🌾 Paramecium wheat grass medium

ATCC medium 802: Sonneborn's Paramecium Medium

🚛 Reagents needed 📦 :

Protocol

1️⃣ Add 2.5 g of wheat grass to 1 Liter sterile water and boil for 5 minutes.

2️⃣ Add 100 ml sterile water to compensate for evaporation.
3️⃣ Filter through Whatman #1 filter paper.

Color of the medium will change from green  to yellow hue, similar to LB medium.

4️⃣ Add 0.5 g Na2HPO4.
5️⃣ Adjust pH to ~7.0 with NaOH or HCl
6️⃣ Autoclave for 15 minutes at 121 ̊C.

Store in 4°C in dark until you want to inoculate with bacteria.

💪 Preparation of steroid supplement

Paramecium requires a steroid as a growth factor.
β-sitosterol is here used to supplement the medium with a growth factor.

β-sitosterol has extremely poor solubility in water and needs 99% ethanol (95% is not good enough).
Make a stock solution of 4mg/mL in pure ethanol and keep in the dark at 4°C 🌌.
In a 15 mL Falcon tube add:

It will take some time for the steroid to fully dissolve. Once dissolved store in 4°C away from light and add to medium just after bacterial inocculation.

🧫 Inoculation with Klebsiella pneumoniae

Inoculation takes 2 days. First day is spent on culturing the bacteria on agar plates then it takes 1-2 days to inoculate the bacteria in the wheat grass medium.
It is important to let the bacteria bloom before adding the Paramecium.

🚛 Reagents needed 📦 :

1️⃣ Smear samples on agar plates and incubate at 37°C overnight.
2️⃣ In the morning verify culture.

3️⃣ Take a loop and collect some of the recently grown bacteria onto the loop and swirl around in 5 mL sterile wheat grass medium.
4️⃣ Place the tubes inside incubator on shaker (200 rpm) and shake at 37°C overnight.
The medium should go from clear yellow to opaque. 

Klebsiella pneumoniae neutralization of components in the wheat grass medium that is toxic to Paramecium. Don't incubate the paramecium in the WGP medium until you have a good bacterial bloom.

5️⃣ Bring the β-sitosterol (567152-5GM) stock solution 4mg/mL out from the cold room and add to the culture in a final concentration of 0.8 μg/mL
(i.e. if 50 mL culture, remove 10 μL of medium and add 10 μL β-sitosterol (4mg/mL) for a final concentration of 0.8 μg/mL).

6️⃣  Add 100 μL of Paramecium from an ongoing culture. Incubate without shaking at 27 °C. 

7️⃣ Check the culture by taking 20 μL from the center just under the water surface and smear out on a microscope slide. Check daily for 2-3 days and you should see the culture rapidly expanding in cell density. 1000-2000 cells/mL is what you want to aim for.