Ah Friday, the last day of the week, the promise of the weekend and we were to carry on with Thursday’s work. More streak plates and agar were to be created, with the numbers made over the last few days ranging into hundreds and hundreds.

For Hayley and Sarah, it was time to let the E. coli loose on antibiotic plates. They had made four different types of antibiotic agar; kendomycin, tetracyclin, ampicillin and and cloramphenicol. There were however, only 12 (excluding a plate which had a clump of cotton wool embedded in it) antibiotic plates, meaning there were three plates of each type. This therefore meant that each test tube containing a strain of E.coli wouldn’t be able to have an entire antibiotic plate of its own. Because of this, each plate was divided into quarters, allowing there to be enough space for all the available (and living) strains to be tested. The bunsen burner was lit, sterilising surrounding air within a close proximity and the wire loop was flamed; it was time to complete antibiotic streak plates…

Sophie and Tammy arrived full of enthusiasm to continue the work on the frozen-dried samples of E. faecium. They produced some more M17 agar to produce further agar plates to be used in the afternoon.  The agar plates previously made yesterday, we used to produce the streak plates.

When working in microbiology aseptic techniques are employed. The Bunsen burner was turned on to sterilise the air around where we worked. All of the materials were within reach to prevent any accidents occurring.

Streak plating is a technique microbiologists use to attain single colonies of an organism to determine the colony and cell morphology used for identification. Their job was to establish whether the samples of Enterococci faecium were alive or dead.

Firstly, using a permanent marker pen, they wrote their initials, the date, the culture medium (M17) and the organism cultured (Enterococci faecium). Next, a wire loop was used to ensure a single colony was obtained. Prior to this the wire loop has been flamed by putting it into the hottest part of the Bunsen burner flame to sterilise it until it glowed red-hot. The M17 agar plate was briefly opened and the cells were smeared five times moving down the plate diagonally. The loop was flamed after each streak to reduce the bacterial load and ensure that single colonies were produced by streak five. The wire loop had to be cooled briefly before the next streak was completed to avoid the agar being burnt. The other streaks  passed through the initial inoculum and the rest of the procedure was the same as above.

We then had a look at the previous day’s streak plates produced observing any cell colony growth.  We found a number had shown successful signs of growth and recoded this information onto the table.  A number showed no clear signs of growth; this shows that further investigating will need to be looked into to more accurately catalogue the microbiology collection within the School of Life Sciences.

Day four started off with everyone splitting into their separate groups. There was a flurry of activity to start with, until we had to wait for agar and broths to be mixed.

Laura and Keiran were set to work with P.acnes (the bacterium that causes Acne rashes)  on TYG agar in an anaerobic environment (this involved using a cabinet which was kept anaerobic (under a nitrogen and not an oxygen environment).

Hayley and Sarah prepared agar plates infused with different antibiotics in order to test the resistance of the E.coli strains in the lab. A new type of agar was to be used instead of the standard nutrient agar which was called plate count agar (PCA). This was to ensure a greater growth of bacteria once the antibiotics had been mixed in and left to set, with the E. coli streaked out and ready to grow. The antibiotics of choice for this experiment were kendomycin (Km), tetracyclin (Tc), ampicillin (Ap) and cloramphenicol (Cm). This preparation involved some very delicate weighing techniques; for Km and Ap, only 0.5 grams was needed for every 10ml of squeeky clean, distilled water. In order to keep everything as sterile as possible, a sterile syringe and a filter were also used. Tc and Cm, however, were even more tricky, this was due to the fact that it was necessary to dissolve their complex structures in 70% ethanol (not to be used for a tasty tipple, nightcap or anything along those lines), they also required only a mere 0.1 grams per 10ml of ethanol. A slight problem arose when Cm accidentally had water added to it instead of ethanol (no idea who did that…), but this was swiftly corrected by adding another 0.1 grams of Cm and 1oml of ethanol to the test tube. After all this work though, the agar was not quite ready to pour, so it was time for a break. But here is a picture of the antibiotic tetracyclin, in its neon yellow glory:

Sophie and Tammy were given the task of investigating if samples of   E. faecium, which had been  stored in glycerol at the university,  were still alive and could be regrown and stored.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2010.04932.x/full

To achieve this we first needed to check each sample to see if the organism was alive. This required us to first grow them on agar plates suitable for the nutrient requirements of the microorganism, in this case M17 agar. The samples were then incubated and checked for signs of growth. We could then recorded the findings and the live samples were then stored correctly in a mixture of M17 agar broth and glycerol before being frozen and stored for at -86 degrees centigrade in a special low temperature refridgerator for future use.

