Protocols: Laboratory Processing

Laboratory Processing Protocols

This document consists of the protocols for laboratory processing of the various sample types collected as a part of the SaniPath Exposure Assessment Tool. We recommend that sample collection occur in the mornings so that samples can be transported to the laboratory by lunch time to allow for adequate time for the laboratory to process and analyze the samples. Due to the time and resource constraints of conducting a rapid assessment we do not recommend processing replicates of samples.

Processing of drinking water or bathing water samples

Drinking water samples should have very low levels of contamination but may have some contamination depending on the area. No special handling should be required to prepare samples for microbial assays. We recommend testing 100 ml and 10 ml.

Processing of non-drinking water samples from oceans, lakes, rivers, and other surface waters

Non-drinking water samples from oceans, lakes, rivers and other surface waters could have moderate to high levels of contamination. We recommend testing 10 ml and 1 ml volumes. Microbial analyses of 100 ml sample volumes will likely result in assays that are too numerous to count (TNTC) and are not recommended.

Processing of non-drinking water samples from flood areas or open drains

Non-drinking water samples from drains and flood areas are likely to have high levels of contamination. We recommend preparing serial (i.e. back-to-back or consecutive) ten -fold dilutions for microbial analysis. Refer to the section on Dilution Protocols on Page 7. By testing 1 ml volumes of the diluted samples, you can determine concentrations of fecal bacteria up to 2.0 x 10⁹ (membrane filtration) or 2.4 x 10¹⁰ (Colilert and Quantitray 2000) fecal bacteria per 100 ml. Microbial analyses of undiluted samples will likely result in assays that are too numerous to count (TNTC) and are not recommended.

Processing of raw produce samples

We measure contamination on produce by rinsing whole pieces of produce and testing the rinse solution for E. coli. Produce samples tend to have a wide range of contamination levels. We recommend testing 10 ml and 1 ml volumes of the undiluted, rinse solution, as well as 1 ml of a 1:10 dilution. This will allow you to accurately measure a wide range of contamination levels. See the Dilution Protocols on page 7. Follow the directions below to prepare the produce rinses.

Materials and Equipment:

● Sterile (autoclaved or sterile filtered) water or PBS

● PBST (1L phosphate buffered saline, pH 7.2 with 0.05mL Tween-80)

● 15mL conical tube (for dilution)

● Sterile graduated cylinder that can measure 500 mls

● Gloves

● 70% ethanol

● Felt tip pen

● Produce sample in bag

● 37°C incubator

● 4°C refrigerator

● Produce Laboratory Form

Procedure:

1. Put on gloves and spray hands with 70% ethanol. Rub hands together to sanitize all surfaces of the gloves.

2. Check that the sample ID on the Produce Laboratory Form and the sample are the same and enter the sample ID into the form. Record the date and time of sample processing on the Produce Laboratory Form.

3. Prepare your work surface by cleaning it with 70% ethanol.

4. Samples should arrive at the lab in a large Whirl-Pak bag. Spray the outside of the bag with 70% ethanol and rub it well. Inspect the bag to determine whether liquid can be added without overflowing. The bag should be no more than 2/3rds full with produce. You should be able to add 500 mL of PBST and still close the bag without it overflowing. If you suspect that the bag is too full, open the bag by untwisting the ties and pulling them gently outwards until the mouth of the bag opens. Remove the extra produce items one at a time by pressing upward underneath them on the outside of the bag, and move them to the top, leaving the bag about 2/3rds full of produce. Never stick your hands into the bag. Discard the removed produce. Only record the produce remaining in the bag on the Data Recording Form.

5. Add 500 mL of PBST to the bag. Seal the bag, trapping minimal amounts of air inside. Incubate for 10 minutes at 37°C.

6. Vigorously shake the bag with the produce for 30 seconds. Next gently massage the surface of each piece of produce item through the bag for 60 seconds. For delicate items like lettuce or onions, try to rub at least the outer leaves. Try not to break open any items. Shake the bag again for 30 seconds.

