Hello! Zhang’e here. I’m going to share with you how fecal extraction is done.
Feces were first dried using a savant rotator evaporator for 2-3days and were then pounded into powder form. 0.200g of each powdered feces was weighed out in labelled 16 X 125mm glass tubes and 4.5ml of ethanol and 0.5ml of distilled water (90% ethanol) were added to each tube. The tubes were vortex briefly and placed in a water bath at 96° for 20 minutes. The rack was kept from touching the bottom of the water bath to prevent splattering of tube contents. 100% of ethanol was added continuously to the tubes during boiling to prevent boiling dry. After boiling, 100% ethanol was added to all the tubes to bring the volume of the extract up to approximately pre-boil levels. The tubes were centrifuged at 2500rpm for 20 minutes and the extract from each tube was poured off into a second set of identically labelled 16 X 125mm tubes. 4.5ml of ethanol and 0.5ml of distilled water (90% ethanol) were added to the original set of tubes containing the fecal pellet. The tubes were vortex for 30 seconds and centrifuged at 2500rpm for 20 minutes. The extracts were again poured into the second set of tubes containing the first extract and the second set of tubes was dried down under air (nitrogen) in a warm water bath (80°). The dried down extracts were re-suspended with 1ml of buffer ‘S’, vortex briefly, and sonicated for 15 minutes. The extracts were poured into labeled 12 X 75mm plastic tubes and were kept for storage.
-In this extraction, ethanol is the solvent used and through vortexing and boiling, the hormones are extracted out of the feces.
- Centrifugation separates the extract (containing the hormones) from the fecal pellet.
-The purpose of extraction is to concentrate the amount of hormones by using ethanol to absorb/pick up the hormones and separates them from interfering compounds (such as steroid-binding proteins) and the extracts obtained are used for hormone assays. (Rather than using the whole feces to perform the assay)
Thats all! It is simple but it takes a day to finish the whole process!
Zhang'e
0704086H
TG02
Zhang E,
ReplyDeleteWhat are the hormones are present in the faeces?
Li Yinliang Alex 0704894E
Group 2 TG02
22 September 2009
Hey Zhang'E,
ReplyDeleteAs it is faeces, will there be like other organism (e.g. Virus) present in the extracted sample?
Is there anyway to prevent the microorganism from contaminating the faeces samples?
Cheers,
Tiong Han
Tg01
hi , can i know what is buffer S? and why do you sonicate the extract?
ReplyDelete(:
LIM JIA HUI (joey)
TGO1 0703605F GROUP 2
Hi Zhang'e,
ReplyDeleteCan I know how ethanol pick up or absorb hormones and separates them from interfering compounds?
Thanks =)
Lok Pui
People! Sorry for the late reply!!!
ReplyDeleteReply to Alex
HI..! To your question, most of the reproductive hormones are present in the feces. Examples such as progesterone, cortisol, estrone, estradiol, testosterone and their metabolites.
Reply to Tiong Han
Hi hi..!!
Yes, there will be microorganisms present in the extracted samples.
Prevention
When the zoo people collect the feces, microorganisms such as bacteria will grow on it. Certain virus may also be excreted out and found in the feces. However, the zoo people will store the feces in -80°C freezer straight away and at this temperature, most of the microorganisms are killed. When the feces are transferred to our lab, we will freeze-dry them for a few days before pounding the feces into powder form. During freeze-drying, the presence of microorganisms will also be reduced. (Freeze-drying at -80°C and water is totally absorbed out). And during extraction, there is boiling. Boiling will also reduce the presence of microorganisms. These are actually the prevention for the growth of microorganisms.
It is also impossible to get rid of the microorganisms completely. There must be certain microorganisms that will be present in the extracted samples. There are articles which show that microorganisms did interfere with the results, however, to only a certain extent. Normally in our lab, we use animal feces to measure cortisol levels and I did some research saying that the interference of these microorganisms to the measurement of cortisol is very minor. So it won’t be a problem performing the assay.
Reply to Joey
Hello Joey!
Buffer ‘S’ is actually PBS + chelatin (an additive). Chelatin helps to prevent compounds from sticking onto the wall of the tube.
The purpose of sonication is to use sound wave to remove air bubbles and mix the mixture well (some extract may stick onto the wall of the tube thus sonication allow Buffer ‘S’ and the extract to mix well).
Reply to Lok Pui
Hello Lok Pui!
As told by my mentor, ethanol pick up hormones is through the polarity of compounds. It is like “like dissolves like”. As different compounds contain different types of functional group or have different structures, they have different polarity. Ethanol is an organic solvent which is polar and thus able to pick up the steroid hormones which are polar. There are actually different solvents which can be used to extract hormones. It depends on the type of samples we have, the hormone that we want to extract and the extraction efficiency of the solvent. As reported, ethanol is able to extract cortisol in feces sample at a relatively high percentage.
Thats all..!
If you still have doubts, feel free to ask! =D
Zhang'e
0704086H
TG02