Category: Uncategorized

Afternoon Today myself and Jo removed the plugs…

Amy Thompson / / Uncategorized

Afternoon,

Today, myself and Jo removed the plugs from the molds into a universal, added lysozyme and mutanolysin and incubated the plugs for 4 hours at 37ºC. During that time we began filming interviews with other students in the lab, discussing their projects/work, positives, negatives and what they had enjoyed and found challenging. We then removed the lysozyme and mutanolsyin and added proteinase K, leaving the plugs to incubate overnight at 50ºC.

Tomorrow we will be washing the plugs, hopefully running a PFGE gel with non restriction enzyme plugs and treating other plugs with the restriction enzyme, Spe1. We will also hopefully be filming another interview in the lab! 🙂

Just had a check on the agar plates…

Dan Firth / / Uncategorized

Just had a check on the agar plates from yesterday. There is zero growth thus far on the bones that were alcohol swabbed and only minimal growth on the bones handled with gloves. This is what i expected to see and hopefully when we compare to the plates we start growing tomorrow, we will be able to say whether or not wearing gloves makes a difference.

However due to the state of the incubators and the incubation room, as well as working shoulder to shoulder with other people, i think its important to note that any results we get are only indicators and not a definitive answer, so don’t expect to just go downstairs and start fondling with one of the bodies without protection!!!!!

Glad we are in our final week, i would like to enjoy some of the summer sun before we lose it!!!

We’re getting toward a point where it would…

Michael Shaw / / Uncategorized

We’re getting toward a point where it would be sensible to start thinking about report writing. For all of those on funded projects (UROS, GenSoc, SGM, etc.) you will need to put together some sort of report and/or poster to finish your project. The specific requirements may vary, but we thought a group discussion about some of the more general points might be beneficial to you.
Nicki and I are happy to sit down (with coffee) THURSDAY (26th) afternoon and talk about what we expect to see in a report, what sort of writing style to use, how to present your data, and how to produce a high quality A0 poster.
I’d suggest you start thinking about this sort of thing and check the specific requirements from your funding body and bring that along if you like.
I think it would be really beneficial if you could all help each other out with these- particularly in making your Posters look awesome.

THUR afternoon in the Post-grad room

Hi all Interesting day today the broths i…

Deborah / / Uncategorized

Hi all,
Interesting day today, the broths i innoculated friday were ready today so i made them into stabs and slopes. Finally my workspace is clearing, made up a few more bijou jars ready for the end of the week when i can hopefully add Enterobacter spp and Citrobacter spp to my completed list. I then went through the cell competency procedure with Nicola and she assaulted my brain with maths (hate maths mondays), we then went over plasmid stability and what we were to achive this week. in preparation we made up 400ml LB agar, 400ml LB agar with chl10, 200ml LB agar with Ampicillin 100 and 200ml LB agar with Ampicillin 50. Tomorrow serial dilution!

Week 5 update Monday Gillian sent me a…

Jacob Matthew Abbott / / Uncategorized

Week 5 update
Monday – Gillian sent me a 12 page skeleton evaluation form which detailed things like age, sex and height. I spent the day doing that.
Tuesday – The previous set of photographs looked too dark (though they looked ok though the camera’s screen) and so I had to do them again. This time I used the flash with both the diffuser on and set on low in order to get the photographs light enough without being washed out.
Wednesday – I went up to the analytical labs to ask how I would analyse the soil/clay I got out of the skull. Unfortunately the tests would take much longer then the remaining time left on the project, and so this will have to be done by the next person. I also measured the skull using the method in ‘Standards for Data Collection from Human Skeletal Remains’.
Thursday – I re-measured the skull using a set of digital callipers to make sure they were right, I then planned my UROS report.
Friday – Friday was a short day, I used the measurements in a program called Foredisk 3.1 in order to determine the race of the skull. Using both the Forensic and Howell databases it was determined that the skull belonged to a white male, which is what I expected it to be.

Evening All Didn’t come into the lab till…

Amy Thompson / / Uncategorized

Evening All,

Didn’t come into the lab till 4pm today, in order to allow our Propionibacterium acnes 6919 strain to grow sufficiently. We took 7ml of the Propionibacterium acnes 6919 strain added 14ul of chloramphenicol and incubated for an hour. Then we pipetted 1ml into 2 eppendorfs and spun them down for 5 mins at full speed. Once pellet, we added 250ul of cell suspension buffer into both, shook and then combined. We then added 500ul of melted agarose and pipetted into 5 plug molds. We then put them into the fridge to set.

