Most summers we have around 20 students working in the Microbiology and Molecular Biology lab, SB209, in the Science Building on the Brayford Pool campus. This is a place for those students to share their experience.
//so… not much progress from the last time… although I am now a master in pipetting and spitting in a tube. found a program called Tandem Repeats Finder on line but don’t know how to interpret the findings so no use of it until someone will be able to show me. Absolutely no research on line for some help in this field.
//Groundhog day!! The plates i incubated yesterday showed no growth at all, Spent the day repeating everything from yesterday, double checking every stage. The plates i innoculated today are now in the 37C incubater, fingers crossed all goes well.
//Really quiet day in the lab, today was the first day of my Uros, plasmid stability experiment. Started the day by growing E.coli C2110 to an OD of 0.35, tough to guess when this happens, 0.38 was the final reading. Whilst waiting for the cells to grow i then innoculated another flask of LB broth with C2110 and placed it in the 37C shaker.The next task was to make the cells competant and then transform them, using the plasmids pOG04 NJC and pOG04 DRW. The final step was to innoculate an LB agar plate (containing Ampicillin 50) and incubate overnight at 37C.
//Hey,
Today was a strange day, Did my final Tend plate count for the week. I inputted all of my weeks figures into a table and Nicola showed me some impressive maths to get to the percent stability of the plasmid used, in this case pOG 04. The percentage was 63, which was waaay out from the expected 3-5%. I am going to redo this plasmid next week and hopefully (wearing my lucky pants) everything will work out as expected. Did a little research into my eight final bacerial cultures still on my bench, i will not be defeated!!!
Also sad to see Sophie and Dan finish their project, both will be missed. Also going to be a little quiet without Amy and Jo whilst they’re n hols. Happy summer guys x
//So…the last post on the blog by me! Finally finished our UROS project today! Excited to have a bit of summer left! Had an amazing time and it’s been a great experience and i’ve learnt alot. I’ve learnt all the bones in the human body, and many anthropology skills and techniques to age sex and determine stature and these skills i will keep with me to help in my final year! I’ve also met lovely people in the micro lab that i probably wouldn’t have got chance to if i hadn’t done this project so for that i am thankful! Being able to work with my peers and lecturers has been a good experience and being able to work in a lab for a long length of time!
On to the project results…this week we swabbed the bones after being cleaned with ethanol and then we handled one set of bones with gloves on and one set without gloves and swabbed these too to come to some kind of conclusion as to wearing gloves affects contamination on the bone. The results mainly pointed in this direction with the swabs taken after handling without gloves gave more bacterial growth than that with gloves on.
We weren’t able to identify any of the bacteria in this project but it will make for an interesting project next summer maybe!
Thanks again everyone involved, its been great! 🙂 Enjoy your summers!!!
//Hey all!
Today we removed, stained and viewed our gel. We saw some very promising results! Although the separation wasn’t great, we saw a large blurred band near the bottom of the gel, near the middle of the low range marker bands present (which unfortunately were quite blurred and difficult to see). This confirmed the presence of DNA in the SpeI treated plug, if not very much, as estimated previously. However, enough DNA was present within the whole plug to show separation, confirming that our plug making procedure and settings were near enough on the right track!
Given the set backs we have been experiencing with the plugs this week and last, this was a real boost to get us excited to start putting our modifications into practise for plug making – increasing cell density/DNA and slightly modifying the PFGE settings – increasing the gel conc. from 1% to 1.4% in order to condense the ladder bands nearer the top, allowing further reference to the Propionbacterium acnes restriction enzyme treated plugs near the middle of the ladder.
We filmed our last videoblog for a week today, as i am away from the lab next week, this will also be my last blog for a week 🙁
Jo will also be away for the next 3 weeks, so we will not be able to put these new modifications into practise just yet! But i am very excited to get started, on what will hopefully be modifications that get us our best results yet for restriction enzyme treated and non restriction enzyme treated plugs on PFGE 🙂
See you all soon!
//Hey,
Started the day by making 400ml of LB agar and 200ml with AMP100 and 500ml of LB broth. Then i counted the T0 replica plates i did yesterday. Next i counted yesterdays T end plates and chose the ones with approx. 100 colonies and replica plated them onto LB agar and LB agar with AMP100. Then went to the presentation for poster presentation and project write up. Then i streaked a fresh LB agar plate with C2110 colony and then another LB agar plate with DH5alpha (for Rosie) and incubated them at 37C overnight, so that we have fresh colonies to work with next week. Nicola gave me my plasmids, popped them in the fridge. Nearly all prepped for next week. Also managed to make up stabs and slopes of E.coli e0791 that i brothed up yesterday. Also BIG thanks to Dan for his anthropology presentation and showing me the difference between boys and girls (on the skeletons). Very interesting seeing the ossicles and learning how to age and sex the bones.
//Hello,
Today we removed our latest gel, stained it and viewed it. Unfortunately we didn’t get very good results – there was alot of blurring on one the low range PFG marker samples, lack of clear band separation on the low range PFG marker samples and no expected band just below the wells for the non restriction enzyme plugs.
