Hello everyone,
I'm currently collecting Eimeria parasites for in vitro tests and would like some advice on improving the process.
Firstly, can I replace chemical-grade NaCl with table salt for flotation, and if so, at what concentration (35% or 40%)?
After flotation, I wash to remove NaCl by adding distilled water at a 4:1 ratio. However, some oocysts remain in the supernatant despite centrifugation. What is the ideal speed and duration for this wash?
Additionally, the oocyst load in the collected intestines is low. Is there a way to maximize the number of oocysts in a minimal volume ? Can I concentrate the oocysts by resuspending isolated pellets from flotation?
After isolation, I proceed to sporulation and purification. Should I add antibiotics or antifungals, or is potassium dichromate sufficient? Some use sodium hypochlorite for purification – is it necessary? Are there other effective purification methods?
Before conducting in vitro tests, I need to wash the oocysts to remove the dichromate. Is suspending them in PBS the correct method for this?
Thank you in advance for your responses and advice.
Regards,
I am sharing some points that I experienced during my work on similar objectives.
- 2% or 2.5% potassium dichromate solution is sufficient, for it facilitates oocyst sporulation and, on top of that, prevents microbial growth because of its antimicrobial activities.
- After washing the oocysts to remove potassium dichromate solution particles, storing them in PBS at 4 degrees Celcius is the best method.
Kindly consult the article mentioned; it may help address your queries to a greater extent. Methods in coccidiosis research: separation of oocysts from faeces by Ryley et al., 1976.
Hi Amira,
1. It is safer to use chemical grade NaCl rather than normal table salt because of impurities the latter might have which will affect the specific gravity of the resulting solution. Although, I have had some level of success using table salt at 40% concentration - but I won’t advise you use the harvested oocysts for exprimental studies.
2. Recovering most of the oocysts after each wash can be challenging. The standard protocol would be to spin at 2000-2500 rpm for 10mins, but this may still leave a considerable amount of oocysts trapped in the supernatant. Your best bet would be to further dilute the supernatant and spin again until you are able to isolate most of the oocysts.
3. Have you considered ezymatic digestion of the intestinal tissue to see if you are able to recover more oocysts?
5. Potassium dichromate is sufficient for sporulation and can be used for its antibacterial properties. However, standard protocols would further purify oocysts with sodium hypochlorite. In addition to its application as a disinfectant, sodium hypochlorite helps to degrade faecal debris.
6. In conclusion, to increase the amount of oocysts, you might want to propagate them in-vivo.
Hi Oluwayomi Adeyemi ,
I will therefore choose chemical grade NaCl to ensure the purity of the solution and the reliability of the experimental results.
For point 2, that's what I do, but it takes me an enormous amount of time, especially since I use a centrifuge with 15 ml tubes.
Thank you for the suggestion in point 3.I haven't tried this method yet. Could you share more details about the recommended enzymes?
Thank you again for your valuable advice.
Best regards,
You are welcome, Amira.
You might want to use 50 ml Falcon tubes - that should help you to conserve more time.
Please forward your request to [email protected]; I should be able to share the tissue digest protocol with you.
Kind regards,
Yomi.
Thank you Oluwayomi Adeyemi
I'll make sure to use them. I'll also send my request for the tissue digest protocol.
Kind regards,
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