Quiet day in the lab today A few…

Quiet day in the lab today!
A few of the yeast cultures i had did not grow after their first innoculation onto MEA plates. I have re-streaked Mucor, Aspergillus niger and Saccromyces spp onto fresh MEA plates and incubated them at 30C. Penicillin notatum and Aspergillus parasitucus successfully grew and i have re-streaked them onto fresh MEA plates and incubated them for two days at 30C, from this i hope to gain a pure strain of the target funghi. Confirmed Chromobacteruim CV026 and have made a broth culture so that i can make up long term storage stabs and slopes. Also made up a batch of TAE with Rosie and Fran. Now to research exactly what it is and does.

Good Evening Everyone Busy day today Myself Jo…

Good Evening Everyone!

Busy day today! Myself, Jo and Mike discussed possible modifications to the plug making method when using Propionibacterium acnes. We decided that we would need to use mutanolysin, which aids in cell lysis, as Propionibacterium acnes can be difficult to break into! We also decided to prolong the incubation time after the addition of mutanolysin and lysozyme from 2 hours to 3 hours at 37°C, in order to give them plenty to time to work on the Propionibacterium acnes.

We then began running through our first attempt at making the plugs! We started by adding chloramphenicol to our Propionibacterium acnes TYGE broth (that Mike inoculated for us). Once that had incubated for an hour we centrifuged it at full speed for 5mins, until we obtained a pellet. We then eluted this pellet in cell suspension buffer. We melted agarose and added these together. We then pipetted this into plug molds, which required a bit of practise! (we made 10 plugs) and left them at 4°C, to solidify. Once set we removed them into a universal tube and added lysozyme, it’s buffer and mutanolysin, it was then incubated at 37°C for 3 hours. We then removed the plugs and rinsed the plugs with pure water. We then added proteinase K and it’s buffer and left it to incubate at 50°C, until tomorrow morning.

We will be washing the plugs A LOT tomorrow! 4 times per plug 1 hour each, with wash buffer! We will then be working through the restriction enzyme digestion of the plugs info, and hopefully get ready to set up our first run with our hand made plugs! 🙂

See you all tomorrow!

Hi all Restreaked a few nutrient agar plates…

Hi all,
Restreaked a few nutrient agar plates, hoping to find pure colonies of the target bacterial cultures that were incubating. One of the cultures didnt need to be streaked, as it was a confirmed bacteria, chromobacterium violeceum. Confirmed as it produces low, convex colonies with a dark purple/metallic sheen (violacein production). This culture, using aseptic techniques, was innoculated into nutrient broth and incubated at 30C for a coulpe of days. Then it will be made into stabs and slopes for long term storage. Researching more on selective agars to confirm more of the cultures on my lab bench. Made up more nutrient agar plates, counted colonies from Sharons Brayford pool project, cleaned lab – quite theraputic. Can’t wait to see what tomorrow brings.

Hey all I made some competent DH5 alpha…

Hey all!
I made some competent DH5 alpha E. coli cells today (which took 5 and a half hours to grow to the optical density of 0.350!) and used the colorimeter to see if they had reached the optical density . I have also transformed them so I can do alkaline lysis tomorrow! Still in the lab currently waiting as the transformed cells are currently in the incubator for 45 minutes then I need to spread plate them on to LB agar plates that have ampicillin in them and put them in a incubator over night. I’m using the same plasmids again (pALA1029, pOG4, pOG04, pOG4.003).
See you all tomorrow!
Soph 🙂

Good evening I haven’t really wrote about what…

Good evening! 🙂
I haven’t really wrote about what we have done so far on our project but after reading Dan and Jacob’s entries I can see they have pretty much covered everything.

