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Surfrider Foundation SLO Chapter

“Keeping It Blue” Project

Blue Water Task Force

Procedures manual

For

Water Sampling and Lab Testing

Compiled* by Niel Dilworth

August 2020

COVID-19 ed.

*This is an abbreviated description of our sampling and lab testing procedures.

For complete information, please refer to the Water Sampling Instructions and

BWTF Lab Instructions, which are available at the following link:

www.bwtf.surfrider.org/resources

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ONE: COLLECTING THE SAMPLE

1. Go to the sample collection site.

2. Depending on the collection method, prepare one

sample baggy or 100ml vial for each site where a

sample is to be collected.

3. Write the time, date, and location of the sample on

the baggy or the cap of the 100 ml. vial.

4. Take the baggy/vial to the location where the sample

is to be taken.

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5. If you are using a baggy to collect the sample –

At the location, carefully open

the baggy by pulling the two

tabs apart. Do not touch the

mouth of the baggy.

6. Walk at least knee deep into the water. Hold the

sample baggy by the tabs and submerge the baggy at

least 6” (six inches). Fill the baggy to the water line

remembering not to touch the mouth or the inside of

the baggy.

7. Close the baggy carefully without touching the mouth

of the baggy.

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8. If you are using a 100ml. vial to collect the sample –

Write the location and time on the bottle cap.

9. Place the bottle in the dipper making sure that the

rubber band is up on the shoulders of the vial. This will

keep the vial from popping out of the dipper when it

hits the water. Remove the bottle cap and place it in a

clean location.

10. Lower the dipper into the water with enough force to

submerge the vial. Once the vial is filled retrieve it and

recap the vial.

Take the sample(s) back to the lab immediately and

prepare the sample for testing. NOTE: If you cannot

immediately test the sample, you must refrigerate it or

put it on ice in a cooler, but in no case for longer than 6

(six) hours.

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TWO: TESTING THE SAMPLE

COVID-19 REQUIREMENTS –

All volunteers must wear a mask or face covering at all

times when they are in the aquarium building and maintain

6’ social distancing with other BWTF volunteers and

aquarium staff.

Overview of Lab Area

The Quanti-Tray Sealer

The Incubator

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1. If you need to unlock the exterior door to enter the

aquarium, upon entering check to see if the alarm is

armed. If it is not armed proceed to Step 2. If it is

armed (the system will be beeping) enter the alarm

code on the keypad and press ENT., NOT COMMAND.

If you have trouble with the alarm system or if the

alarm sounds, call Mace Security at their number on

the keypad. Explain who you are and that you are

having trouble with the alarm and ask for help to reset

the system.

2. Enter your name, date and time in the Log in the front

of the binder that holds the location sample sheets.

3. If they are not already turned on, turn on the tray

sealer and the incubator and confirm that the incubator

is set to the correct temperature.

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4. In the Sample Logs in the binder write in the time,

date, and location of each of your samples, as well as

your name, the tide and the weather.

5. Sterilize the workspace and sink area: take a paper

towel and clean the surfaces with the 10% Clorox

bleach solution. Let dry.

6. Put on clean lab gloves.

7. Pull out these items and place on the sink area: THE SAMPLE(S) THAT YOU COLLECTED

THE DISTILLED WATER THE PIPETTE TOOL (The Extractor) and for each sample: A 100 ml PLASTIC SAMPLE BOTTLE A sterile PLASTIC PIPETTE AN ENTEROLERT PACKET FROM THE REFRIGERATOR A QUANTI-TRAY®

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8. Label the Quanti-Tray and the 100 ml plastic bottle

with your sample’s location, date, time.

9. Open plastic sample bottle taking care not to

contaminate the inside of the bottle cap.

10. Add 100 ml of distilled water to the bottle.

11. Insert a sterile pipette into

the green and white extractor

tool and use it to remove 10 ml of

the distilled water from the

plastic bottle, leaving 90 ml

inside the bottle.

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12. Discard the 10 ml of distilled water and set the

pipette and extractor aside. Don’t let the pipette touch

anything.

13. Add the Enterolert to the 90 ml of distilled water in

the plastic bottle by breaking the capsule over the

mouth of the vial. Replace the bottle cap. Shake bottle

gently back and forth for 40

seconds or until all visible

chunks are dissolved.

14. Without touching the

pipette to the baggy or the

mouth of the 100ml vial,

extract 10 ml of your sample

from the baggy/vial.

15. Add the 10 ml of sample

water to the plastic bottle

containing the Enterolert

mixture. Gently shake for 20

seconds.

16. Dispose of the plastic baggy, the plastic pipette, and

the Enterolert container. We repurpose the 100ml.

vials. Place the lids on the empty 100ml. vials and place

them in the recycling box that is under the fish tanks.

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17. Open the Quanti-Tray by squeezing it near the top.

18. Pour the entire contents from

the plastic bottle into the

Quanti-Tray. It should fill most

of the cells.

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19. Check to see that the green light on the Sealer is on.

20. Place the Quanti-Tray onto the red rubber mat and

feed it into the Sealer closed end first.

21. After the Quanti-Tray is sealed, confirm that the

Incubator is at 106° F or 41° C. Then place the sealed

Quanti-Tray in the Incubator.

22. Let the sample sit for 24-28 hours in the Incubator.

23. Dispose of the used gloves, bags and pipettes, rinse

and cap the 120ml bottles and put them in the plastic

lined box for repurposing. Put the green and white

extractor that fits on the pipette back in the box of

pipettes.

Quanti-Tray Filled with Complete Contents of Sample Bottle

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THREE: ANALYZING and RECORDING DATA

1. After the incubation period is complete (24-28 hours),

remove the Quanti-Tray(s) from the Incubator.

2. Turn on the

Black Light

and scan the

Quanti-Tray

with the

Black Light.

3. Count the cells, both large and small, that light up or

“glow.”

4. Consult the chart in the binder to find the test result.

Find the number of large cells in the vertical column

down the left side, and the number of small cells in the

horizontal row across the top. At the intersection of

the appropriate column and row is the result or the

MPN (Most Probable Number). Multiply that number

by 10 (ten) to determine the actual level of

enterococcus in the original sample of ocean water in

cfu/100ml; this is the actual result of the test.

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5. On the log sheet, record the following: (1) the number

of large cells; (2) the number of small cells; (3) the

MPN, and (4) the actual test result.

6. Communicate the results to the person who is

responsible for posting the results on the BWTF web

site. Currently this is done by taking a photo of each

completed log sheet and sending them by email to the

volunteer who is responsible for uploading the data to

the BWTF website.

7. Dispose of the Quanti-Trays. Clean up the lab area.

Turn off the Sealer and Incubator if not in use by

another BWTF volunteer.

8. If you are the last BWTF volunteer to leave and the

aquarium is open to the public, just leave. If the

aquarium is closed to the public check to determine if

there are any aquarium volunteers or staff in the

building. If staff are present, leave and make sure that

the door locks behind you. If no other volunteers or

staff are present, make sure that all exterior doors are

shut tightly and set the alarm by entering the alarm

code on the key pad and pressing ENT. The warning

beep will begin. You have 60 seconds to leave the

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building before the alarm arms. If you have trouble and

the alarm sounds, see Section 2, Item 1.