The microbes in the hay infusion may come from the dead grass that was put in the cup. Some of them came through the air that was channeled to the surface of the water table.
In the hay or on other dry plants, leaf litter etc. there are numerous resting stages of bacteria (e.g. spores of the so-called “hay bacterium” Bacillus subtilis), algae, other protozoa or rotifers and tardigrades . After a few days in the water, these organisms come back to life.
After about a week, bacteria and protozoa should be visible under a light microscope. Most organisms can be observed at 200x magnification. The sugars in hay form the basis for a food web. Bacteria feed on the sugars and protozoa feed on the bacteria and each other.
The hay infusion contained an abundance of different species of protists, including flagellates, amoebas, and fungi (Figure 3 A, Figure S1). Most of the diversity was found in the family Ciliophora, so we designed and optimized a ciliate-specific ssu rRNA probe.
Take a handful of dried grass or hay (free from pesticides or herbicides) and cut the grass into smaller pieces. Put the cut grass in the cup and about 0.5-1 liter of water. Add 1-2 drops of milk. The water becomes slightly cloudy.
A good way to catch them is to turn a container upside down until it is positioned just above the surface of the mud. Then slowly let the air out so that the top one layer is sucked into the glass. You can move the glass slowly as you tilt it, allowing you to collect from a larger area.
Boil a liter of pond, spring or rain water. When the water is boiling, add a small handful of hay (ideally timothy grass hay) and boil for another ten minutes . Boiling breaks down the hay and creates an ideal medium for bacteria to grow. Leave this mixture for two to three days.
Next, why did we add the yeast? Yeasts are tiny fungi that multiply rapidly, feeding on the nutrients from dead grass. Yeast is excellent food for protozoa. Initially, the population will be unbalanced.
During incubation, check the infusion and add more pond water as it evaporates. In 5 to 10 days, the broth should turn dark and cloudy. Examination under a microscope reveals a large number of microorganisms.
Hay infusions are nutrient media for growing ciliates, such as Paramecium, which are interesting microscopy specimens. A little dry grass (e.g. hay) is boiled with a little water. The water turns brownish-green and looks a bit like tea.
The easiest way to collect small pond life and organisms is to squeeze water from plants or pond sludge into a container. Scrapping the growth of aquatic plants or anything covered in green or brown vegetation usually results in high levels of microscopic pond life.