But in my own words, in week 1 we learnt how to fill out inventory forms correctly when looking over skeletal remains which included noting whether bones were present or absent. This was quite easy to get the hang of after the first skeleton. There were 4 skeletons laid out for us to look at and get to bases with the names of all the bones properly and where they lay in the human structure; so our next task was to lay out a skeleton for ourselves and understand how to differ between left and right bones etc. After getting to grips with the skeletons we started looking into how you work out the stature of the skeleton, by looking at the leg bones (femur & tibia), and how to determine age (using the pubic symphises on the pelvis and teeth if any were present) and to determine sex (using 5 main features on the skull and also the pelvis). Jacob, Dan and I all went to visit St. Katherine’s which is a church converted to a small museum. We went here as a lot of the bones we have in the lab are from St Katherine’s, however, it didn’t prove to be necessary as there wasn’t much information on the actual bones.

Last week was spent mostly doing inventory of all the bones in the lab and deciding which ones needed cleaning and so learnt the cleaning methods, which are quite simple for mud covered bones, being warm water and a toothbrush! But I imagine cleaning densely covered bones is more difficult. The rest of last week was spent doing our own research to prepare for our start doing the microbiology work, such as researching the best sampling method and the best agar to transfer it too along with looking at what types of bacteria are already present on bones and our hands to see if these can cause degradation.

Now comes the exciting stuff and will keep you all up to date at the end of next week!
Have a good week! 🙂

Afternoon all Just to let you know the…

Afternoon all…Just to let you know the hard work that Amy and Jo have been doing to VLOG (Video blog) as well as blog their progress is now live on Youtube. Both this blog and the vlog are new experiments for Nicki and myself to see just how useful/effective different means of web-sharing can be; maybe it’ll become something we embed into teaching from hereon in, maybe it won’t…only one way to find out.

UROS VLOG on Youtube

Evening All Today myself and Jo came into…

Evening All! 🙂

Today myself and Jo came into the lab at 9am, and Mike gave us some new stuff to play with! – the newly arrived BioRad plug making kit and restriction enzyme digestion 🙂
We worked through the kit and information provided, made some notes and researched anything we didn’t understand online and through a discussion with Mike. We then produced a new COSHH assessment for our new procedure of making plugs and researched any modifications that journals mentioned when using Propionibacterium acnes specically.

We then made up a stock solution of Chloramphenicol (90 mg/ml), that we will need during plug production. It is a antibiotic, that is bacterial static and stops replication. We will also be using lysozyme, to break down cell membranes, Proteinase K, to break down protein and restriction enzymes, to cut up DNA specifically. Each also has a specific buffer, this is due to enzymes specific nature (pH, Cofactors). We also discussed why it was important to remove remaining EDTA, as it removes cofactors, which are very important to enzyme activity! We also discussed how obtaining the proper cell concentration within the plugs is most likely to be the most difficult part of production. In order to get the most accurate concentration we will be using an OD and CFU/ml against time to produce two sigmoid curves in order to work concentration out.

Excited to start practising these new techniques, so bring on tomorrow morning! 🙂

Right have been a little late setting this…

Right have been a little late setting this up so here goes,

My names Dan and i’m working on a UROS project alongside Sophie Webber, under the superfision of Gillian and Clare. The project is looking at whether or not wearing gloves when handling skeletal material matters. Obviously in forensic cases gloves should always be worn, but with archaeological remains there is great debate about the need to wear gloves. Hopefully by the end of the project we will be able to see whether or not there a scientifically significant transfer of microbiota from handlers to the bones if not wearing gloves. If there is a significant difference in the quantities of microbiota on skeletal material when not where wearing gloves (In comparison to wearing gloves), the next step is to see whether or not the microbiota transeferred from hands actually degrade the bones themselves.

Thats the basics behind our project. now to bring you up to speed with whats happened in the first 2 weeks.

In the first week, i met with Gillian, Clare and Sophie to discuss the project and what we would be doing. Gillian set us off familiarising ourselves with the bones of the body- learning their names, their anatomical positions, and how to sex, age and obtain stature estimations from the bones concerning the individual- this was a great start point and also really boosts my chances of passing the anthropology exam in October/November.

Following this we were then asked to identify which bones in the universities skeletal collection required cleaning, and then to clean those bones. Cleaning bones is a slow process, but oddly hypnotizing so time flies by.

So thats the important stuff thats happened over the past 2 weeks, which doesn’t sound much but the time spent just examining the bones and learning from Gillian has greatly increased my knowledge. Next week Sophie and i will be joining you in the microlab and the project can really get underway.

The two of us have been working closely with another of Gillian’s UROS students, Jacob Abbott, who has been tasked with cleaning and analysing a skull found in Fiskerton LIncolnshire, that is believed to be Iron age!!!!!! How cool is that!!!!!!!! Unfortunately he first has to work out how to remove a huge lump of clay from inside the skull without damaging it, i wish him luck with that.

So i think i have said everything that needs to be said, i’m really enjoying my project thus far, and it staves off the dull boringness of being alone in Lincoln for the summer hols. See you all next week hopefully

Busy day today Started the day going through…

Busy day today,
Started the day going through wednesday and thursday’s results with Rachael, so she knows where we are with our cultures, and so we can plan what we’re doing next. Taught Hammad how to do water filtration, made up 950ml of M17agar base with 10g Lactose suppliment (suspended in 100ml distilled water), both were autolaved before mixing. After lunch Rachael poured the plates. Made up 2800ml (7 x 400ml) of LB agar and poured plates with five of these. The other two were left for Nicola to add ampicillin and then pour the plates.I also made up 250ml of 0.85% w/v NaCl, this also needed to be autoclaved. Made up two stabs and slopes with the PS aeruginosa broth that i incubated overnight. Counted colony cultures from Sharon’s Brayfod water project.

Afternoon Everyone Pretty short day today Myself and…

Afternoon Everyone!

Pretty short day today, Myself and Jo came in at 11am to film our latest videoblog (I’m starting to really enjoy making them!) and talk through our previously filmed material from last week with Mike. Our footage won’t be up for you all to view for a little while yet, but I will let you know when Mike makes it available!

We then removed our latest gel from the PFGE, stained it and viewed it. We have made some initial observations; that the separations unfortunately ran off the end of the gel and we also sized up the bands using the information within the NEB yeast package.

Myself and Jo decided on some modifications between us, and we will be reviewing these with Mike on Monday morning.

Can’t believe week 2 of the project is over already! See you all next week! 🙂

A really successful day Yesterday i streaked 4…

A really successful day!
Yesterday i streaked 4 Pseudomonas CFC agar plates with cultures of PS aeruginosa.Today the plates showed excellent growth and the cultures glowed florescent green. Gram staining showed that the cultures were Gram negative and rod shaped. All this was evidence that i had a pure strain of PS aeruginosa, I innoculated nutrient broth and incubated it overnight at 37C. Tomorrow i will make up two stabs and two slopes. One confirmed bacterial culture down twenty seven more to go!!! Using the Qcapture pro (microscope attached to a computor) i saved images of my Gram stained cultures to usb, hopefully i will post pictures soon.

Hello Everyone Sorry I Haven’t done a blog…

Hello Everyone! Sorry I Haven’t done a blog in a while… But i will start doing them now!

So here is a basic low down on what is going on in my project!
First I had been extracting E.coli from the Brayford and growing them on MSLA agar plates. These are the red plates and they will only grow the Ecoli from my sample as yellow, well rounded colonies. With the whole streak plate/broth/agar situation.. I have never really had much practice with it.. But now i know how to do it all! 🙂 (Thanks BioMeds)

