Worm Collection

Worm collection

    Stuffs to bring

• 2 x 1 l PBS  buffer (make this in advance and store it at 37 C)

• Table

• Boots

• Scissors to cut intestines

• Cooler (5 GAL)

• Thermometer

• Gloves

• Boning knife

    PBS buffer pH 7.0 (4 l), check pH by pH indicator paper

• Na2HPO4    9.44 g

• NaH2PO4 2H2O    5.24 g

• NaCl    35 g

Making activator

1. Dissect out vas deferens from Ascaris tails. 

2. Put glass homogenizer on ice, put each vas deferens in homogenizer after dissected. 

3. Add 1 ml HKB buffer to every 10 vas deferens. 

4. Homogenized well. 

5. Centrifuge at max speed at 4 C for 10 min. 

6. Aliquot supernatant; 50 ul into 1.5 ml microcentrifuge tubes and store at -70 C.  

    In our lab we use 5 - 10 ul of activator at full strength to activate cells in a 1.5 ml tube.  It is also okay to used a drop of diluted activator (1:20) for a 1.5 ml tube.  

    HKB buffer pH 7.0 (500 ml)

• HEPES    5.95 g

• KCl    2.6 g

• NaHCO3    0.42 g

        Add KOH pellets (about 7 pellets) to bring pH to 7.6.  Then use CO2/Nitrogen gas to bring pH to 7.0.  

        Using fresh HBK is really important for sperm activation. 

Fixing crawling sperm cells

    It is bit tricky to fix MSP filaments in crawling sperms since it disassembles very quickly.  The best way to do this so far is using a perfusion chamber so that you can exchange HKB buffer to fixative quickly.  

1. Make a perfusion chamber by assembling two cover slips (22 x 40, 22 x 35 mm) using a double sided tape as a spacer. 

2. Infuse activated cells to the chamber and observe it with a microscope.  The chamber needs to be warmed by a dryer to help them crawl.

3. Perfuse 2 ml fixative from one side and suck buffer from the other side using paper filter. 

4. Incubate for 20 min at r.t.

5. if you used GTS fixative wash out fit by perfusing HKB.  GTS fixative makes precipitation that bothers EM or light microscopy experiment. 

6. Keep fixed cell at 4 C.  

    GTS-Fixative (2.5 % Glutaraldehyde, 2 mg/ml tannic acid, 0.5 mg/ml Saponin) in HKB (40 ml)

        Glutaraldehyde     4 ml 

        Tannic acid           80 mg

        Saponin                 20 mg

KOH-washed cover slip

    I would recommend using KOH-washed coverslip for crawling assay.  More cells stick to the surface. 

1. Place coverslips in a glass container and incubate with 1M KOH for over nigh.Rin

2. Rinse coverslipss with ddH20 for ten times

3. Fill the container with ddH20 and sonicate in water bath for 30 min

4. Fill the container with 50 % Ethanol and 30-min sonication

5. Fill the container with 75 % Ethanol and 30-min sonication

6. Fill the container with 95 % Ethanol and 30-min sonication

7. Fill the container with 95 % Ethanol and store at r.t.