7. To remove the produce, open the Whirl-Pak bag, gently press upwards underneath the item and move it to the top. Take care not to lose any water or smash any produce. Remove the produce at the top of the bag and set aside

8. Close the Whirl-Pak bag.

9. Weigh the produce (using aluminum foil) and record the weight on a Produce Laboratory Form.

10. Store all samples at 4°C until they are ready for processing.

Processing of street food samples

We measure contamination on street food according to the protocol for food processing from the WASH Benefits Study—Bangladesh, World Bank Add-on (refer to LabSOP_20August.doc). Street food is processed by homogenizing the street food and testing the solution for E. coli. Street food samples may have a wide range of contamination levels. Our preliminary recommendation for dilutions is to measure 10 ml and 1 ml volumes of the homogenized solution, as well as 1 ml of a 1:10 dilution. This will allow you to accurately measure a wide range of contamination levels. See the Dilution Protocols on page 7. Follow the directions below to prepare the produce rinses.

Materials and Equipment:

● BagMixer bag

● Sterile (autoclaved or sterile filtered) water or PBST (phosphate buffered saline, pH 7.2 with 0.04% Tween-80)

● Gloves

● 70% ethanol

● Felt tip pen

● Street food sample in bag

● 37°C incubator

● 4°C refrigerator

● Street food Laboratory Form

Procedure:

1. Shake the food sample to mix it well. Weigh the entire street food sample and record the weight on a lab processing form.

2. On aluminum foil, weigh out 10 g of food (acceptable range: 9.50 to 10.50 g).

3. Add 10 g of food into a sterile BagMixer bag.

4. Using a sterile graduated cylinder, add 100 mL of distilled water into BagMixer bag.

5. Place bag into the BagMixer and mix for 1 minute at speed 4 with gap at -3 mm.

6. Using a sterile disposable pipette, immediately remove homogenized solution from the bottom of the bag (do not allow particles to settle) and add to dilutions outlined in the Dilution Protocols table.

Processing of swab samples

Swab samples have a wide range of contamination levels. It is not uncommon for many swab samples to have no detectable contamination and a few to have very high contamination. This protocol will elute bacteria from one swabs used on latrine surfaces. We recommend testing 10 ml and 1 ml volumes of the undiluted swab elute, as well as 1 ml of a 1:10 dilution. Please note the 1mL volume of undiluted swab elute is optional. This will allow you to accurately capture a wide range of potential contamination levels. See the Dilution Protocol on page 7. The resulting bacterial counts will represent the total bacteria per object or surface area.

Materials and Equipment:

● PBST (phosphate buffered saline, pH 7.2 with 0.04% Tween-80)

● One 15 ml OR 50mL sterile conical tube

● Felt tip Pen

● Gloves

● 70% ethanol

● 10 ml serological pipettes (or other disposable pipet with 10ml capacity)

● Vortex

● 4°C refrigerator

● Pipet Aid

● Swab Laboratory Form

Procedure:

1. Put on gloves and spray hands with 70% ethanol. Rub hands together to sanitize all surfaces of the gloves.

2. Check that the sample ID on the swabs and the sample ID on the Swab Laboratory Forms are the same. Record the sample processing date and time.

3. Prepare your work surface by cleaning it with 70% ethanol.

4. Label a sterile 15 ml conical tube or small autoclave-sterilized container with the Sample ID, the date, and your initials.

5. Carefully unscrew the cap of the tube with the swab, taking care not to touch the swab itself, nor any other part of the swab aside from the lid.

6. Using a sterile 10 ml pipet, add 7mls PBST to the swab container, and place the swab back into the tube, screwing the lid down well.

7. Repeat steps 4-6 with the second swab for the same sample.

8. Vortex the tubes containing the swabs for 30 seconds, then incubate for 5 minutes at room temperature and vortex for another 30 seconds.

9. Reopen each swab container and pour the swab elute into the empty labeled 15 ml conical tube.

10. Repeat steps 5 to 8 for each swab. Press the swab against the side of the tube to squeeze out as much remaining wash solution as possible, and discard the swab. Transfer the second elute solution to the 15 ml conical tube (there should be a total of ~12-14mls of swab elute).

11. Store all samples at 4°C until they are ready for processing.

Processing of soil, sand, and sediment samples

Generally, soil, sand and sediment samples are moderately contaminated with fecal bacteria, but some samples will have much higher contamination levels. These samples are rinsed with 20 mL of buffer to wash the bacteria off of the soil particles. The soil is allowed to settle and the clarified rinse is tested for E. coli. We recommend diluting this rinse solution 1:10 and 1:100 prior to membrane filtration or IDEXX testing, using the Dilution Protocol on Page 7.