In the last couple of days I have filtered 100ml of Brayford Water onto MSLA plates containing 100ug Ampicillin. This is a very high concentration of antibiotic, therefore the colonies I obtained on these plates were very resistant and pure. I then proceeded to make several LB broths with Ampicillin with the colonies from the plates. Today I used the QIAcube with my plasmid broths, and I also did alkaline lysis by hand and ran the samples side by side on a gel to observe the differences/similarity between the different methods and to see whether I had any plasmids and to interpret the banding patterns seen on the gel.
I found that in 2 out of my 6 samples I had a very distinct interesting band pattern occuring, so I will keep these and do futher work with them later and all the rest showed nothing so they have been disposed of!
I will now start making agar plates with different antibiotics on – starting with Ceftriaxone.
Once I have gathered all of this information from the different antibiotics I will see the range and frequency of plasmid stability systems present in plasmids conferring antibiotic resistance in the Brayford allowing understanding of their persistence in the environment!

This will also enable me to have an understanding of plasmids from E.coli that confer antibiotic resistance, focussing on the CTX-M ESBL gene which is found in E.coli which are resistant to third generation cephalosporins such as Ceftriaxone and cefotaxime. This is why I will be doing the above procedure just with these antibiotics on the plates and in the broths!

See you all tomorrow 🙂 Xxx

Hey all Today I have transformed some competent…

Hey all!
Today I have transformed some competent cells (DH5 alpha E.coli cells) and used four different plasmids pOG04, pOG4, pALA1029 and pOG4101 (pOG 4.003) and spread plated all the plasmids once they had been transformed and I have put them in a incubator overnight at 37 degrees! Tomorrow I am going to do alkaline lysis (plasmid DNA extraction) in the morning and then learn how to use restriction enzymes and do a restriction enzyme digest. I also plan to do gel electrophoresis with the different plasmids. I finally feel I’ve got the knack for using pipettes (A bit late I know) but i feel alot more confident with them.
Hope to see you all tomorrow! 🙂

Hello Everyone Myself and Jo got into the…

Hello Everyone!

Myself and Jo got into the lab early this morning, in order to set up our next PFGE run with our new modifications. We made up 3L of 0.5X TAE buffer and 150ml of agarose (in order to reduce error), then left the gel to set. While it was setting we made up 50ml of agarose to seal the plugs into the wells. We then cut up 4 samples of NEB yeast, pushed them into the wells and sealed them up with agarose. We then set up the PFGE equipment to run at 6°C, for 15 hours at 6 V/cm with a 70 second switch interval and then for 11 hours at 6 V/cm with a 120 second switch interval.

The run is to finish at 12.15pm tomorrow, so myself and jo will be in the lab from 11am to film our second videoblog section! 🙂

See you all tomorrow morning!

Today I have seen some really good growth…

Today, I have seen some really good growth on my streaked agar plates. Pseudomonas is growing nicely on CFC agar, and i am going to make up some agar to selectively grow Lactobacillus. Tomorrows task is to Gram stain the agar plates that have good single colony growth. My friend google will help me to identify the Gram stained bacterial cultures.

Evening all Myself and Jo spent this morning…

Evening all!

Myself and Jo spent this morning researching the next stages of our project, then we came into the lab for 3pm, in order to take our gel out of the PFGE. We then stained it up and viewed it with Mike. We saw much better separation than before, with the bands reaching all the way to the bottom of the gel, and running off. Both yeast samples, BioRad and NEB, also appeared consistent with each other. The 1Kb ladder was not visible, which was expected. However, the separations slightly shifted to the right, producing wonky bands, that unfortunately weren’t very defined. Mike said this was most likely due to imperfections in the agarose, and we could rectify this by producing 150ml of agarose in order to reduce error. In order to try and stop the yeast sample running off the gel we decided to change our switch interval as well. We also decided that as the NEB yeast has been the most consistent and successful sample, we would stop running the BioRad yeast and 1Kb ladder. We will keep the size of the plugs the same, and well as sealing them with agarose.

Reviewing the NEB yeast information we decided to try running our next gel following the guidelines provided, 1% agarose, 15 hours at 6 V/cm with a 70 second switch interval and then 11 hours at 6 V/cm with a 120 second switch interval. Using 0.5X TAE buffer, 2 samples of NEB yeast and at 6°C as before.

