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To begin with, Dr Rosmilah, the assigned lecturer for this course, SSS1024 - Integrated Seminar in Science, has introduced us to the basics, including the expected outcomes in taking the course. We were briefed about our assignments, including a group project which carried a heavy weightage for this course. We were to work with different lecturers other than Dr Rosmilah for this project, who will be supervising the groups for the course of 12-13 weeks. My group was assigned Dr Nurhaida, which was fortunate as we had previously taken a course under her for our last semester, so the communication process was expected to be a smooth one throughout conducting this project.

My group leader had contacted Dr Nurhaida as soon as we were advised to do so by Dr Rosmilah to discuss about the aspects of this project. By the end of discussing with Dr Nurhaida, we had agreed upon a day and time of formally meeting each other to discuss about the project she had in mind in greater detail. For the rest of the week, we had looked up videos on the techniques of isolating microbes from soil, as instructed by Dr Nurhaida.

I hadn’t had a difficult time grasping the techniques displayed in the videos I had looked up as the techniques were something I had previously done with success during my last semester with Dr Nurhaida. I was excited to be given the chance to try something related to this technique again, although, the last time, i had worked with prepared bacterium instead of soil. I looked forward to the meeting with my mentor/supervisor.

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Dr Nurhaida and my group members had agreed upon meeting in her office on Wednesday, 24/01/2024. The meeting started with Dr Nurhaida proposing the project we will be project on, which is to isolate fungi and yeast from the soil sample she had collected on an expedition last year. She had explained that soil contains a wide range of microbes, and if we were to isolate a specific microbe, we would have to use a suitable medium that would allow the growth of these microbes. Dr Nurhaida then introduced my members and I to the rose bengal + Chloramphenicol agar powder. The rose bengal in this powder has the ability to promote fungal and yeast growth, and the Chloramphenicol is used to inhibit the growth of bacteria, which is exactly what we need in order to focus on isolating just the fungi and yeast.

We then went to the biotech lab located in the first block. Dr Nurhaida had briefed us on the materials and apparatus’ uses and locations in the lab. We had agreed on starting all lab works starting Friday, 26/01/2024. The meeting ended shortly after that.

As much as I was looking forward to conducting this project, I was a little nervous as we all came to accept the fact that it may require a lengthy trial and error process in order to isolate the fungi and yeast from the soil. Soil home up to hundreds if not thousands of microbes, and thus utmost accuracy in preparing the medium and curating it it required in order to succeed. We also had to mostly work on this project on our own, which meant the experimenting process might take longer, as this was something neither me nor my members had never done before. Though it could be very insightful if we manage to achieve our goals in this experiment.

Regardless, we had went in to the lab as agreed upon on Friday, and did preparations of materials and apparatus for the experiment. The experiment was set to begin on Monday, 29/01/2024. I nervously looked forward to Monday.

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We begun the week by going to the lab on the 30/01/2024 to start with the his trial of the experiment. We had previously prepared a potato dextrose agar solution which was autoclaved along with the rose bengal + Chloramphenicol agar solution.

The isolation process was done using a serial dilution process, in which the specific soil sample ‘S8_D3’ was selected and 1ml of the soil sample was diluted with 10ml of sterile distilled water in a test tube in a laminar airflow. Then the serial dilution process was carried out with a total of 4 dilutions, with 1ml of the previous dilution and 9ml of sterile distilled water, and the results from each dilution were poured into a petri dish where my group mates and I added 1ml of the dilutions with a sufficient amount of the rose bengal + Chloramphenicol agar solution on a petri dish. We had also prepared several small and big petri dishes of the potato dextrose agar solution, and put everything in an incubator set at 37° Celsius.

My only worry about the first trial is that we had missed a missed wherein we were required to close the top of the test tube with a cotton wool after each dilution, but had overlooked that part. I was hoping it would not affect the results of the experiment. We were set to go to the lab again in a few days. On 01/02/2024, I went back to the lab to check in on the results of the experiment. There were a few growth on all the agar with different colony densities, and the results were shown to Dr Nurhaida.

Unfortunately Dr Nurhaida mentioned that the colonies were too densely packed, and so we repeated the entire process with our sample ‘S8_D3’ again by using the same process. The results would be checked again in a day’s time. To our disappointment, the second batch of samples had yet to show any fungal growth from any of the dilutions. We were told to leave the petri dishes in the incubator for a few more days. This showed me that time and patience is necessary in this experiment in order to witness the full growth of the fungi and yeast.

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On the 5th of February 2024, I went back to the lab with my group mate to check the results of the second run of the sample ‘S8_D3’. The growth on the agar looked promising enough, and so after showing the results to Dr Nurhaida for approval, we began to prepare to subculture our samples on the petri dishes prepared with potato dextrose agar previously.

