Apr 12, 2022
Version 2
Pasteuria farm protocol V.2
This document is a draft, published without a DOI.
- Meghan Duffy1,
- Katherine Hunsberger1,
- Rebecca Bilich1
- 1University of Michigan - Ann Arbor
Document Citation: Meghan Duffy, Katherine Hunsberger, Rebecca Bilich 2022. Pasteuria farm protocol. protocols.io https://protocols.io/view/pasteuria-farm-protocol-b7g8rjzwVersion created by Kira Monell
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: April 12, 2022
Last Modified: April 12, 2022
Document Integer ID: 60672
Abstract
This is a protocol to infect and maintain Daphnia with Pasteuria ramosa for use in laboratory experiments.
Pasteuria farm protocol:
Written by Katherine Hunsberger
Additions made by Rebecca Bilich on 3/2/2021
Set up: start a farm with ~75 Mid 37 animals (young animals, if possible) and a dose of 2000 sp/mL of Pasteuria G/18
Use a 1000 mL Erlenmeyer flask and add Daphnia from a beaker of 75 by running them down the side of the flask, then rinsing the inner sides of the beaker and flask to dislodge any stuck animals. Small juveniles 0-2 days old are the most likely to be infected although adding variety of ages will help the farm last longer.
Add water to bring the final volume of the farm up to 900mL.
Select Mid 37s already infected with Pasteuria G-18 to create the spore slurry.
Estimate the number of animals needed by supposing that each infected animal contains 60,000 Pasteuria spores.
Grind the animals up in microcentrifuge tubes using an electric pestle from 60 seconds, making sure to make a homogenous mixture with no obvious Daphnia parts. Then remove the slurry to a small (5 mL) beaker. Then rinse each tube with about 0.5 mL of filtered lake water, vortex the tubes for a few seconds, and add this water to the slurry as well.
Mix the slurry well with a pipet for 60 seconds and then add 10 mL to four wells in a Neubauer Hemocytometer. Do 4 cell counts, excluding spores that do not appear viable (degraded or immature spores.) If the grid is very dense, you can estimate by counting the 5 diagonal squares and multiplying the total by 5.
Choose 3 or more counts that are within 85% of each other, take the average of this number, and multiply it by 10,000 to get the concentration of the slurry in spores/mL.
Use the equation M1*V1=M2*V2 to find the volume of slurry to add to each farm. Add this amount with a pipet and then feed each farm only 10 mL of food (after adding the spores, to mix them around.) Tap the flask with a small amount of cetyl alcohol. Also feed only 10 mL on the next feeding day to encourage Daphnia to consume pasteuria spores. On subsequent feeding days feed 20 mL.
Maintenance: check farms for first time after ~30 days; then check every 7-10 days
- Follow similar rules as Metsch farm maintenance for adding more animals and/or spores or taking down the farms
- If you start checking a farm and the animals look pretty lightly infected, I would hold off another week or so before checking them to let the spores grow up
Storage: store in freezer indefinitely, but ramp up for experimental needs as far in advance as possible