Hi, thanks for looking in on the Uni of Surrey Veterinary Biosciences blog. This blog is no longer updated because we now have the Green Room student blog where Jade will be keeping us up-to-date with life as a vet bio student.

My last week in the labs has been a fairly quiet week compared to some of the others. I have still been busy doing PCR and gel electrophoresis and now feel confident in both of these techniques. This will help me greatly in my next year at University as well as in my future career.  I have been carrying out PCR and gels on a variety of sample types including DNA from tissues, DNA from blood and DNA from serum from a variety of hosts including humans, cats and dogs.

I will be sad to leave the labs as it has been a really good experience on the whole and I have learnt so much during my time here, but I am ready to go home and see my family and friends again. This weekend we are heading up to the mountains for a final flourish before we leave.

Again this week I have been continuing with PCR and gels for various studies.

I have been testing samples from the study previously described looking for the competency of the cat flea as a vector. In this study Bartonella henselae is used as the infectious agent and to see if the fleas have passed it from one animal to the next. This means that all of the positive results we get should be B.henselae positive. However, for a few of the samples I have tested I received positives for Bartonella koehlerae, they also can back from sequencing as B. koehlerae. This means that somewhere during the experiment contamination with B. koehlerae has occurred. If this is the case it means that all of the experimental data will have to be scraped and it may have to start again. They will have to work out how B.koehlerae has entered the experiment and on first thoughts it is believed that it may be due to the fleas which could have been infected with B. koehlerae before the experiment started.  

Our adventures

This week we did a Segway tour of Raleigh as we thought it would be the perfect way to see the sights of the city and downtown area. We took an hour tour which took us down the main street, to the governors’ mansion and to other important areas of the city. On the same day, Bugfest was going on in the city. This is a celebration of bugs and there is even a bug cafe which serves chocolate coated crickets and mealworms in sauce. Me and Jade did not try any of the things as it didn’t look like the most pleasant thing in the world. However, we did get a funnel cake, basically a giant sugary doughnut!! Sparkfest was also going on in downtown and this is a celebration of the arts, people were doing chalk drawings on the street and there was various music and dancing happening down the main street it was a really good atmosphere although trying to dodge all the people and artwork whilst on a Segway was difficult to say the least.

On Sunday we decided to go to the cheesecake factory for one last time before we left. We decided that we would just have cheesecake this time as in previous occasions we have had other food and been too full for cheesecake even if we have only shared a starter!! We even got a piece to take home for the next day so we could try as many flavours as possible!!

 

This week in the labs I have been continuing to test various samples using PCR and gel electrophoresis.  This includes samples from the study I have previously mentioned, where I was testing for competency of the cat flea as a vector for Bartonella Henselae. This week I have also carried out manual DNA extractions on a few pieces of tissue. This process involved lysis of the tissue using protinase K, then cycles of adding various buffers and centrifugation. One of the samples yielded a very high DNA concentration as measured by the nanodrop spectrophotometer, so I had to repeat the last step in the process in order to dilute the DNA concentration as otherwise it would be too high to complete PCR and Gel electrophoresis and the results gained would not have been accurate.

This week I have also received the DNA sequencing results back from the positive samples I had obtained from the Canadian dog blood. Unfortunately, all the sequencing reactions failed to yield sequences that matched what we were looking for. This is a shame as it would have been nice to have been got one positive from all of my samples tested. However, this could be a good thing in the long run as this may mean that Bartonella species, especially Bartonella vinsonni subspecies berkhoffii type 4 has been eliminated from this area. However, more information is required before this assumption can be substantiated, including repeating the DNA sequencing.

Monday morning was the second time I’d been down to the necropsy lab. Several post mortems of pigs were underway!! I was able to watch the removal of the dorsal root ganglia. This process took a few hours, but was incredible to watch from an anatomical perspective.  I also watched a cow post mortem. The cow had died from septic mastitis, according to the veterinary pathologist I was assisting.

For the rest of the week I did more Western Blots with the hope of finally obtaining some results by changing yet more variables and altering the concentrations of the primary and secondary concentrations- but, still with no luck!! The results seem to be getting worse, which may suggest that the mucosal scrape samples have degraded over time. I think next week will be our last week of trying to get this technique to work. To try and obtain results we can also perform an ELISA which again uses antibodies to bind to protein. This method can not only detect the presence of a protein, but also quantify it. It is a much more sensitive procedure, but cost three times as much as a Western Blot!!