To our dismay, we had made a mistake of leaving the potato dextrose agar in the incubator for far too long. The agar was dehydrated by the time we were ready to subculture our samples, and so the process was halted as we had to prepare a fresh batch of potato dextrose agar medium. With the help of the lab assistant, we left them in the autoclave machine before we were bound to start the subculture process.

The progress for this week had been the slowest, as the Chines New Year holidays allowed us to return home for a short break, and so we planned to continue the experiment on week 6. I am very disappointed for overlooking the first batch of the potato dextrose agar medium, as this had pushed back our experiment quite a bit. Though soon after, I had come to terms with the fact that trial and error was necessary in this experiment in order to achieve satisfactory results. This would also help me prevent from doing the same mistake twice, and learn how to efficiently run an experiment.

Despite the lack of much progress this week, it was also one of the most insightful as I had learned a lot about the agar medium used and it’s efficient storage method to use in the upcoming weeks, as well as for any other experiments i may do in the future. I excitedly looked forward to the coming week to prepare the petri dishes with the subculture medium so we could gain our results sooner.

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On the 14th of February, my group mates and I went to the lab again to subculture our samples into the prepared potato dextrose agar. It was a smooth process as Dr Nurhaida had previously explained to us the correct method of subculturing our samples.

We were expected to transfer the growth that were seen on the Rose Bengal + Chloramphenicol agar according to their colonies. The growth noticeable on the agar mostly looked similar in shape, size and colour, which is white, except for a few which were as small as tiny dots, but still white in colour. After subculturing and incubating our samples, we had a meeting with Dr Nurhaida on the 15th of February. During the meeting, we discussed many beneficial information, including possibly identifying the types of growth that were seen on the agar. Dr Nurhaida had also made us start on new samples so that the progress would be much quicker.

So the entire process from preparing Rose Bengal + Chloramphenicol agar, potato dextrose agar to preparing and streaking serial diluted samples were made for other new samples. We had also prepared extra potato dextrose agar in advance for future uses for all the samples we were working on. Dr Nurhaida had also wanted to eventually check whether the growth on the subcultured plates were fungi/yeast or not.

This was a rather productive week as we had managed on work very quickly on as many samples as possible in a short period of time. It had also taught me how to work quickly and precisely without breaking the sterile field in the laminar air flow chamber. I have high hopes that we will soon get to move the next part of the experiment, which is to create a stressful enough environment for the fungi/yeast that it will produce lipid on its own as a survival mechanism.

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Moving on to week 6, on the 19th of February, we had yet another meeting with Dr Nurhaida. She had briefed us on the next step of this project, which is to perform a technique called Single Spore Isolation (SSI). This technique is usually performed to obtain a pure culture of a fungus. This process involves isolating and cultivating a single spore from the fungal spore mass we had subcultured for all samples in order to establish a genetically homogeneous culture. Dr Nurhaida had taught us a step by step guide as to how to perform this technique during our meeting. The result will be called a pure isolate.

And so we began to single spore isolate from all of our samples. With great care and precision we had done this process in order to prevent cross contamination and provide the best results for our samples. We also meticulously labeled each and every sample with lengthy but necessary unique IDs in order to make it easier for us to figure out the master sample as well as to update it in an organised way in a spreadsheet for reference.

The activities done throughout this week was a rather repetitive one, but it has proven to me that I am capable of and very much enjoy doing stuff that are repetitive in nature such as this one, and has given me a slight idea of the types of job I would enjoy working in the future. It has also made me strictly follow all rules  we initially agreed on to keep the workplace sterilised as well as to accurately isolate our samples. Communication between my group mates also improved through this as we had to interact frequently given we had separated our tasks accordingly.

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Another meeting was held with Dr Nurhaida at our usual microbiology lab, during which we moved to one of the final, but also crucial part of this project. As expected from the objectives we had initially stated in the beginning of this project, we were expected to study the morphology of the fungal samples he had collected from Behrang Reserved Forest.  In simple words, this means to examine and study the form and physical structure of the fungi we had sampled and isolated. The purpose of this is get a deeper understanding of fungal biology.

This was done under a microscope, as these fungi are far too small to be observed with naked eyes. Hemacytometer, which is a specialised gridded glass slide used to count cells and microorganisms accurately, was used throughout this process in order to measure the fungal population from the isolated samples. To observe on the hemacytometer, lactophenol cotton blue (LCB), which is a mounting medium commonly used for the microscopic identification of fungi, was also provided by Dr Nurhaida, and we were guided on ways to use it to stain the sample and slide to proper observation under the microscope.

The actual observation process ended up being a very thorough and exhausting one, as it deemed to be rather difficult to find extremely clear images which highlights the morphological nature of the fungi including the spores, hyphae, and fruiting bodies. We had to repeat the observation process several times for all successful isolated samples, and this process stretched out until the end of the week.