See you all tomorrow morning as we set up our next, and possible last gel before starting on our next stage in the project! 🙂

Today Rachael and I started to isolate single…

Today Rachael and I started to isolate single colonies from the agar plates we incubated yeserday and streak them onto fresh nutrient agar plates. These have been placed in the 37C incubator overnight. Hopefully if there is growth, we can start to Gram stain them, further research will help us to correctly identify them and make up more stabs and slopes. We also streaked a number of yeasts onto MEA and placed them into the 25C incubator. I learned that it is better to incubate some cultures at a lower temperature for longer than at a higher temperature as this could possibly kill some cultures, especially if the higher temperature is at the top end of their tolerance range. Whilst looking at the agar plates from yesterday the P.S. aeruginosa looked cloudy and smeary, very similar to B. cereus and not the expectation of P.S. aeruginosa. We Gram stained a couple of slides using P.S. aeruginosa they were Gram positive, our research showed that it should have been Gram negative, rod shaped. Because of this we are going to streak P.S aeruginosa on Pseudimonas CFC selective agar. Hopefully tomorrow we shall see some results. Thanks to Nicola and Mike i am learning to tag,italics and google chrome.

Afternoon Everyone Myself and Jo came in at…

Afternoon Everyone!

Myself and Jo came in at 9am this morning, and began filling out our COSHH and risk assessment forms. Although they were a bit of a challenge we managed to get them finished before our PFGE gel came out at 11.30am. Filling out the COSHH form involved making a list of all the chemicals we have used so far within our project, looking up their hazard category (using MDSS – which was very useful!), assessing their exposure potential (glacial acetic acid and ethidium bromide being our highest), steps we could take to protect ourselves (containment level), first aid and how to dispose of them. Our containment level for procedure was 1. The Risk assessment form involved identifying possible hazards within the lab, reviewing control measures we could take to reduce this risk and giving an overall risk assessment rating, ours was medium. I felt that filling out these forms was good practise as next year, during our final year projects, we will be required to do this ourselves for our own projects.

We also watched Mike re culture the Propionibacterium acnes in the anaerobic cabinet, which was really interesting to see!

We then removed, stained and viewed our gel. We managed to obtain much clearer bands and we no longer had very dark areas (large amounts of DNA) within the wells. However we did notice slight band elongation, which is caused by increased temperature. This is expected, as unfortunately the PFGE is still running at 25/26°C, instead of the 14°C it really should be running at.

We therefore decided to keep the smaller amount of DNA we were putting in the wells, keep sealing in the plugs, keep the buffer at 0.5X and not include the initial switch interval as before. In order to reduce the temperature and obtain clearer bands we decided to move the PFGE equipment into the fridge (to run at 6°C). In order to obtain more separation we decided to increase the voltage to 6.0 V/cm. We therefore set up and started a new run with these changes for 24 hours at 6.0 V/cm and with a 45 second switch interval, still using 2 samples of NEB yeast, 2 samples of BioRad yeast and 2 samples of 1Kb ladder. With a second block for 2 hours at 0.6 V/cm and with a 45 second switch interval, in case we weren’t ready to remove the gel.

Really excited to see how the massive change in temperature effects our separations! 🙂

See you all tomorrow!

Quiet day in the lab today made nutrient…

Quiet day in the lab today, made nutrient agar and MEA plates ready for tomorrow. Todays task was to try and streak plates with bacteria that looked dead. The dried bacterial sample were ‘wetted’ by adding a little Ringers solution and then streaked on nutrient agar (with the exception of the fungi, they were streaked on MEA). Once this was done, a little research was needed to find out the incubation periods for the target bacterial streak plates. On to more research to find out what each colony should look like and whether it is Gram positive or negative. Hopefully we can positively isolate and identify each bacterial culture and create stabs and slopes.