Materials and Equipment:

● PBS (phosphate buffered saline, pH 7.2)

● One 50 ml sterile conical tube

● Two 15ml sterile conical tubes (for processing and dilutions)

● Gloves

● 70% ethanol

● Felt tip pen

● Autoclaved metal spatula, or sterile-wrapped disposable spatula

● 1 autoclave-sterilized graduated cylinder or 10 ml serological pipet per sample

● Pipet or other type of dropper

● 0.1 N NaOH

● pH meter or pH test strips

● Shaker

● Scale

● Vortex

● 4°C refrigerator

● Pipet Aid

● 1 ml pipet

● Soil Laboratory Form

Procedure:

1. Put on gloves and spray hands with 70% ethanol. Rub hands together to sanitize all surfaces of the gloves. Check that the sample ID on the sample and the sample ID on the Soil Laboratory Form are the same. Record the date and time of processing.

2. Prepare your work surface by cleaning it with 70% ethanol.

3. Samples should arrive at the lab in a small Whirl-Pak bag. Spray the outside of the bag with 70% ethanol and rub it well.

4. Label the sterile 50 ml conical tube and the 15ml conical tube with the Sample ID on the barcode label of the bag, the date, and your initials.

5. Rotate the Whirl-Pak bag with the sample five times to mix the sample.

6. Using a weighing scale and a sterile spatula, measure 10 grams of sample into the one of the labeled 50 ml tubes. Using a graduated cylinder or pipet, add 20 ml of PBS to the measured sample.

7. Screw the cap on the tube firmly and vortex for 30 seconds, then slowly adjust pH to 9.0 by adding drops of 0.1N NaOH. Test the pH after each series of drops using a pH meter or pH test strips.

8. Shake vigorously on a rotator or shaker for 30 minutes at room temperature. If a rotator or shaker is not available, shake manually every 5 minutes for 1 minute, for a total of 30 minutes.

9. Let the sample settle for 30 minutes. With a sterile pipette, carefully remove 12 mL of supernatant from the top and transfer it to the labeled 15 ml tube. Take care to not suck up particulate matter or debris.

10. Store all samples at 4°C until they are ready for processing.

Dilution Protocols

Below, we have provided the recommended dilutions for the SaniPath Exposure Assessment Lab Standard Operating Procedures. Depending on the sample type and the expected contamination level, we recommend between one and four dilutions. While these are our recommendations based on previous experience with the tool, they may not be the best dilutions for your setting. Therefore, we encourage users of this protocol to adapt the dilution methods and use any two to three dilutions on the lab data entry form for their work.

Materials and Equipment:

● Autoclaved or sterile filtered water/distilled water

● Up to 4 sterile 15 ml conical tubes, depending on number of dilutions to be prepared. Gloves

● 70% ethanol

● Felt tip pen

● Pipet with 1 ml tips (preferred but not required)

● Pipet Aid

● 10 ml serological pipettes

● 4°C refrigerator

Procedure:

1. Prepare your work surface by cleaning it with 70% ethanol.

2. Put on gloves and spray hands with 70% ethanol. Rub hands together to sanitize all surfaces of the gloves.

3. Prepare your dilution containers.

a. For each sample dilution, label a 15 ml conical tube with the sample ID and the dilution.

b. Using a 10 ml serological pipet, add 9 ml volume of distilled water to each of the 15 ml tubes.

4. Samples should have been collected in a Whirl-Pak bag. Spray the outside of the packaging/bag with 70% ethanol and rub it well.

5. Mix the contents of the bag by turning it end over end 5 times.

6. Open your bag of sample water, and transfer 1 ml volume of sample to the first dilution tube. This will make a 1:10 dilution.

7. Mix the diluted sample by swirling gently.

8. Transfer 1 ml of the diluted sample to the next dilution tube to prepare 1:100 dilution.

9. Repeat steps 7 and 8 for each subsequent dilution, as shown in the table below.

10. If the samples will not be processed immediately, store all samples at 4°C until they are ready for processing. For all diluted samples, 1 ml of sample will be mixed with 99 ml volume of sterile distilled water for processing by IDEXX.

Recommended tray volumes by sample type*: