Hay (Con)fusion

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linuxusr
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Hay (Con)fusion

#1 Post by linuxusr » Thu May 27, 2021 2:46 pm

Years ago, first scope, I got great specimens. Now, years later, I'm re-visiting hay infusion:

a. "Infuse" means "to soak," as you would infuse loose tea in boiling water, so a hay infusion is soaking/soaked hay.
b. Any infusion, in time, will be loaded with bacteria. As in our normal flora, most bacteria are not pathogenic. However, those that are, as we well know, can wreak havoc. So the hay infusion is not an innocent procedure. Assume pathogens as you cannot prove otherwise. Wash hands. Clean work area with bleach/alcohol, etc.
c. In time, there will be a food chain, prokaryotes < eukaryotes; and eukaryotes < eukaryotes. The succession is pretty much pre-determined but what that succession is, is not clear in my mind. I'm thinking bacteria (maybe a biofilm) < ciliates < rotifers. Can someone with more experience detail this a little more?
d. Some say to boil the organic material; others make no mention of this. Boiling will diffuse the substances inside the hay to the water as you will see a color change, but will not boiling kill many of the same organisms we wish to view?? And this entails another question. From where do the protists come? The general answer is that they are everywhere. I am thinking they must be in the hay, maybe dormant and encysted, and some might be in the atmosphere but if the specimen jar is covered ??
e. Assume a specimen jar has 10^5 drops. Really? You are going to find organisms in only one drop? If so, there must be millions. Or what if you find none? Do you sample on different levels?

I now have two specimen jars going, one with soil from my chicken coop, the other with alfalfa hay. When these projects are said and done, I'm going to start over and be more scientific about the process. I will start with the same sets of media but once per day I will chart whether there is biofilm or no, protista or no, and do so checking on three levels, top, middle, bottom. I will keep track of what appears as a function of time per medium. That should give me a clearer picture.
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lorez2
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Re: Hay (Con)fusion

#2 Post by lorez2 » Fri May 28, 2021 3:06 am

Regarding "what's in the sample", I had an unusual experience yesterday wherein I picked a cup of water from a duck weed covered pond to take a quick look at with a stereo microscope. There were no moving critters at all... at the 10X and 30X magnifications of the scope. This is a first for me. Of course, a lot was unseen using only a simple stereo scope, but seeing nothing of the usual suspects was a bit of a surprise. Having a simple stereo microscope permanently set up on top of a file cabinet is a great way for some interesting ad hoc viewing.

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charlie g
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Re: Hay (Con)fusion

#3 Post by charlie g » Fri May 28, 2021 7:00 pm

Hi 'linuxur'...why not try native organisms about where you live...rather than a totally 'human induced scenario of cooked organics left to ferment/ to spoil/ whatever terms we humans use to describe our gathered food stocks spoilage.

Please go outdoors and collect a clump of moist moss in a paper cup, a clump of aquatic vegetation from a pond shore...or stream shore ( again in a simple paper cup). If this outdoor collection hike ( good for you...good for a doggie you might have in your family)...if outdoor collection for microscopy is not in your plans...sample the various zones in a home aquarium, sample waters from indoor or out door potted plants.


Cooked organica, fouled milk, molded breads..these are so so 'unnatural circumstances' for the meiofauna, and the protists which are our helpful and essential neighbors.


Please consider a native clump of moss or water sample from your home locale...rather than 'cooked organics left to fester'. charlie g, finger lakes?US

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linuxusr
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Re: Hay (Con)fusion

#4 Post by linuxusr » Sat May 29, 2021 3:57 pm

Your point is well made. Given that I'm dead center in the city, there are no lakes, rivers, ponds, streams, etc. But you have given me an idea. Instead of buying alfalfa from Amazon and shipping it here, I should go to a park with trees, bushes, and grasses and find stuff there. I think I will try that.

Now I am using soil from my chicken coop . . . nearly a week has past and no organisms in my two specimen jars. Do you think I should dump and start over or wait some more?
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Re: Hay (Con)fusion

#5 Post by BramHuntingNematodes » Sat May 29, 2021 4:53 pm

A potted plant even in an urban setting is likely rife with nematodes if they are of interest
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linuxusr
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Re: Hay (Con)fusion

#6 Post by linuxusr » Tue Jun 01, 2021 12:38 am

OP Add-on:

I've been reading about culture media for protozoa at microscope-microscope.org There is one item in these directions that I don't understand:
[The "hay infusion" is perhaps the most well known culturing technique. Boil one liter of pond, spring or rain water. As the water comes to a boil, add a small handful of hay (ideally, timothy hay) and boil for ten additional minutes. The boiling will break down the hay and set up an ideal medium for the growth of bacteria. Allow this mixture to stand for two to three days. Add 25-50 milliliters (2 to 4 T.) of your sample (this is called "inoculation"). In a few days, small protozoans such as Chilomonas will populate your culture. If Paramecium are present, they will feed on the Chilomonas and eventually increase in number (in 10 to 14 days). The organism at the top of the food chain will become the most common but will quickly die off as the food supply is exhausted. You may be successful in maintaining one organism for long periods of time by sub-culturing into newly prepared media./quote]

OK, I have prepped my hay infusion and it's sat for a few days. Fine. Then I'm told to add a small quantity of my sample (inoculation). What sample? I don't have a sample. I only have the hay infusion. Unless the meaning is that the hay infusion is my first culture and I want to propagate it by inoculating to the infusion?? But this is never explained, so I'm confused.
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Peter
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Re: Hay (Con)fusion

#7 Post by Peter » Tue Jun 01, 2021 5:48 pm

What they are intending for you to do is introduce a small sample from a local: pond, stream, swamp, river, lake, pudle, or whatever "natural" water source you wish to experiment with.
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Harold
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Re: Hay (Con)fusion

#8 Post by Harold » Sun Aug 22, 2021 11:19 pm

I too was a bit confused by the common instructions about preparing a hay infusion. What you're really doing by boiling Timothy hay in non-treated water is preparing a culture medium to receive a sample of whatever. Once you've done that, the culturing possibilities are endless. Throw a small handful of grass clippings into the prepared medium and you'll be amazed at the critters that show up in a few days to a week.

enricosavazzi
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Re: Hay (Con)fusion

#9 Post by enricosavazzi » Fri Sep 17, 2021 5:52 pm

I had good results by plucking a handful of dry moss off a granite boulder in a public area in Stockholm, Sweden, in the autumn or early winter. I placed it in a small translucent polypropylene jar and filled 3/4 of it with tap water, then left the closed jar a few weeks on the window sill where it could get indirect sunlight (but no direct sunlight). Within one week the moss started growing off the little soil that came with the sample. The best find was a piece of biofilm of 2-3 sq mm with plenty of testate amoebae, cyanobacteria, occasional nemerteans and dozens of different species of "microalgae" and bacteria (but no diatoms and no moving protozoans). After six months the biocommunity was still quite complex.

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DonSchaeffer
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Re: Hay (Con)fusion

#10 Post by DonSchaeffer » Sat Sep 18, 2021 3:01 am

Take the loose grass and leaves from the park. Ciliata are carried as cysts by birds and animals as well as the wind. In a few days they will start to appear.

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Re: Hay (Con)fusion

#11 Post by DonSchaeffer » Sat Sep 18, 2021 3:11 am

lorez2 wrote:
Fri May 28, 2021 3:06 am
Regarding "what's in the sample", I had an unusual experience yesterday wherein I picked a cup of water from a duck weed covered pond to take a quick look at with a stereo microscope. There were no moving critters at all... at the 10X and 30X magnifications of the scope. This is a first for me. Of course, a lot was unseen using only a simple stereo scope, but seeing nothing of the usual suspects was a bit of a surprise. Having a simple stereo microscope permanently set up on top of a file cabinet is a great way for some interesting ad hoc viewing.

lorez
Most ciliates are too small to be well seen with a stereo microscope. You may see little moving dots on a dark background but nothing on a bright background. Often what you can't see with astereo microscope will be viable at 100x or more.

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