Today we pippetted out the agar into plastic tubes we autoclaved to maintain a sterile environment and avoid contamination of the bacteria. (WARNING; agar sets quickly-had to make agar again), Second attempt agar once cooled BUT NOT SET we continued to pippete into the plastic tubes. Once the agar was set we could then follow the correct sterile techniques to produce “stabs” into the agar, using the successfully grown streak plates. We correctly labelled each one to identify them according to strain used, and from which culture. These we recorded as successfully grown from the originally stored samples, and stored as “stabs”.
Sophie in the lab
A "Stab" culture
We were disappointed we ran out of time and was unable to produce the glycerol mixture with the samples in the broth. But we want to come back (during term time or a a fuure holiday period) to complete the job.
The remainder of this day was spent finishing off any jobs that we still had to complete, this included the rest of the “stab” cultures and checking the P.acnes plates as well as gram staining some friendly E.coli to confirm thier identity. We also made sure that the technical staff knew exactly what had been completed over the past two weeks and the records were up to date in order for others to carry on after our work.
From left to right: Gemma Bowey, Sarah Beane, Sophie Cowper, Dr Tim Bates, Tammy Wiltshire, Laura Brown and Hayley Brudzinska
It’s often the way of the world that a new electronic toy will always encounter teething problems, whether it be a new phone or a computer. It’s a learning process, figuring out what all those mysterious buttons do. This is especially true with “The Stig”, the latest piece of equipment to grace one of the laboratories of the University of Lincoln. On Wednesday morning, “The Stig” dug its heels into the ground and refused to cooperate, so it was back to the safety of the microbiology labs, where the technology was slightly more cooperative.
It was a more sedate morning than the one before, where we had been dissecting the various organs of a pig (a.k.a the “glamorous” work). Microbes require time to adapt and grow on their new homes, such as the various agar plates being used for specific organisms, so it was the task for that morning to check how they were growing, if they grew at all, and to plan the next course of action, especially for their storage for future use by other students at another time.
In the afternoon, we were visited by Professor Paul Stewart, who is Pro Vice Chancellor Research and was Founding Head of the new Engineering Hub for the School of Engineering.
He was given a tour of the main microbiology teaching lab whilst Dr. Bates and Dr. Williams explained the work we had been doing, and how it provided us with additional valuable experience that we were unable to obtain elswhere, while we students shuffled around trying to avoid the Professor Stewart’s camera. Unfortunately, pictures were eventually taken…
Laura in the anaerobic cabinetSophie counting colonies
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 Enterococcifaecium were alive or dead.
Firstly, using a permanent marker pen, they wrote their initials, the date, the culture medium (M17) and the organism cultured (Enterococcifaecium). 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:
"Ooh yellow" - tetracyclin
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.
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.
Dry ice sublimating
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:
What makes a good cancer drug? Efficacy, specificity, ease of formulation and delivery as well as having a high bioavailability. During the development of a new novel anticancer drug these criteria must all be met. The steps that Dr Westwell and his team had to take to produce their most effective drug to date included the development of a group of 2-4(aminophenyl)benzothiazole compounds and consideration of the metabolic changes that would occur to the drug in vivo in man, for example, hydroxylation which can change the properties of the drug. After manipulation which included adding fluorine to the molecule to prevent these changes – PHORTRESS was synthesised. This then went into investigation at the Cancer Research UK labs in Newcastle and London where the first pass metabolism (in the liver) came to their attention. The researchers there avoided this problem by developing a range of amide "prodrugs" which enabled the drug to be water soluble and administered by injection.
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”.
