SIP Week 8 Sharing

Subject: Lab Technique

Name of Test: Faecal Extraction

Hi people! Because I am in a research lab, I don’t get a chance of rotating around the different labs. And so, yes, I am still having my attachment at the endocrine lab.

Well, most of the time I am doing my MP. So, in this post, I am going to talk about a lab technique that is frequently being carried out in the lab for my MP.

Faecal Extraction. Yup. You guys didn’t read wrongly. Its faecal extraction. It is not really that smelly.

But before I go into that, let me discuss about my MP first. My MP has got to do with the clouded leopards. Clouded leopards are presently being classified as vulnerable and their actual numbers are unknown as there hasn’t been much research done on them. And because their numbers are declining, there are many conservation projects that are being undertaken by the zoos. However, the breeding process shows little success. This has been thought to be due to stress. Hence, for my MP, my main aim would be to establish their stress level and hope to be able to provide information to help reduce the stress brought about to the leopards.

To help analyse the stress levels, the hormones that we are measuring are glucocorticoids - cortisol and corticosterone and the samples used were faeces.

Why faeces?
This is because it can allow a more accurate result obtained. The collection of faeces for testing is a non-invasive method. Hence, it does not induce stress to the animal which could eventually lead to bias results. Urine could be another sample option but as we all know, it’s kind of hard to collect urine especially from such a fierce animal. Blood samples are not used as this is an invasive method in which it will tend to induce stress to the clouded leopards and thus could result in biasness in the results obtained. Furthermore, it is impractical to collect blood regularly.

Before we can measure the hormones, we need to extract steroids from the faeces first. This is done using the boiling method.

Method:
1. Dry the faeces using a lyophilizer for about 2 days.
2. Pound the faeces into powdered form using a pestle.
3. Weigh out 0.200 ± 0.002g of faeces into the test tubes.
4. Add 4.5 ml of ethanol and 0.5 ml of distilled water to the test tubes.
5. Mark the levels of the solution in the test tubes.
6. Vortex briefly.
7. Boiled the test tubes in the water bath at 95˚C for 20 minutes.
8. Add 100% ethanol as needed to prevent the test tube from boiling dry.
9. Bring the volume up to pre-boiled level using 100% ethanol.
10. Centrifuge the samples at 2500 rpm for 20 minutes.
11. Pour off the supernatant into another set of clean test tubes.
12. Add 4.5 ml of ethanol and 0.5 ml of distilled water to the test tubes.
13. Centrifuge the samples at 2500 rpm for 20 minutes.
14. Pour off the supernatant into the test tube containing the first set of supernatant.
15. Dry down the second set of test tube using a water bath and purified compressed air.
16. Add 1 ml of PBS and vortex briefly.
17. Sonicate for 15 minutes.
18. Pour the extract into 12 x 75 mm plastic tubes and store them at -80˚C.

Hope you guys understand. =) How to go about analysing the hormones level will be explained in the next post.

Enjoy.

Xin Yi
TG02