with and without carbonization