More Western blots this week, and still no success!!! All of this lack of success has highlighted how much work and patience goes into every aspect of a given research project.  Even though the Westerns have not woirk, the work is still incredibly interesting; it’s just a shame that recently there has been such a lack of success!! I was talking to one of the people in my lab today and she said that each Western blot costs about $200 which is a huge amount considering how many I have done!!!  It took someone in my lab three months to perfect a Tryptase Western which I repeated a few weeks ago.

In addition to the blots, which I have done for most of the week, I have been down to the necropsy room.  The necropsy room is where all the animal post mortems are done.  Watching these was fascinating, especially as I want to be a veterinary pathologist.

Early this week I also finished my cell counts- finally! I can start analysing the data next week.

Outside the lab:

This week has been pretty eventful! There was an earthquake in Virginia, which is 3 hours from here, but we felt it in Raleigh. I was sitting in the microscope room when I felt it, and it was a very odd sensation, I thought I was going insane. Hurricane Irene also hit North Carolina on Friday evening/Saturday, we are about two and a half hours from the coast which was hit hard, we just got lots of rain and winds heavy enough for blow over a few trees. It wasn’t as bad as it was forecast to be, we had a thunderstorm a few nights after the hurricane which bought heavier rain and threats of tornados.

On the Thursday before all this, we went to another Durham Bulls game, unfortunately they lost…again!!! The only two games they lost all season, and Bella and I saw them both, we figured we must be bad luck!! The second time we went, we even wore our Durham Bulls t-shirts! Again it was $1 concessions night, so we filled up on hotdogs and chips J

On Friday night we went to the Raleigh Little Theatre to see a musical called Ruthless. It was pretty weird, but still good. The theatre is tiny but the show was good. It is a spoof of musicals in general with songs and acting that are deliberately bad. We had a great evening!

On the Sunday after the hurricane the weather was amazing, so we went and explored Cameron Village some more, just to find that all the shops are boutiques and incredibly expensive, so that was a brief trip. However, we did have lunch at the Mexican restaurant again, which was delicious.

I started the week by manually extracting DNA from 3 tissue samples. This involved leaving them in protease K for around 45 minutes which enabled the tissue to be broken down so the DNA could be accessed. The samples then had to be washed and centrifuged with various different washes and buffers in order for the DNA to be extracted. I had to be careful not to cause any cross contamination of these samples so had to open the tubes individually and handle them carefully. I am still waiting on the streptokinase enzyme and for it to be tested to make sure that it is suitable for use on the samples, so my project has been put on hold until this happens. To fill in my time again I have been helping on various projects running PCRs and gels for different people within the lab. This includes testing dogs blood who are involved in a study testing the competency of the cat flea, C.felis, in transmission of Bartonella henselae  from cats to dogs.

Our adventures

We have had a busy weekend this weekend and have jam packed it full of stuff. On Friday evening as it was the first Friday of the month the art galleries, restaurants and bars downtown were all open until late so we decided this would be a good opportunity to go downtown and have a look around. There was live music in some of the venues and we had the opportunity to have a look at some art, jewellery and ceramics made by local artists. After this we then went out for dinner to a Japanese/thai restaurant. On Saturday were supposed to be going to the cinema to see The Help but we got the viewing time wrong so this has had to be rearranged instead we had a look around the mall and went out for lunch. In the evening it was the first American football game of the season and we were lucky enough to have tickets. We went tailgating for a little while before the game. This is where people sit by their cars and listen to music, eat food and play games before the football game actually starts. It was a really good experience and something that would not happen at home. The game was really good; there was cheerleaders, a marching band, cannons when we scored and the atmosphere was amazing. Even at professional sports in the UK you would not have as much going on before and during the game and this was only a college game. In the end NC state won which was really good as otherwise we would have not been happy.

On Sunday, we went to the beach. Beth, from Jade’s lab offered to take us as her family has a boat so we were able to go to beaches that were more secluded. Unfortunately after we had been on the boat for half an hour the engine overheated so we had to get sea tow to take us back to the marina as the boat couldn’t go any further. Luckily, some of their friends who also have a boat and live by the beach  saved the day and took us out to cape lookout on their boat. We stopped off at the beach and went looking for shells and from the beach we saw a pod of wild dolphins swim past the beach which was lovely. We then took a smaller boat out to what was supposed to be Shark Island. However, we think that Hurricane Irene had washed the island away as it had been a sand bar so this was possible. We had a lovely day with Beth and her family and after a tiring day we were glad to have Monday off because of Labor day.

