“I know it has been sometime since I last posted. In fact the blog post I’ve written below was done close to 1 ½ weeks back. For some reason I just couldn’t find the time to post it. I have moved forward quite a lot as at now, which I will blog about within the next few days. I don’t want to add it up here because it will only make the post lengthier and boring. Currently I’m running lots of bioassays, TLC separations and will be running a column soon”
There was a lot going on during the past couple of months that I can't believe I couldn't find the time to sit down with my blog. Final year semester exams, an ongoing research project and a family wedding to add to it where I was the bridesmaid, was not all so easy to go through at once. Thus my free time was stolen.
So, let me quickly wrap up on what's going on with my research. In fact I have advanced a lot further from the point where I blogged about last. I remember I ended by complaining about the long hours of "roto-vapping" required to isolate the crude extracts in solid form from the four fungal strains I obtained by culturing. These extracts were then air dried and further vacuum dried before weighing them. Then the weighed products were obtained for the EtOAc and MeOH/CH2
The next step was to test for the activity of these isolated compound mixtures against bacteria and bioassays were prepared subsequently. These tests were done against four main bacterial strains namely; MRSA, E. coli, B. subtilis
and Pseudomonas sp.
The crude extracts were mixed with EtOH up to a particular pre-calculated concentration and was loaded onto autoclaved (sterilized) paper disks using a micro pipette. The usual amount a disk could hold is 20 micro liters. Four disks loaded with the same compound was placed on prepared bacterial culture plates in nutrient agar medium and each trial was duplicated. This method was followed for all four fungal extractions and for each bacterial culture. Positive and negative controls were also used here. Gentamycin was used as the positive control. I first tested the EtOAc fractions and Whoa!!! I got positive results for extracts obtained from three fungal strains out of the four I cultured. One of them showed mild activity (strain3-using terminology from previous posts), and strain4 and strain2 showed considerable activity at a concentration of 150 micro grams per disk. I don't think I need to tell you how thrilled and relieved I was to finally possess two fungal strains with considerable antibacterial activity. I'm certain it was a great pay off for all the hard work I put in.
In order to catch up with the time, I was advised by my supervisor to continue the project forward, therefore instead of testing for the remaining fractions of MeOH/CH2
I started concentrating on separating the compound mixtures in strain4 and strain2 EtOAc extract into simpler ones. So, I ran couple of TLCs (Thin Layer Chromatography- A method of separation of a compound chemical mixture in small scale) at first in order to select the better fungal strain out of the two I have chosen, strain4 and 2. But strain4 showed better activity than strain2 and I had plans to continue on it anyway. Nevertheless I thought to check on the complexity of each extract before making a decision. The initial TLCs showed that the extract from strain 2 was more complex and having many compounds than the one from strain4. Therefore with a clear mind for higher activity and easy separation I picked fungal strain4 for continuation. For a detailed guide on how to run TLCs and what they are about, I'm attaching a link here from MIT Open Course Ware which I found quite short and sweet. Those of you who need to know what TLC is can take a look at this.
The selected fungal strain (strain4) was then grown in large scale, this time in 40 Pyrex bottles in PDB (Potato Dextrose Broth) medium just as before. Previously, handling 8 Pyrex bottles for each fungal strain was like torturing and I can't believe I held my nerve right through with 40 bottles. And this time for some unknown reason it was better than before. Same procedures were followed as in the case of small scale culturing and the fungi were left over a period of 42 days till they reached maturity. Luckily this time period overlapped with the exam time and I could happily take a break for my last minute express study routine. I have become such a last minute person that I reach my maximum productivity only when at the last min. I seriously have some personal issues here! Ha ha!
Gosh, I know I have already written too much but before I stop, let me quickly tell you about the harvesting of the fungal compounds. Here, the same method was followed but this time even the compounds from the mycelia were extracted into EtOAc and the fraction from the broth and the mycelia were mixed together. This step was taken as the positive activity was shown for the EtOAc fraction when previously tested. I have finished all the filtering, separating, and roto-vapping. The samples are now left to dry in order to collect their weights.
Also in the meantime, to get an idea about which specific portion of the mixture in the extract is responsible for the antibacterial activity, further TLC analysis is being conducted. I started by testing the separation for 100%CH2
, 1% MeOH/CH2
and 5% MeOH/CH2
solvent systems. Then I tried Hexane/EtOAc mixtures. Still I wasn't able to get a good separation. This means that the compounds are quite polar (ionizability is high) in nature and therefore I tested with MeOH/EtOAc mixtures. Here the 20% and 30% MeOH/EtOAc mixtures gave fairly good separations. Currently I'm testing further in reverse phase with MeOH and water as the solvent system. I had a good shot at it with 65% MeOH/H2
O just before leaving the university labs today.
Here I am right afterwards, typing this post at the IT unit in the science library. They will be closing soon. I need to go get something to eat now.
Greedily visualizing a hotdog and a hot Nescafé,
(I’m attaching the presentation I made for the weekly research group session here. Please check it out, all the pictures and illustrations are included there.)