Their first job was to make the M17 agar for bacteria to grow on. This was achieved by suspending 48.25 grams of M17 powderd agar into 950ml of distilled water and then bring it to the boil on a hot plate with a magnetic stirrer to blend the mixture. The mixture was sterilised by autoclaving and then left to cool. Just before use 50ml of sterile lactose solution (10%w/v) was added and they were good to go.  They poured the agar solution into the agar plates and left to cool, to be used later for producing streak plates.

After lunch, the PCA  made by Sarah and Hayley was ready to be poured. A small amount of antibiotic was pipetted into an empty petri dish, and it was then covered with a layer of molten PCA. After this, it was time for the swirling to begin, thoroughly mixing the antibiotic through the plate. This task took relatively little time, so whilst the plates were left to set, it was time for the dry ice fun to begin!

Dry ice is actually the solid form of carbon dioxide, and as seen in the picture on the left, the ice has been placed in a beaker of water and undergoes the process of sublimation (changing directly from a solid to a gas – without being a liquid), producing the strange fog/mist favoured by progressive rock bands the world over.

For our third day of laboratory work experience, the day started at 9 o’clock (again) with an inspection of the previous day’s work, i.e. looking at the “streak” plates of all the bacterial cultures that the group had made. It was fair to say we had mixed success, with some microorganisms like E.coli growing like weeds and providing a good full plate and some other types and strains of bacteria appearing to be dead or at least taking more time grow. So for those bacteria that hadn’t grown much, it was back into the incubator at 37ºC to be given a further chance to grow.

After this, we set about working in smaller groups, with one group producing streak plates on M17 agar, whilst another made an initial spreadsheet in order to catalog the microorganisms that we’d worked with. Finally, the last group set about making M17 broth, M17 agar and TYG agar. After these tasks were done, at around 10:30am, we had a break before we were to learn all about the world of blogging from Joss Winn who is a senior lecturer in the Centre for Excellence in Research and Development (CERD), which is based at the University of Lincoln in the MHT building, and headed by Professor Mike Neary the Pro-Vice Chancellor for teaching and learning.

At 12:30pm, we met Joss who provided with some training in the use of WORDPRESS “blogging” software and also showed us how to build the website that we would use to create and publish this very blog on. Turns out that there is a lot more to blogging than meets the eye!

Afterwards, we attended a lecture given Dr Andrew Westwell, a scientific collaborator of Dr. Tim Bates (who organised the lab. work experience described in this blog). Dr. Westwell is a Senior Lecturer in Medicinal Chemistry in the Welsh School of Pharmacy at the University of Cardiff. He was describing experiments using a new drug that had been synthesised by his team called ‘Phortress’ which is designed to act against breast cancer associated with an enzyme called E3 ubiquitin ligase (BCA2). His work is so much more complex than the undergraduate lectures on breast cancer, which meant that we were left feeling somewhat confused by the highly technical terms. But it must be said that Dr. Westwell and his peers have created a very important compound which is currently undergoing the first stage of clinical trials, in association with Cancer Research U.K.. Below is a summary of the lecture giving more detials on the developing novel drug:

PHORTRESS has a novel structure and mechanism with a high potency and specificity. It involves only one step conversion so there is ease of manufacture. Fluorescence and other techniques such as Positron emisssion tomography can used as imaging modalities to see where the drug is deposited in the body, as the molecule includes fluorine which makes the molecule fluorescent, and fluorine can also be used ot make positron emitting version of the PHORTRESS molecules..