On Monday, we met up with Jade’s friend from home ,Shekinah, who has been travelling around America this summer and has now stopped off in North Carolina. We went to the mall although we had a little trouble getting there as we did not realise the buses did not run on labor day so had to get a lift from Jades flatmate instead. We went to the cheesecake factory for lunch and had a look around the shops, it was a lot more relaxing than the other days which was nice and it was good to see someone from home.

I am still waiting for the streptokinase enzyme to arrive so I can extract the DNA from the rest of my samples. We did try placing the samples in the water bath to see if they would lyse as this had previously worked with the others.  However, unfortunately this did not work so I am having to wait until the enzyme arrives before I can continue with the project.

This week I set up PCR and running gels for various other projects and scientists within the lab as they are all extremely busy with their respective projects.

This week I have continued with my project testing dogs blood from Canada for Bartonella. In total I have extracted DNA from 32 samples , so I went on to perform PCR’s and gel electrophoresis on these samples.  From these samples 10 showed positive results so these were kept in order to be sent for sequencing. 

The samples which had previously been shown to be positive and had been sent for sequencing came back as failed sequencing which meant that the DNA that produced the band on the could not be identified.

I also tried to extract DNA from the final 22 samples, but the blood would not lyse so it would have difficult to aliquot. This meant that I am having to wait for a streptokinase enzyme which can lyse the blood so that DNA extraction can be done on these samples.

Finally this week I had to re test all of the samples which had shown positive results to make sure that we had not received false positives for them and we would not be wasting money sending them off for sequencing. I have had 13 positives so far so retested the DNA with PCR and gel electrophoresis. Out of the 13 positives I had, 10 of them showed positive for the second time so were sent off for sequencing I am waiting for the results to see if we can confirm the presence of any Bartonella species.

I have also been helping other people this week by running various PCR and gels in my spare time.

Our adventures

This week we wanted something to remind us of home so we decided to cook a roast dinner with all the trimmings, roast chicken, roast potatoes and vegetables. This took slightly longer than we had anticipated as we didn’t realise the oven temperatures were in Fahrenheit so had the oven on too low for a while!!

At the weekend we decided to head down to the buck 50 movie theatre, where you can watch movies for only $1.50!! They are slightly older movies that may have come out a couple of months ago but definitely worth waiting to only pay that price! We went to see Kung Fu Panda 2 and were both laughing throughout the film!! We had also gone out to dinner to a place called Mitch’s tavern which is so cheap. We ended up getting dinner and a movie for around $10 which would never happen at home.

We decided to make the most out of the lovely weather and went to the pool on Saturday. In the evening we went to see Back to the Future at the art museum open air cinema. So we sat under the stars to watch the movie which was a really nice experience and the atmosphere was brilliant. Sunday again we made the most of the fantastic weather and had a BBQ at my house.

This week has again been much of the same work. We continue to attempt to perfect the Western Blot, which is proving increasingly difficult as the variables that we can change are now becoming limited. We have been getting results, however these are few and far between and do not appear to be reproducible, so more work needs to be done. Even the positive control is proving to be difficult to obtain results from.

On Wednesday and Friday I continued with my cell counts which are getting there and should be finished in the next week which will be good, then we have to analyse that data, which will be tricky as there will be over 700 bits of data to organise.

On Thursday I carried out another protein assay in preparation for more Western Blots next week which was pretty straight forward as I’ve done a few in the lab before, however when you have to do the assay on 40 samples, it does become a little complicated. Also on Thursday I went downstairs to learn about the work that another lab does with Zebra fish. One of the people I work with in the lab wants to do future work with these fish because they have a similar GI tract anatomy as humans in that the GI tract is in different sections. Zebra fish are also transparent which makes it possible to use fluorescent markers to mark the tract, in addition, they have rapid growth and are easy to genetically modify in large numbers. Aside from the GI work that can be carried out on these fish, Zebra fish also are one of the few animals with heart cells that can regenerate, and therefore they are becoming essential in research to cure heart disease.