This document describes the protocols to be followed by volunteers aboard the Ka'imimoana for producing chlorophyll profiles and running the underway mapping system.

Essentially, there are two main tasks:

	sampling the CTD (Conductivity, Temperature, Depth) profiles for extracted chlorophyll and nutrients
	Maintaining an underway mapping system which measures in vivo fluorescence of chlorophyll and photosynthetically available radiation (PAR).

The CTD "rosette", which includes the conductivity, temperature and pressure sensors, and the 12 or more sample bottles (subsequently referred to as Niskin bottles) usually sits outside of the wet lab doors, and this is where it is deployed from. Under normal circumstances, the CTD will be deployed and lowered to 1000m, then brought back to the surface. On it's way to the surface, the Niskin bottles will be "fired" at certain depths, thus trapping water from that depth inside the bottle ready to be sampled on deck.

The depths at which chlorophyll samples will be taken are shown in the table below. There are 4 or more other bottles on the rosette which the Survey Technician uses for deeper samples, but depths greater than 200m are of no real interest biologically, because of the low chlorophyll concentrations there. The Survey Technician, Dennis Sweeney is already aware of this sampling regime but it might pay to discuss this prior to the first CTD. There will be occasions where the Survey Technician will want to use all depths for a calibration cast, but for the most part this chlorophyll sampling regime can be accommodated:

The following sample bottles will be required:

	8 x 500 mL for the regular chlorophyll samples, labeled with their % light depths as in the table above.
	1 x 1000 mL for the surface A* sample, labeled A*.
	2 x 250 mL for the surface size fractionation samples, labeled 1m and 5m .
	8 x 20 mL plastic nutrient vials.

Once the CTD is on deck, tied down etc, you can begin sampling; the sooner the samples are drawn, the better, but keep out of the way of the Survey tech until the rosette is secured. Always "break the seal" on the Niskin bottle by pushing in the bottom spigot first, check to see that there are no leaks, and note any such problems on the log sheet provided. To start the flow of water from the Niskin bottle, release the top valve, and then use the bottom spigot to turn flow on and off. Be sure to follow any recommendations of the Survey Tech when doing anything related to CTD sampling. Depending on how much water is being taken for other purposes, it may be necessary to conserve water from the bottles, and so turn the flow off between rinses, fills etc so as not to waste the sample water.

Rinse each of the eight chlorophyll bottles (500mL) briefly (just a splash, or about a 1/4 cup) three times and fill to the bottom of the neck from the appropriate Niskin bottle.

Rinse the A* bottle briefly three times and fill it from the bottle fired closest to the surface (usually bottle 12).

Rinse and fill the 1m and 5m size fractionation bottles from the bottle fired closest to the surface (usually bottle 12).

Fill the nutrient vials from the same bottles as the chlorophyll samples came from. It is important to rinse the vial and the cap at least three times each, and make sure that the vial is only filled about 1/2 way - it is going to be frozen, and if the vial is too full, leaks may occur. Things like dirt, grease or sweat on your hands have the potential to contaminate the nutrient samples, so avoid sticking your fingers inside the cap or vial, and make sure you rinse 3 times. The sample vials have most likely been used before (they may have some seawater in them, which you should of course empty) and so will have some salt crystals on them - be sure they are properly rinsed off. Once the nutrient vials are filled, you just need to freeze them - no filtering.

For each of the eight chlorophyll samples, filter 500 mLs (actually 528 mLs) onto a GFF filter. The GFF filters have a slightly different pattern on each side; one side has a kind of cross-hatch pattern, while the other side appears to have lines in one direction only. Place the filter on the filter rack with the cross-hatch pattern facing up. Filter at a vacuum pressure of 5-7 mm Hg. If you are using the Venturi seawater suction system, you can judge the right pressure by timing how long it takes to filter one full filter cup (200 mls). It should take about 90 seconds, so adjust the seawater flow (which governs the vacuum pressure) accordingly.

Now filter the 1m and 5m size fractionation samples, but use the appropriate membrane filters. These filters also have a particular orientation; whichever side is facing up when they are sitting in the filter box is the side that should face up when they are placed on the filter rack. Use the same vacuum pressure for the 1m and 5m samples - they will filter more quickly than the GFF samples, especially the 5m sample.

With forceps, place each of the eight chlorophyll filters, and the two size fractionation filters in a separate scintillation vial with 10 mls of 90% Acetone. Shake and place in the freezer in the science hold. It is important that the samples are not left in bright light or at room temperature for too long, so try to put them in the freezer soon after filtering. Samples should be left for 24-48 hrs in the freezer for the chlorophyll pigments to extract, then read them in the Turner benchtop fluorometer.

