From notes I've accumulated:
Cell Fractionation Based On Density Gradient
https://user.eng.umd.edu/~nsw/ench485/lab10.htm
Determination of the Specific Gravity of Certain Helminth Eggs Using Sucrose Density Gradient Centrifugation
https://www.jstor.org/stable/3281005
Separation of Cryptosporidium Oocysts from Fecal Debris by Density Gradient Centrifugation and Glass Bead Columns
https://www.ncbi.nlm.nih.gov/pmc/articl ... 0-0127.pdf
There are sucrose density tables online and probably in the CRC Handbook.
And notes I wrote from having done it:
Documents upon which this procedure is based:
http://www.ncbi.nlm.nih.gov/pmc/article ... 0-0127.pdf
Stored locally as Microscopy/Techniques/Centrifugation/jcm00240-0127.pdf
http://www.jstor.org/action/showArticle ... %2F3281005
Stored locally as Microscopy/Techniques/Centrifugation/dtc.182.tif.gif
1) Put 8 ml of saline in a 15 ml centrifuge tube.
2) Collect 3 fecal samples from distant points along stool's length.
adding to centrifuge tube to bring total volume to 10 ml.
3) Vortex centrifuge tube to suspend sample.
4) Strain sample through nylon hose.
5) Strain sample through Keurig coffee filter.
6) Wash
6.1) Split the 10 ml sample between 2, 10 ml centrifuge tubes.
6.2) Wash twice by centrifuging with 10 ml saline at 650g for 5 minutes. Decant supernatant, examining it for eggs.
7) Separate
7.1) Re-suspend pellet in 2 ml saline.
7.2) Create a density gradient of 1 ml each: 13, 24, 35, 54 and 70% by weight of sucrose in saline, by under-laying
each successively higher-density sucrose solution using a blunt hypodermic needle. The 70% layer is to act as
a cushion.
7.3) Moving top-down, extract each layer's band with a dull hypodermic needle, place the band contents in a 10 ml centrifuge
tube containing 8 ml saline, centrifuge at 650g for 5 minutes, decant, re-suspend in 1 ml saline, use to create wet mount
microscope slides.
Giardia:
- giardia cysts should be above the 24% layer
- 10µm wide:
http://www.dpd.cdc.gov/dpdx/html/MorphologyTables.htm