Jul 19, 2022

Public workspaceProtocol for counting pathogen spores on hemocytometer V.3

This document is a draft, published without a DOI.
  • 1University of Michigan - Ann Arbor
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Document Citation: Meghan Duffy 2022. Protocol for counting pathogen spores on hemocytometer. protocols.io https://protocols.io/view/protocol-for-counting-pathogen-spores-on-hemocytom-cdras52eVersion 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: July 19, 2022
Last Modified: July 19, 2022
Document Integer ID: 67074
Abstract
This is a protocol to quantify the number of pathogen spores you have in a sample using a hemocytometer.
Protocol for counting pathogen spores on hemocytometer
  1. Rinse hemocytometer and glass covering with 90% ethanol and dry off with kimwipe.
  2. Place dead infected Daphnia in 1.5 ml centrifuge tube, and suck out all excess water with a glass pipette.
  3. Fill Pipette with a known volume of DI water. If you are doing spore counts for a single Daphnia, 0.1 ml of water is most appropriate. If you are doing spore counts for multiple Daphnia at a time, then 0.5 ml is most appropriate. Do not fill to 1 ml or above, as this will lead to water splashing and overflow during homogenization process.
  4. With a motorized pestle (Fisher Scientific catalog number: 12-1413-61), grind up Daphnia in DI water for approximately one minute. Throughout process, use pestle to push water collecting on sides of tube into the bottom so that the entire mixture can be properly homogenized.
  5. Pipette between 10 and 20 µl of solution into one side of hemocytometer. Pipette enough that the fluid covers the counting chamber, but not so much that you dislodge the cover plate. The exact amount of fluid needed depends on the exact positioning on of cover plate.
  6. Wait ~ 1 minute for the spores to settle. If you look through the microscope at 40x magnification and see different sets of cells when you slightly adjust the focus, then you need to wait longer.
  7. In the middle of the hemocytometer is a 3 by 3 grid of 9 1mm x 1mm squares, each subdivided into smaller grids. You will focus on the corner squares that are each subdivided into 16 0.25 mm x 0.25 mm squares. (consult diagram of hemocytometer). You will count spores within each of these corner squares, resulting in 4 counts.
  8. Calculation for spores per Daphnia is then
spore/daphnia=average count*10,000*solution volume(ml)