Filter the A* sample as for the GFF chlorophyll samples, but take care not to let it "run dry" (ie try to stop the filtering as soon as the last of the water has passed through the filter). Using forceps, fold the filter in half and place it in an appropriately labeled "cryovial" and drop into the liquid nitrogen. Note that for the A* samples only, you simply place the filter in the vial and freeze - NO ACETONE.

Place the nutrient vials (in a separate flat to the chlorophyll samples) in the freezer in the science hold.

The MBARI samples will identified by the Program Name, CTD ID, and depth.

The MBARI program name for each cruise will be N (for NOAA) followed by 00 (the year), followed by a sequential letter, A, B, C... where A would be the first cruise of the year, that is GP1-00-KA.

The Chief Survey Technician identifies the CTDs using a letter/number combination like KA10011. Where KA1 would mean Ka'imimoana, 1^st cruise of the year. The next three digits refer to the cast number, so 001 is the 1^st CTD station. The last digit refers to the number of the cast done at that station, so the final 1 indicates the 1^st CTD cast at that station. Occasionally, more than one CTD cast will be done at a particular station, in which case the last digit is incremented. All that is required is to copy the name of the CTD from the Survey Tech's log sheet.

The depths will usually be as described in the table above, so just label the scintillation vials 0m, 10m, 25m, 40m, 60m, 100m, 150m, 200m. However, when filling out the depths on the data sheet, copy down the exact depth from the survey Tech's log sheet. For example, this would probably be something like 10.5m for the 10m sample. This is the actual depth it was fired at.

So, for the first CTD on cruise KA1, the chlorophyll samples will be labeled as:

N00A N00A ... N00A

KA10011 KA10011 ... KA10011

0m 10m ... 200m

The 1m and 5m size fractionation samples will be labeled:

N00A N00A

KA10011 KA10011

1m m 5m m

The nutrient samples will be labeled in the same way as the chlorophyll samples (except you won't have a 1m m and 5m m sample). It is important to label both the cap of the nutrient vial, and the side of the vial, because the labels on the cap can be rubbed off in transit. Remove all previous labels by wiping with Acetone prior to sampling.

The A* sample should be labeled:

N00A

KA10011

0m A*

The room where the fluorometer is used is shared with the Salinometer, so try to sort something out with the Survey Tech, so that both of you are not trying to use the room at the same time - it's pretty cramped in there.

Before reading, remove samples from freezer, shake gently, and allow to warm up for 30-60 min in dark or at least dim light - placing them under a shelf or putting a clipboard over the flat that holds the samples works well. Also turn on the fluorometer to warm up. It is important that the samples are at least close to room temperature, otherwise misting will occur on the cuvette, which will affect the readings obtained. It may work best to take the samples out of the freezer at about dinner time, let the samples and the fluorometer warm up while you eat, and then come back ~30-60 minutes later to read them.

There are two major gain settings on the fluorometer (x1 and x100), and 4 minor gain settings (min sens, which is actually equal to 1, 3.16, 10 and 31.6). With the black knob/lever set to "x1" and the red light indicating "min sens", the effective gain is 1. From there, if you step up through the gains using the switch, you will go to 3.16 (3.16 x 1), 10 (10 x 1) and 31.6 (31.6 x 1). To increase the instrument's sensitivity past 31.6, change the black knob/lever to "x100" , and set the minor gain to "min sens", this is an effective gain of 100 (1 x 100). If you step up through the gains from there you will go to 316 (3.16 x 100), 1000 (10 x 100) and 3160 (31.6 x 100).

Thus the full range of gains, in order from least to most sensitive goes like this:

1, 3.16, 10, 31.6, 100, 316, 1000, 3160.

1 is the minimum sensitivity, which would be used to measure very concentrated chlorophyll solutions, and 3160 is the maximum sensitivity, which would be used to measure very dilute samples. Most of the CTD samples will probably be read at around gain 31.6 to 316, but some may need a gain of up to 3160, and others may need much less sensitive gains, but it would be rare to go below 10 for the equator.

Zero the fluorometer with 90% acetone, with sensitivity at 100 x 31.6. To do this, rinse the cuvette to be used with 90% acetone several times, dry it inside and out with a tissue, then fill about 3/4 of the way, and place it in the fluorometer. Unlock (using the outer ring) and adjust the blank knob, but NOT THE SPAN, until it reads zero.

