Samuel J Gurr, PhD

SOP Feeding larvae

by Samuel J Gurr PhD 6 min read

SOP! Feeding larvae in static system

Objective:

Batch feed larvae using all precautions necessary for a static system (1) assess the current algal density of both the culture and static larval system using flow cytometry (2) add the correct volume of algae to a target cell density.

Materials:

  • ~6-10 stirrers

  • new 22um cell strainer

  • new 25 ml disposable serological pipette

  • pipette pump (for serological)

  • two cylindrical 3-4L beakers

    • one with hot water
    • one with filtered seawater (tangential filtered!)

  • 1 ml pipette tips (~1 per tank)

  • 1 mL pipette

  • 2 ml tubes compatible with flow cytometer - labeled for tank number (1 per tank)

  • spray bottle filled with filtered seawater (tangential filtered!)

  • tube rack

  • 1 L tripour

  • access to the internet and a compatible device (i.e. phone)

  • 100 - 500 mL graduated cylinder (depends on the algae culture for your master mix in II. below)

Steps:

I. Assess the current algal density

NOTE: This is typically a ‘morning’ task, meaning that your static larval tanks were fed the day (morning/afternoon) prior. The objective here is the, following all proper stands below, sample each tank and run flow cytometry to learn the current cell density. These data will be used to calculate the % removed and the amount of algae to add in II.

(1) Gather all materials. Make sure everything is clean (hot water rinsed). Some materials must be new (cell straine & serological pipette).

(2) Fill the two 3-4L cylindrical beakers, one with hot water and one with tangential-filtered seawater. Position these on a wheeled-cart.

(3) Position your 1 mL pipette and tips, new cell strainer, pre-labelled 2 mL flow cytometry tubes, and tube rack on your cart

(4) Place all your clean stirrers in the hot water beaker

(5) (Optional) To keep track of which tank you have completed, move the lid of each tank slightly ajar as to indicate those that you have not taken

(6) Place all stirrers in the beaker with tangential-filtered seawater

(7) Begin process of taking a sample by assigning your station (cart) with the correct ID - lightly position the cell strainer on the opening of your first pre-labelled 2 mL flow cytometry tube, as to avoid piecing the 22 um mesh

(8) In one hand, remove a stirrer from the filtered seawater beaker. in the other hand, pick up the 1 ml pipette and add a 1 ml pipette tip (make sure your pipette is set for at least 500 ul)

(9) With the stirrer, place the mixing end (flat circle with holes) in the tank correct tank (same ID as your prepped 2 mL tube with cell strainer) and begin mixing by moving up and down in the water column of the bucket. Caution! Larvae are fragile, be careful as to avoid hitting the bottom/sides of the tank and do not splash. We wish to avoid cross-contamination of our treatments/replicates.

(10) While mixing, take a submerge the pipette tip in the seawater surface (not the pipette!) and take your sample.

(11) Keeping your stirrer inside/above the tank dispense the sample through the cell strainer into the pre-labelled 2mL flow cytometry tube. Dispose the pipette tip onto your cart (i.e. a paper towel/waster beaker) and place down the pipette

(12) Pick up your spray bottle filled with tangential-filtered seawater and spray the stirrer into the tank, such that all any submerged parts with clinging larvae are removed of those larvae and all are kept in the tank. Place the stirrer in the hot water beaker

(13) Rinse your cell strainer into the tank with tangential-filtered seawater

(14) (Optional, if completed step 5) Close the lid fully and move to the next tank with aa slightly-ajar lid

(15) Repeat steps 7 - 14 for all remaining tanks

(15) Run you sample on the flow cytometer and calculate the cell density for each tank as cells per milliliter

II. Batch feed the static larval tanks

NOTE: Batch feeding differs depending on the current cell density.

  • Case 1: On a drop day. ‘Drop days’ are when each tank is sieved to investigate larvae before replenished with new water. thus, the flow cytometry results steps covered in I. (above) do not apply and each tank will be fed to the total target cell density

  • Case 2: On a maintained day (all non-drop days) In this case, each tank was fed to the target density the day prior and the objective is to batch feed the difference in cell density. The Difference between the total target and observed cell density will determine the volume to add

(1) Answer the following questions:

  • 1a: What case are you? (read above)

    • If 1, you are adding the total target cell density

    • If 2, you are adding the difference in total - observed

  • 1b: How old are your larvae?

    • very important as this will determine the species of algae, percent composition, and the target cell density

  • 1c: What are the cell densities of the algae cultures?

    • IMPORTANT: To complete this step using the RShiny app, you must know the cell density of the algae cultures (cells per mL). If you do not know them, consult with the lab notebook (if already measured) or measure them yourself using the flow cytometer at a 10x dilution of algae culture and filtered DI water (i.e. 100 ul algae to 900 ul DI) - calculate each culture and label them with this value.

(2) Gather your materials on your benchspace/counter. This includes the following: serological pipette and pump, 22 um cell strainer, clean 1 L tripour, your algae

NOTE: algae are stored in the light room in the second floor. Each should be labelled with the species ID (i.e. Isocrysis genus as T-ISO).

(3) Use my RShiny App to calculate the volume of each algae to make your master mix

*NOTE:* The app will start by asking you a few questions...

- How many tanks am I feeding

- Do they differ in volume, What is/are the volume(s) of the tanks?

- What is my target cell density at this life stage?

- What is the cell composition for the species of species I will feed? (i.e. 75% t-Iso, 25% Pavlova, etc.)

- What are the cell densities of the algae cultures?

Press ‘Feed ‘em!’ and you will get a table for the volumes (in Liters!!) of algae to make your master mix and the volume of the master mix to add to each tank.

IMPORTANT: you will need to fill all dependencies for all tanks that differ in volume and target cell density - if all are the same you are in luck, one and done!

(4) Measure the correct volume of algae for each species using the graduated cylinder. Pour the contents of each species in the designated 1 L clean tripour as your master mix

(5) Gather the master mix, serological pipette and pump, and cell strainer to your cart.

(5) (Optional) To keep track of which tank you fed, move the lid of each tank slightly ajar as to indicate those that you have not taken

(6) Begin process of batch feeding a tank by taking and dispensing ~5-10 mL of master mix 5 times - you can also swirl the serological pipette in the master mix careful to not damage it.

(7) Dispense the calculated volume of master mix through the 22um and into the tank

(8) (Optional, if completed step 5) Close the lid fully and move to the next tank with aa slightly-ajar lid

(8) Repeat steps 6-8 for each tank

(9) Wait one hour and complete all steps in I. (above) to measure the current algae density of each tank