We have left them in the fridge till tomorrow, when we will treat the plugs with lysozyme, mutanolysin and proteinase K.

See you all tomorrow! 🙂

Going over the results we gathered from our…

Dan Firth / / Uncategorized

Going over the results we gathered from our background swab of the bones and can’t help thinking how frustrating research is!!!! Why are all my bones growing different stuff on them, and why is it every time i do a repeat i get a different result!!!! Why can’t it all be as expected and work out for a nice simple conclusion????

I guess thats just the nature of research, and we can’t make the evidence fit the conclusions we want, the conclusions must fit the evidence.

Don’t get me wrong i’m still enjoying the research and my experiances this summer, its just the lazy part of me wishing it was all black and white and i could get a simple logical answer.

Evening all Started out the day by performing…

Deborah / / Uncategorized

Evening all,
Started out the day by performing an indole test, confirmed Kl oxytoca i have made a broth so that on monday i will make up stabs and slopes. Once the Kovacs reagent was added to Kl oxytoca a cherry red ring immediately formed on the top of the broth, meaning that it was a positive result and that Kl oxytoca has the ability to cleave indole from the amino acid Tryptophan. Also i have brothed up Lactobacillus and incubated at 25C so that it will be ready to make up into stabs and slopes on monday. My A.baumanni stabs and slopes are incubating at 37C until tomorrow. I have restreaked a couple of plates Ent. cloace, Enterbacter spp and Serratia marascens hopefully i will be able to confirm these next week. My work bench is clearing!!!

Just a quick reminder to all users to…

Michael Shaw / / Uncategorized

Just a quick reminder to all users to utilise the ‘Tag’ function on the Blog. Tagging key words on every one of your posts will make it much easier for us to review the use of the blog and will allow you to easily collate all the posts from your project, or quickly review all the posts on someone else’s project.
I’m pretty certain you can go back and add tags to your previous posts using the ‘edit’ button (top right of your post).
Cheers

Right our project has finally begun We started…

Dan Firth / / Uncategorized

Right our project has finally begun!!!!!! We started swabbing the bones as they were ie. no cleaning or handling- these swabs were placed in ringers and pipetted out onto nutrient agar and left to grow at 37 degrees for 48 hours. The result showed very little growth on the majority of plates 1-3 colonies on most or none at all!!! Except a few plates which had become overrun by a “Fuzzy mass” We are now using gram staining and subculturing to identify the colonies and the “Fuzzy mass”

-Post edited by Admin –

Hello Filmed our newest videoblog this morning in…

Amy Thompson / / Uncategorized

Hello,

Filmed our newest videoblog this morning, in 5 mins! Our quickest yet – we are becoming pros! 🙂
Removed, stained and viewed our newest gel today. Unfortunately we got pretty conclusive results that our plugs were the problem. No separation was present from our old or new plugs, therefore next week we need to completely remake plugs.

We will be making 5 plugs all from Propionibacterium acnes 6919 strain, taking extra care to pipette very carefully, in order to produce better plugs. We will also only be producing our plugs up until treatment with lysozyme, mutanolysin and proteinase K, then running them on PFGE, to see if they are viable, before treating and running them with restriction enzyme, Spe1.

We are expecting to see one very clear band just below the wells, with no separations underneath and possible clear bands between the band and wells, as DNA should all still be intact and not multiple variable size fragments. If we see this, we will then treat the plugs with Spe1 and run them. We are expecting to see bands forming from the wells and towards the bottom of the gel, with clear bands throughout, but most dense around halfway down the gel, adjacent to our new low range PFG ladder, and the band – 100kb, as DNA will be lysed into approx. 86 fragments ranging from 817bp-13702bp, mostly around 10000bp (100kb).