The activities carried out this week gave me beneficial insights and knowledge of how to handle fungal samples with their specific staining agents and apparatus. It had also taught me a lot on the morphology of fungi, as we had all discovered many different shapes and structures of fungi throughout the observation with different samples. This had inly further increased my interest and love for microbiology, and I definitely intend to put this knowledge to use in the future by pursuing a degree related to microbiology.

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The ninth week of the project was carried out by once again diving deep into the single spore isolation technique. During a meeting with Dr Nurhaida, we went through the aspects of how to use the single spore isolation (SSI) technique to separate the fungal spores individually from the mass sample in order to create uniform cultures. This includes counting the spores and making accurate calculation in order to dilute them to the correct concentration in order to isolate them individually.

Though it took us several tries, we had eventually understood the formula and calculation method in order to accurately determine the spore density to be distributed to prepared plates with potato dextrose agar (PDA) on them. Dr Nurhaida had mentioned that we would be aiming for around 30 spores per agar plate, so we had adjusted our calculations accordingly in order to avoid overcrowding the plate with spores.

As previously mentioned, it took a few trial and error to get to an accurate result, but with the constant hard work of my group mates and our dedication to not giving up, we made it. This technique proved to be crucial for many scientific and industrial process, and I am glad I had the chance to experience the usage of this technique as preparation for the future. I also learned the importance of applying the formula and calculating correctly in order to get the most accurate count because dealing with living microorganisms such as fungi, can easily go out of hand with a small yet simple mistake.

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Throughout week 10, we had been continuously observing the fungi under the microscope as we had done before.

We were also dedicatedly preparing for the seminar, which had eventually been turned into a simple class presentation. As we reached the final stages of this project, we had begun to work on the poster and report for the presentation.

My group mates and I made sure to incorporate simple yet detailed information so as not to overwhelm and confuse the listeners. We wanted to make as many people understand our project as we could, so that they would also take interest in the fascinating identification and study of the fungal species.

We made sure to regularly check with Dr Nurhaida for the information we decide to include in the poster. As for the report itself, we had included everything we had done, from A to Z, so that we could truly show just how much effort we had put into this project.

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The update for this week would be rather short, as my group mates and I went home for the Raya holidays. We with the lack of people to observe the fungi, we had made sure to place the samples and the agar plates in the freezer before leaving, to prevent them from dying so that we could continue to observe them once we got back from the holidays.

While at home, my friends and I still continued to work on the report in hopes to produce a satisfactory report that would reflect our hard work.

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Same as the previous week, we were still away from the college and laboratory, so the most I could do was continue to prepare for the presentation.

My group mates and I had divided our work for the report writing accordingly and updated every single picture and information we could input into the report. This might have been the most detail and satisfied I had been with writing a report.

I anticipated returning to the laboratory and wrapping up our work once the Raya holidays were over. While at home, I looked up videos and articles about fungal species. This genuinely shows how this project had piqued a new interest in me, and I am much grateful for this course, and Dr Rosmilah for being the catalyst for this newfound interest.

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We were all truly back to the laboratory after the holidays. My group mates and I got back to the freezer, and fortunately, the fungi were very much still alive.

We had continued to observe them under the microscope, but to our dismay, not many ultra clear pictures could be captured from the objective lens of the microscope, and this posed a new problem, as Dr Nurhaida had been hoping to get a clear image of the 2 different types of fungi we had successfully managed to identify, so that we could study the morphology well.

To overcome this problem, we had decided to use a new microscope attached to a screen, also called a digital microscope, in order to portray clearer pictures of the fungi. But this would only be done during week 14.

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It was the final week of the project. As I prepared to present our results to my peers and Dr Rosmilah alongside my group mates, we had also began to observe the fungi under a digital microscope. We had to prepare small amounts of agar on glass slides and inject tiny amounts of the fungal sample into the agar, and let it sit in the incubator for 3 days in order to observe the growth on the agar under the microscope. The preparation was done on Monday, and we went to the lab back on Thursday to observe the results.

On the morning of Thursday though, I and my friends had to first present our work of labour to our friends and lecturer. It was rather nerve wracking, but I believe I had done a good job with the presentation. My group mates and I were very satisfied by the end of the presentation.

We then went back to the lab, and fortunately, we had managed to capture a few clear pictures which had satisfied Dr Nurhaida.

With that, we wrapped up our project of 3 months for good. It had been bittersweet to part with Dr Nurhaida and the lab I had been religiously going to every weekday, but I am also very happy for being able to learn an abundance of new knowledge from this project.

I could not thank Dr Rosmilah enough for giving us this opportunity, I will always be grateful. I hope that, in the future, my juniors would be able to experiment more with the fungi we had left for future use, had discover more fascinating information later.