The Ubiiquitin-protease system is targeted using this molecule, as there is a selective net uptake of the chemical which ultimately leads to cell damage and death via inhibition of the ubiquiting-proteosomal pathway.

BCA2 is only expressed in cancerous tissues, it can be isolated in the cytoplasm and nucleus, this is correlated with positive oestrogen recetor status and allows another target of the drug. This molecule has a significant ring domain structure involving cytene and histamine structure, this unique shape along with computer modelling can help scientists like those at the Cardiff university and at the Cancer Research Facility in Nottingham to adapt and engineer molecules with specificity with the BCA2 structure.

The Rab-7 molecule is very important in trafficking and decreases expression of the BCA2 which in turn reduces Rab-7 and consequently an increase in EGF-R(Growth factor degredaton). This means that the Rab-7 molecule has a negative effect on the growth of the tissue and therefore prevents proliferation of the tissue.

Dr Westwell mentioned the importance of disulphide bonds, these have a high affinity for zinc and inhibit expression of BCA2 which leads to growth inhibition. As this effect is only apparent with cells expressing the BCA2 gene, normal cells are not affected.

Although this drug has opened up a new point of entry into the battle against cancer, as scientists we must assess the overall effects of this molecule and continue research to determine the actual mprecise molecular mechanisms of thi and other anti-cancer molecules, as is happening in the laboratory of Dr. Bates, at the University of Lincoln).

Following this research lecture, which left many of us in a slight “daze”, we retreated back to the microbiology research laboratory, in order to sort out who would be working on what task for the following morning:

So to finish this blog, dear reader, here is a joke loved by scientists the world over:

An electron and a proton went into a bar. The proton says to the electron, “your round”, the electron replies “you sure?”, so the proton says “I’m positive”.

Oh dear…

Day two essentially follows on from where day one left off,  but the early mornings are still a shock to our “student systems” as we rather quietly arrive at the University of Lincoln Science Building. The first task of the day is to track down the bottles of agar we made on the previous afternoon and transfer them to an autoclave (a pressurised high temperature steriliser) where any unwanted “baddies” (such as bacteria) would be completely destroyed, leaving only clean, fresh and nutritious agar to pour onto yet more Petri dishes. Unfortunately though, autoclaving can be something of a time-consuming process, seeing as the contents of the machine are heated at a toasty 121°C for quarter of an hour. So it is swiftly onto task number two; using the now set agar plates made from yesterday’s batch and producing “streak” plates for the vials of microbes that call the research laboratory their home, and there seems to be quite a few bottles of them…

With the number of available plates, the group of us was subdivided into two pairs and a “three” and set to work on producing a “streak” plate for every microbial sample. But whilst we were in the full flow of producing streak plates, several vials of the same microorganism were discovered, with the condition of each vial ranging from the appearance of being pale and healthy to dark and dry. Because of this, a new form of agar was introduced to Lactococcus lactis; M17, an agar that was far better suited for our numerous microbial samples and one that would be further used as our microbial cataloging project went on. As time went on, the number of available plates dwindled, meaning that it was then a waiting game for the other flasks of agar to cool down and be ready for handling and pouring. So, lunch time it was. But not before we had made a note of the names of the microbes on the vials in order to catalogue them at a later date.

After lunch it seemed that it was then the time to set about the large task of producing yet more nutrient agar plates. The agar had to be in what can only be described as the ‘goldilocks zone’, meaning that it was neither too hot to handle or too cold (“said baby bear”) to pour. When the agar temperature at around 55ºC is the best time to start the assembly of agar plates, and for this project there was to many plates made. 1 Litre of agar solution would comfortably  make around 40 plates, so with 6 Litres there was to be around 240 plates sitting on lab. benches by the time we had finished. And so it was that we began pouring with as much care and as steady a hold as possible, leaving ourselves surrounded by plates of agar:

Once the hundreds of plates had set, it was back to the world of sterilised wire loops, deciphering labels and letting nothing touch the desk that could possibly be contaminated, as microbes such as fungal spores like to float in the air; waiting for their chance to find a nice home and grow to their “hearts content” (a lovely thought on which to finish today’s post).