Hannah Preedy


In the Lab

This week I finished the DNA extractions and so now they are going to be put in the PCR machine to amplify millions of copies of DNA. We will first PCR the samples for E. Hirae as we think this is the most likely bacteria we are going to detect. We know for sure that the bacteria are Gram positive cocci adhering to the gut epithelium.

If some samples come back negative for E. Hirae then we will run them through a PCR for E. faecalis/E. faecium. Then any samples negative for this will be run through a SOD PCR. SOD is super oxide dismutase, which is a gene found in all bacteria. We will then send off the DNA for sequencing to determine what bacteria species were present.

We then finished off the F.I.S.H for E. coli in order to show that E. coli is adhering to the epithelium. We originally decided to perform F.I.S.H for E. coli as ram stains for these samples demonstrated Gram negative rods adhering to the epithelium and so we believed these were most likely E. coli. In order to confirm this we used an E. coli probe which binds to a sequence of DNA that is specific to E. coli.

Out of the 9 samples we observed Gram negative rods adhering in 8/9 samples and these fluoresced for E. coli when tested using FISH and then examined using a florescent microscope. Also these 8 samples showed that the E. coli was adhering to the tissue. Interestingly, all 9 samples were taken from unhealthy kittens that had been euthanized or died due to intestinal problems such as severe diarrhea.

Finally, this week I went to a rounds meeting one morning. It is where qualified vets who want to specialize in a specific area meet to discuss cases that have been seen at the hospital with the qualified specialist neurologists, cardiologists etc.

They go through the tests results carried out and the symptoms presented by the patient. They then list various differential diagnoses as to what could cause the symptoms and decide how to treat it.

The qualified specialists are there to ensure the residents are correct in what they think and say and to ask them challenging questions. It was really interesting to see how many different differentials could cause polyarthritis (a whole white board full!!!).

From these possible diagnoses they then decided what is most probable and relevant, relating back to the results, symptoms and history of the patient.

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This week we discovered that the DNA extraction from the paraffin embedded blocks was successful. We therefore managed to extract E. Hirae bacteria successfully from the tissue sections. Compared to previous times we used a microwave to melt the paraffin together into a ring around the tube rather than using xylene, as it is thought that the xylene damages the DNA and may inhibit the PCR.

We also carried out F.I.S.H on 5 of the slides which corresponded to 5 different animals, presumably healthy. F.I.S.H works using specific fluorescent probes which bind to a specific sequence of the organism that is being searched for. On these sections we added an E. coli probe which fluoresces red under UV light. We discovered that all 5 animals had E. coli present and more importantly they were seen adhering to the epithelial cells.

More F.I.S.H is going to be carried out on more slides from individual animal this week.

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My 6th week (Hannah)

We are trying a different protocol in order to extract good quality DNA from the tissue section slides. Instead of scraping the slides with the mounted tissue, we shaved off 10, 15 and 20 sections of the tissue which is embedded in paraffin, rather than getting histology to mount them onto slides.

We think that the large amount of xylene that we add to remove the paraffin may be damaging the tissue, so we placed the tissue embedded in paraffin into the microwave to clump all the paraffin together. We also cut out the specific section of the tissue where we saw large amounts of bacteria adhering to it and again added this to a tube and micro-waved it. The results for the gel that we run will be ready in the next blog.

Our Adventures

My parents came to visit me for a week last week. At the weekend we flew out to Washington on a 36 seater plane, it only took 30 minutes but as the plane was so small it was a little scary when there was turbulence. We spent the weekend just looking around and sight-seeing. We saw the white house and all the various war memorials and president memorials. It reminded me of a European city. Then during the week they had a tour of the vet school given by Jody Gookin and I got to show them what I’ve been doing so far over the summer.

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In the lab this week, I’ve been extracting tissue from slides and carrying out PCR followed by gel electrophoresis in order to determine if Enterococcus species are present in the sick and healthy kittens.

I had a tour of the small and large animal hospital this week too. Derek, who works in my lab, is also a large animal vet and he took me down to the cow barn along with some vet students to learn all about cows. I learnt how to listen to the heart and lungs and learnt what signs are abnormal in an ill or unhealthy cow. I also got to palpate a cow which I’ve always wanted to do. It was very warm and the contractions of the muscles were so strong, it made my arm ache a little.

Our adventures

This week Anna and I went to a shopping mall. It had lots of expensive shops and even sold Hunter wellies for $120! So they are a lot cheaper in England. There is also an outdoor pool nearby so we spent one day sunbathing and chilling in the pool. It was very relaxing and a nice way to cool down as it’s so hot at the moment. Saturday night we went to watch Salt featuring Angelina Jolie at the local cinema.

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In the lab this week I have been carrying out F.I.S.H on some slides in order to show where and if bacteria are adhering to the intestinal wall of infected animals. In this technique, fluorescent probes attach to the bacteria, and they can be identified under different wavelengths of light.

I’ve also been carrying out PCRs on the slides with the tissue and adherent bacteria in order to amplify the bacterial DNA, giving us lots of copies to work with. The DNA was analysed by gel electrophoresis to identify what species of bacteria was present.

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This week I have read lots of research papers, in order to come up with a protocol for my experiment. I’m going to grow bacteria isolated from approximately 100 samples and use them to infect some polystyrene wells, in order to determine if they can form biofilms. I have also been looking at Gram stains using a microscope which have shown that in these 100 samples, Enterococci and E. coli bacteria have adhered to the intestinal wall, and in many cases, these adherent bacteria may have caused diarrhoeea in the samples.

Unfortunately, the stems cells didn’t grow so next week we are going to attempt to culture some more by changing the solutions used, amount added etc.

I have watched gel electrophoresis being carried out and it’s very interesting to see what happens and the results in real life compared to learning and reading about it in lectures. It all makes a lot more sense now.

The people I am working with in the lab and I went for an ice cream where they have over 90 flavours! There was one called ‘cold sweat’, which contains lots of chillis and peppers, unfortunately, no one tried it.  A waiver form has to be signed in order to eat it and it made the morning news here!!!!!

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On Monday I had a lab meeting where I met with everyone who works in the gastrointestinal physiology lab which is run by Dr Jody Gookin. The personnel in the lab all discussed what their various projects were with me and any plans they had to improve their projects. That lunchtime we all went out for a Mexican in a restaurant; I had never eaten in a Mexican restaurant before, so I had beef fajitas which were lovely.

During the week, I observed how to run a PCR to detect Tritrichomonas foetus in cat faeces. The lab has various pieces of equipment to diagnose Tritrichomonas foetus, a protozoan parasite of cats which causes diarrhoeea and can be easily spread to other cats, especially in shelters and multiple cat households. I also learnt how to passage pig cells, and have my own culture of pig intestinal cells growing in the incubator which I need to check on every 2 days and replace the media.

One of the PhD students in the lab is attempting to grow some cat colon stem cells so I observed how she attempted to isolate crypt cells from the colon. The crypt cells are where the stem cells are found in the colon. It was also my birthday on Friday so all those in the lab and I went for an Italian at lunchtime. I had a bag of presents and cards from home which I was strictly told not to open until my birthday.

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