Floatation Fluid for Fecal Examination

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GerryR
Posts: 183
Joined: Sun May 22, 2022 11:44 am
Location: Virginia, USA

Floatation Fluid for Fecal Examination

#1 Post by GerryR » Fri Oct 21, 2022 3:45 pm

Just an idea that maybe some people haven't thought of. Floatation fluids can be purchased having a specific gravity of 1.2 - 1.3, but you can also make your own with Epsom salts, Sodium Nitrate, and I'm sure, a multitude of other combinations that I know nothing about. However, testing them for specific gravity requires a hydrometer or refractometer. The hydrometer requires a larger volume of fluid for testing than a refractometer and both can be expensive if looking for a laboratory grade device. However, it dawned on me ("then came the dawn!"), being an auto enthusiast, that battery hydrometers and refractometers cover the specific gravity range of 1.1 to 1.4 (some only to 1.3). These units can be had relatively inexpensively on ebay and work perfectly for testing floatation fluids. Just a thought. ;)

ldflan
Posts: 134
Joined: Wed May 22, 2019 11:36 pm
Location: Morna Moruna

Re: Floatation Fluid for Fecal Examination

#2 Post by ldflan » Fri Oct 21, 2022 5:10 pm

Used (but classic) B&L 3L abbe refractometers come on sale on eBay for widely variable prices. Liquid refractive index can be measured from a single drop, and it's possible to measure RI for some solids (like dried mounting medium on a coverslip), too. The 3L is pretty easy to work on / repair (it took me two cheap ones to build one good one) but (if possible) try to get one that includes the calibration kit - a bottle of 1 bromonapthalene and a solid test piece.

GerryR
Posts: 183
Joined: Sun May 22, 2022 11:44 am
Location: Virginia, USA

Re: Floatation Fluid for Fecal Examination

#3 Post by GerryR » Fri Oct 21, 2022 6:19 pm

The 3L is definitely a nice unit but overkill for other than a lab environment, unless you can get one really cheap. I was just looking for an inexpensive way to check nd, whether for purchased floatation fluid or home-made. I looked at the 3L and some similar units; they were all in the $200 to $500 range. My battery (and anti-freeze) refractometer was ~$30. and when I measured the floatation fluid that I purchased, it was 1.25 (spec'ed at 1.25-1.30) and fits in a draw when not needed.

ldflan
Posts: 134
Joined: Wed May 22, 2019 11:36 pm
Location: Morna Moruna

Re: Floatation Fluid for Fecal Examination

#4 Post by ldflan » Fri Oct 21, 2022 6:41 pm

Yes, definitely not a cheap as a battery tester, and it takes up bench or shelf space equivalent to a small microscope.

OTOH a lot of folks here have their own laboratories at some level, so I thought I would point out the usefulness of being able accurately to test the RI of solidified mounting media, and very small quantities of liquid, if those are matters of interest. And for a lot of us they are.

I think I put my 3L together for a couple hundred bucks from two that were sold on eBay as "parts"; as I recall, I made a series of lowball offers because some of these things sit unsold for years and eventually a couple sellers agreed. It then took about three hours to repair and calibrate the resulting machine, so it wasn't a quick out of the box solution either. But it has actually proved a lot more useful than I expected, and it was fun to figure out how it works.

DWSmith
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Joined: Sun Nov 06, 2022 12:07 pm

Re: Floatation Fluid for Fecal Examination

#5 Post by DWSmith » Wed Dec 28, 2022 5:01 pm

What should the specific gravity of the floatant be for fecal examinations? Is the 1.25 to 3.00 correct?

dtsh
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Joined: Wed May 01, 2019 6:06 pm
Location: Wisconsin

Re: Floatation Fluid for Fecal Examination

#6 Post by dtsh » Wed Dec 28, 2022 7:25 pm

DWSmith wrote:
Wed Dec 28, 2022 5:01 pm
What should the specific gravity of the floatant be for fecal examinations? Is the 1.25 to 3.00 correct?
It depends on what you're working with, too high and you float debris, too low and you don't float ova.
Quoting from "Diagnostic Parasitology for Veterinary Technicians 4th Ed" p316 'TECHNICIANS NOTE: Fecal floatation solutions with a specific gravity of 1.2 to 1.25 are used to "float" parasite ova, cysts, and larvae while the fecal material sinks to the bottom of the container.'
It states that fecal material tends to have a sp.gr of 1.3 and higher and that most parasite eggs have a sp.gr of between 1.1 and 1.2.

GerryR
Posts: 183
Joined: Sun May 22, 2022 11:44 am
Location: Virginia, USA

Re: Floatation Fluid for Fecal Examination

#7 Post by GerryR » Thu Dec 29, 2022 12:16 pm

DWSmith wrote:
Wed Dec 28, 2022 5:01 pm
What should the specific gravity of the floatant be for fecal examinations? Is the 1.25 to 3.00 correct?
1.25 to 1.3, not 3.0 The book that dtsh referenced, "Diagnostic Parasitology for Veterinary Technicians 4th Ed" is an excellent reference and is fairly inexpensive as a used book.

iPeace
Posts: 28
Joined: Tue Sep 05, 2023 3:06 pm

Re: Floatation Fluid for Fecal Examination

#8 Post by iPeace » Wed Sep 13, 2023 7:23 pm

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

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