Have a good weekend, and see you all on Monday 🙂

Awesome day in the lab today Made up…

Deborah / / Uncategorized

Awesome day in the lab today,
Made up a broth of 1g Tryptone and 0.5g Soduim chloride in 100ml sterilised water. this was then dispensed into universal jars (4ml per universal) and autoclaved at 121C for 15 minutes. Using aseptic techniques i the innoculated the broths with Kl terrigena, Kl pneumoniae and Kl oxytoca these were incubated over night at 30C. So hopefully they will have enough growth so i can add Kovacks reagent tomorrow (indole test) and i can hopefully differentiate the three culures. I have confirmed Listeria monocytogenes and so have made a broth and will hopefully make up stabs and slopes tomorrow. Alice has kindley donated a pure sample of A. baumanni, which i streaked onto a nutrient agar plate and also innoculated a nutrient broth and incubated at 30C overnight. I have also confirmed Streptomyces aureofaciens which i will broth culture tomorrow. Mike was amazing and explained loading dyes in detail, and how pH can alter substances, and how dyes can be used to range DNA banding during electrophresis (bromphenyl blue around 300bp). Then mike sprinkled a red powder (flourescens) into a beaker of water, because of the pH change the water began to glow like a yellow highlighter pen. Mike explained why this happened (outer shell valence and refraction of light) but i was happy with my glowing water xx Next Nicola explained how she was to cut out DNA from the agarose gel she had just ran and the purpose of this. Then i watched as she cut out each band of target DNA so that tomorrow the agarose gel can be dissolved away to leave behind the desired DNA. I Iooked at the kit to do this and it is in no way as simple as it seems. Even had time for an ethics debate with Dan, Sophie, Rosie and Kamilla.

Afternoon Quite a quick day today came into…

Amy Thompson / / Uncategorized

Afternoon,

Quite a quick day today, came into the lab stained and viewed our gel. We saw little/no separation from the Propionibacterium acnes 6919 strain restriction enzyme plugs and the non restriction enzyme plugs. However we did see separation from the NEB yeast.

Myself and Jo decided that this may be due to our newest plugs. As mentioned before one of the pipettes we were using was full of liquid, and therefore may have given us incorrect amounts of substances when being used. We were unsure as to whether this was resulting in no separations from plugs on our latest gel, so we decided to run another gel with 2 samples of our old Propionibacterium acnes 6919 strain restriction enzyme plugs, and 2 samples of our new Propionibacterium acnes 6919 strain restriction enzyme plugs, along with one sample of new non restriction enzyme plug and 1 sample of NEB yeast.

We are hoping that by doing this we can establish if the lack of separation is due to our new plugs being poor, or the settings recommended by the new ladder – low range PFG marker, not being quite right. We set up the gel ad ran it with the same settings as yesterday – 15hrs, 6 V/cm, 1-12 second switch interval ramping, 1% agarose, and then a second block to hold the gel at 0.6 Vcm, 5 second switch interval until tomorrow morning when we will be in to remove it, stain it and view it.

Filming our newest videoblog tomorrow… 🙂

Hello So my Antibiotic Cefotaxime has arrived I…

Rosie Myers / / Uncategorized

Hello,

So my Antibiotic – Cefotaxime has arrived! 🙂

I am now proceeding to follow all of the methods that I used with Ampicillin, just with the Cefotaxime instead and changing them slightly. I made up agar with 4mg/ml of cefotaxime in, and filtered 100ml of Brayford Water onto 2 of the plates. This morning I only had 3 colonies on just one of the plates. This concentration of Antibiotic is quite high for E.coli to be resistant too, so in a way the 3 colonies are highly resistant which is good, but I may have to start doing more plate sto get more out or maybe the rainy weather yesterday affected the amount of E.coli present.
I have now placed these colonies into broths containing cefotaxime again, and tomorrow I will proceed to do Alkaline Lysis with these samples. Once I have a few more, I will run them on a gel and see what banding patterns are going on 🙂

Evening Firstly i checked on the Pseudomonas B263…

Deborah / / Uncategorized

Evening,
Firstly i checked on the Pseudomonas B263 that i had made into stabs and slopes, all are showing excellent growth, another culture cleared from my bench! next Sharon and i made a few slides using lactophenol cotton blue as we still need to practice our basic techniques. Then we Gram stained a few more of the bacterial cultures on my workbench, confirming as much as we can that they are what they should be. Because a few of my bacteria are in th same family and look the same under the microscope an indole test is required to confirm their identity.I have checked that we have everything required to carry out the test, so the job for morning is now to make up the broths ready for the test.

