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.

Hi all quiet day in the lab today…

Deborah / / Uncategorized

Hi all,
quiet day in the lab today. Made up 10X10ml universal bottles of MEA agar, as slopes so that tomorrow i can innoculate them with the confirmed funghi i have, Aspergillus paraciticus, Penicilin notartum and Aspergillus niger. Also made up 50ml of MEA broth so that i can hopefully encourage my remaining funghi to grow. Now to research Listeria

Evening All Today myself and Jo started our…

Amy Thompson / / Uncategorized

Evening All,

Today myself and Jo started our second batch of plugs, made from 10 different Propionibacterium acnes patient samples and a Propionibacterium acnes 6919 strain. We followed the same protocol as before, but made 4 plugs from the Propionibacterium acnes 6919 strain and 2 plugs from each of the 10 different Propionibacterium acnes patient samples. Mike checked their OD to ensure appropriate cell density (0.25). We also centrifuged down 2 eppendorf tubes containing 1ml of the same sample to pellet, we then eluted the pellet in 200ul of suspension buffer each, in order to produce a cell concentration of 2 x 10^8 ,we then removed 100ul.

Having agarose divided into separate eppendorfs was very useful when melting agarose to combine with the centrifuged samples.
However we did experience a few problems, when trying to pipette the agarose and centrifuged sample into the molds some bubbles formed, and some samples appeared to contain less then others resulting in some plugs being half the size of others. We decided that this was most likely due to one of our pipettes being inaccurate due to liquid internally, and that we only produced exactly how much we needed, resulting in some being lost during the procedure. In order to correct this Mike took apart and emptied the pipette for us, and we planned to modify the method to produce slightly more (10%) to allow for losses.

After the plugs had set, we separated them all into separate eppendorf tubes, added lysozyme and buffer, mutanolysin and incubated for 3 hrs at 37ºC. We then added proteinase K and buffer and left overnight to incubate.

We also discussed more appropriate ladders with Mike, he showed us afew that might be better to use, such as http://www.neb.uk.com/productcatalogue/productinfotransfer.aspx?id=/New%20England%20Biolabs/Markers%20and%20Ladders/PFG%20Markers/N0340.
We decided that tomorrow during plug washing, we would use the online genome cutting software we have used before to find exactly how many and what size fragments Spe1 would produce when lysing Propionibacterium acnes so we could make a final decision on ladder size range, and order one asap.

See you all tomorrow! 🙂

Another long update post must remember to do…

Dan Firth / / Uncategorized

Another long update post, must remember to do this more regularly!!!

So polywipes for our project still haven’t come in, but while we wait we are using spare supplies to see how long bacteria prevails on skeletal material. Unfortunately the results were a bit screwed up, so we have had to start again. On the bright side, despite the screwy results it looked like if there was anything on the bones it was gone after 24 hours, hopefully our repeat will show the same.

Also been very busy dealing with open days and work experiance students. On wednesday i think it was (Open day) we could barely do anything because of the prosepective new students and their families buzzing in and out asking questions. Had a workshop earlier last week as well, which was fun, teaching a class about how to build a biological profile from skeletal remains; some of the kids were really enthusiastic and eager, but as always there were a few who messed around and had me going up the wall!!!!!

Had a work experiance kid in with us for one day. His name was Luke, we showed him the bones and had him attempt to age, sex and calculate stature of one of the remains, and then showed him blood spatter and got our friend Hannah (Who is working with Dr Croxton on the composition of fingerprints) to talk about her project before we handed him back to Deborah for the afternoon, hope he had a good time.

All in all a busy and frustrating week, but i keep learning new tricks and tips and am still having a good time. Hopefully come wednesday i will have definitive results on the persistance of microbes on skeletal material (Just waiting overnight while the plates incubate) and possibly have our final supplies for the main project

Hi today Luke spent a little time with…

Deborah / / Uncategorized

Hi,
today Luke spent a little time with Sophie and Dan, Big thanks guys x. Then we made up few slides using Lactophenol cotton blue to verify the fungal cultures i have. Penicillin notatum, Aspergllus niger and Parasiticus,these are now ready for me to innoculate into MEA slopes next week. The only two fungi i have not been able to culture are Sacchromyces and Mucor but thanks to Mike i know where to begin on monday.

Hello Everyone Yesterday we ran a 24 hour…

Amy Thompson / / Uncategorized

Hello Everyone!

Yesterday we ran a 24 hour gel, at 4 V/cm with a 20 second switch interval PFGE gel. We used 3 large restriction enzyme treated plugs and a NEB ladder. Mike also asked us to research the genome for Propionibacterium acnes and the size of the fragments produced when lysed with SpeI. He asked us to do this so that we would be able to sketch a rough drawing of what we would really like to see on a perfect gel. Also to help us look for a more appropriate ladder to use with the Propionibacterium acnes samples, as the average size of a fragment is approx. 100kb, and the NEB yeast we are currently using runs from approx. 1900-225, not quite low enough to compare most fragments.

Today we observed the gel with Mike, which was how he expected it to look, with the NEB bands close together near the top of the gel. However, no bands were visible under the Propionibacterium acnes samples and Mike thinks we really need to have another go at making the plugs, perfecting the cell density and process. We also filmed our latest videoblog!

See you all on Monday 🙂