Once you have set the blank, lock the blank knob using the outer ring on the knob. You are now ready to read the samples, and you will NOT need to re-zero when you change gains. However, if you use a different cuvette, you will have to re-zero the instrument. Bottom line is; zero/blank the instrument each time you start it, and try to always use the same cuvette.

To read samples, first rinse the cuvette with about 1 mL of the sample to be read, and discard. Then fill the cuvette to about 3/4 full with the sample and read total fluorescence (F₀). Set the sensitivity so that readings are between 3.2 and 10 on top scale. Always use top scale, even though it appears that the top and bottom scale should be used depending on what gain you are at. Our equation for chlorophyll takes this into account later.

Record data manually on log sheet.

Add 3 drops of 10% HCl to cuvette while it is still in the fluorometer, but be careful not to spill any acid or acetone inside the fluorometer. Re-read the fluorescence at same sensitivity setting ( this is your F_a value, acid destroys the Chl, so your reading is now phaeophytin, which is included in the first reading). Do not change sensitivity for this reading, even if it is below 3.2, which it could easily be.

Record data manually on log sheet.

Rinse cuvette with acetone. Discard acetone and used chlorophyll solutions into a labeled bottle for later disposal over the side of the ship; vials can also be disposed of over the side, but the caps are plastic and must be disposed of on shore. Acetone is extremely flammable, so be careful.

In the wet lab is a small flow-through fluorometer which measures in vivo fluorescence of phytoplankton in seawater. It is connected by a cable to the old-looking desktop PC in the computer room, and the fluorescence data are displayed as a green trace on the screen and a green number with the label "WETStar". The computer is also connected to two light sensors (mounted next to the balloon launching area) and a GPS system for logging position. These are the yellow and pink traces/data on the screen, but one sensor is not working at the moment.

When the ship has left port and the seawater is turned on, restart the computer (you will have to temporarily remove the floppy for it to boot properly, but remember to reinsert the floppy). A set of instructions will appear on the screen. Hit escape to run the program without the flowmeter (it broke some time ago and has been removed). To judge flow through the system, use a stopwatch and a measuring cylinder at the outlet. Try to get the flow between 1-2 liters/minute (1.5 is good).

Latitude and longitude may not displayed correctly on the computer screen, due to a software/connection problem. Hence, you should get latitude, longitude and GMT date/time (necessary for filling in the log sheets) from another screen somewhere on the ship, like the one in the wet lab. Ask the Survey Tech or the Electronics Technician if you are unsure where to get this information. Note that there are two seawater temperatures displayed on the screen. One says something like "TSG instrument temp" and this is the temperature inside the thermosalinograph in the wet lab. It is usually 0.4°C warmer than the "External TSG temp" which is closer to the real value. Check with Dennis to make sure.

About four times per day, if possible, take some "underway calibration samples". It is best to try to do these at 10 minute intervals of the time displayed on the computer screen (so at 0, 10, 20, 30.. minutes past the hour). This is because the system samples at these times and we want the time that the calibration sample was taken to correspond to the time the computer made a sample.

The calibration samples are taken from the seawater outlet in the wet lab sink. Take a nutrient sample (1/2 fill a plastic vial as you would from the CTD) and a chlorophyll sample (fill a 500 mL bottle, filter and place in a scintillation vial with 10 mLs of 90% acetone as you would for a CTD sample). Freeze the nutrient sample, and place the chlorophyll sample with the CTD samples in the freezer. Leave it for 24-48 hrs and then read in the fluorometer as you would for the CTD samples.

Fill in the data on the log sheets provided, in a folder next to the logging computer. As mentioned above, it is probably best to get date, time, lat and long from elsewhere, but check from time to time that the date/time on the computer screen is close to that displayed elsewhere on the ship. If it is "old", ie displaying the date and time from a few hours or days ago, then reboot the computer as described above and this will hopefully re-establish the connection to the GPS. Julian day (Jday) is just the day number, where Jan 1=1 and Dec 31=365 (for a non-leap year). This will be displayed on the screen but will be incorrect if the date/time/lat/long is not updating correctly.

It would be good if the 4 samples were taken at regular intervals during the day. If nothing else is going on, I usually take them around meal times, and just before going to sleep at night. When things start to get hectic, especially when lots of CTDs are being done in quick succession, then it is ok to take less of these samples, as the surface CTD samples are essentially the same thing.

stpe@mbari.org

831 775 1802 (work, with voice mail)

831 775 1620

Shipping address:

MBARI: Attention Pete Strutton

7700 Sandholdt Rd

Moss Landing, CA, 95039

USA