Hey Everyone Came into the lab this morning…

Amy Thompson / / Uncategorized

Hey Everyone,

Came into the lab this morning to start to set up our next gel. I made up 3 L of TAE 0.5X, incubated 1 Propionibacterium acnes 6919 strain restriction enzyme plug and 1 non restriction enzyme plug in 1X wash buffer and then in 0.5X TAE buffer. I then made up a 1% agarose gel and left it to set. I put the gel and plugs into the fridge and left them until 5pm.

When i came in at 5pm, i cut and loaded 2 samples of Propionibacterium acnes 6919 strain restriction enzyme plug, 1 sample of non restriction enzyme plug and 1 NEB yeast ladder. I sealed the plugs and then ran the gel according to the info recommended on the low range PFG marker ladder, for 15hrs, at 6 V/cm, with a ramped switch interval from 1-12 seconds. I added a second block as the gel is only running for 15hrs, and will finish at 8.30am tomorrow morning, of 0.6V/cm for 2hrs with a 5 second switch interval.

We will hopefully have the new ladder tomorrow, so we can modify our new gel according to the results we get when the run finishes tomorrow and use the new ladder!

See you all tomorrow 🙂

Hi all another quiet day in the lab…

Deborah / / Uncategorized

Hi all,
another quiet day in the lab but very interesting. First started by collecting water from the Brayford, then Sharon spread plated, MEA, Mannitol salt, Baird parker, MLSA and nutrient agar plates. Sharons project was to count colony numbers, now that is complete, we were curious as to the type of colonies. Next i innoculated a couple of MEA slopes with the three confirmed funghi on my work bench and made a couple of MEA broths using the two funghi i have as yet not been able to culture. Saccharomyces spp and Mucor you will not defeat me!!! Nicola had a meeting which clashed with the timing of her cells being ready for competency, so she talked me through the process. Really loved it, want to learn more about anti microbial resistance.

Afternoon Everyone Today we started the looong washing…

Amy Thompson / / Uncategorized

Afternoon Everyone,

Today we started the looong washing phase of plug making! We washed all 24 plugs (2 per sample, and 4 per 6919 strain sample) as before, 4 times for an hour each in 1X wash buffer. We then separated off plugs from samples 2-11, added fresh 1X wash buffer and put them into the fridge for storage. We then washed the 4 6919 strain sample plugs with 0.1X wash buffer twice, and separated off 1 plug (not to be treated with restriction enzyme Spe1). We then added 1X Spe1 buffer to incubate the plugs, before removing this and adding fresh 1X Spe1 buffer, Spe1 enzyme and BSA. We then left the 3 plugs to incubate overnight at 37ºC.

While waiting for washes, we used online REviewer software to work out the number and size of fragments Spe1 will produce – 85 fragments ranging from 132702bp – 817bp, with the most fragments approx. 10000bp (100kb). We therefore decided to order BioLabs (NEB) low range PFG marker that ranges from 194000bp-2030bp (194-2.03kb), that will cover all the larger-medium size fragments only missing 4 smallest fragments. The image produced by REviewer is attached, showing all fragments along genome

Tomorrow, we will be setting up our next gel, using settings recommended by BioLabs (NEB) on the new ladder we have chosen to use. Hopefully we will have the new ladder very soon, so we can run it!

See you all tomorrow!

Late post time week 4 Monday Another day…

Jacob Matthew Abbott / / Uncategorized

Late post time, week 4:
Monday – Another day of cleaning up the skull, making sure there wasn’t any dirt left in between the various bones.
Tuesday – A bit more cleaning. A school came to visit so I helped them age, sex and estimate the height of the skeletons we’d laid out in the previous weeks.
Wednesday – As it was the open day I spent most of my time giving people a run though of my project, I also had enough time to age and sex the skull (turns out it’s most likely male and was around 47 years old when he died).
Thursday – I re-aged the skull using the Meindl and Lovejoy method and calculated the age range to be around 35-45 (approx 40), I also took photographs of the skull in various anatomical positions using the forensic camera kits.
Friday – I spent the day doing a bit more research. I first tried to track down the man who found the skull (Erik Grigg) to ask him a few questions about it. An article I read mentioned he owned a music shop in the Mall, but when I went looking for it it seemed to have been shut down. I instead went to Jew’s House up the Steep Hill and asked them, luckily enough they had a magazine which had is handwritten account in it, meaning I don’t need to trawl the internet looking for a phone number.