We decided that this was most likely due to lack of cell density, and therefore lack of DNA which that could be seen on the gel. We discussed that this could most likely to be fixed by allowing the Propionbacterium acnes samples to grow longer and therefore increase the cell density and DNA, and possibly by centrifuging more of the sample, to reach a higher conc. We also re researched info on cell density within plugs in the manual and journals – we found that the optimum OD was around 0.8-1.0 and we had been working with 0.24. Because of this increase in cell density/conc. we will also need to increase our lysozyme, mutanolysin and proteinase K to compensate.
Mike asked us to run a standard gel, in order to see if there was actually any DNA present at all. We ran a 0.8% gel, for 2 and half hours at 120V/cm with a 1kb ladder. We saw very faint separation, that did confirm that DNA was present, but at very low density/conc. as expected. This also confirmed our modifications which we will put into practise when i come from holiday on Monday 6th August.
After speaking to Mike we decided to attempt to run a new PFGE gel using the whole Propionbacterium acnes restriction treated plug, in one well, in order to use as much DNA as possible, and hopefully see some separation showing multiple bands due to lysis. We ran this 1% gel at 6V/cm, for 15hours with 1-12 sec switch interval ramping, using a whole Propionbacterium acnes restriction treated plug and the low range PFG marker.
Tomorrow we will be viewing this gel and filming our latest videoblog! 🙂
//Hi all,
Started day by completing my serial dilutions from yesterdays broth culture, I spread plated the dilutions of 10-6 and 10-6.5 on LB agar and incubated them overnight at 37C. Then taking the eight plates from yesterday I counted the colonies and chose the four with around 100 colonies. I then replica plated each of these onto a further two LB agar plates (one containing Ampicillin 100). These were then incubated at 37C overnight. I then also broth cultured another confirmed strain of E.coli (e0791).
//Evening,
Today we washed our plugs, as before in 1X wash buffer 4 times for an hour each. We then separated our 5 plugs into separate eppendorfs, and put 3 in storage in fresh 1X wash buffer. To the 2 remaining plugs we added 0.1X wash buffer for an hour.
To one we removed the wash buffer and added 1ml of Spe1 buffer to saturate for an hour. We then added fresh Spe1 buffer, Spe1 enzyme and BSA, and incubated overnight at 37C.
We set up a new gel in order to run the unrestriction treated plug, to ensure that the DNA within the samples are viable for use. If we see one large band just below the wells, that is as clear or clearer as our best previous gel run, then we will run a gel with the same settings, but with restriction enzyme treated plugs.
We made up 3L of TAE 0.5X and a 1% gel. We removed the wash buffer from the other plug and added 1ml of TAE 0.5X buffer to saturate it. Once the gel had set, we cut and placed 2 non restriction enzyme treated plug sections and 2 sections of the new low range PFG marker into the wells and sealed with agarose. We ran this gel at the low range PFG marker settings: 1% agarose gel, 15 hours, 1-12 second switch interval ramping and 6V/cm.
I also filmed with Sophia and Barney, regarding their experiences in the lab so far! 🙂
We will remove this gel tomorrow, stain it and view it. This will help us to decide whether to run the restriction enzyme plug, with the same low range PFG marker settings.
See you all tomorrow!
//…. and I thought the post about Yoda was the best thing I’d read this week… This surpasses it:
http://www.what-if.xkcd.com/4/
//Hi,
Started the day Gram staining E.coli a strain i dont have, this is another cultue confirmed so i then made a broth which i am incubating overnight at 37C. Spent most of the day waiting for cells to grow to an OD of 1, though 0.8 – 1.5 would be within range. Nicola went through serial dilution with me and showed me how to do the first set, i then completed the following three, broth culturing -4 dilution and spread plating -5 and -5.5 dilutions onto LB agar plates and incubated them overnight at 37C. Hopefully tomorrow we will have enough growth to count colonies (100 would be nice) then we can do replica plating. Had another Mike Shaw masterclass and debate (loving lab life xx).
//So, i keep forgetting to post but Dan has been up to date which is good so you can get the idea of what we have been doing!
Week 4 consisted mostly of waiting for our supplies to come in again and taking student as producer literally by experimenting with cotton swabs on some of the bones after handling to see how long the bacteria maintains on the bones. The results for these came in all shapes colours and sizes and so we were unsure if they were viable and not contaminated. We also helped out an an open day and workshops teaching the students how to age, sex and determine stature of the skeletons.
Week 5 we continued the experimenting into the cotton swabs and the bacteria living on the bones. However Wednesday we actually found all our supplies and so the micro side of the project finally began!!!
We plated up agar and made ringers in prep for our swabs. Starting with swabbing the bones with the polywipe sponges for a background check to see what was already on the bones, doing 5 repeats of 5 bones so a total of 25 nutrient agar plates, these were incubated at 37degrees for 48 hours and looked at again on Friday! Obviously as Dan has already said, the results were weird! research for you. They could have been from contamination, if not then our bones have lots of different types of bacteria/fungus present!! We sub-cultured most of the plates onto several different types of agar and we also took samples and gram stained them to see if we could identify what we had…this left us even more confused!
Now we are in our final week of the project and yesterday we cleaned 5 of the bones with ethanol (50%, 75% and 95%) and let the ethanol soak in to destroy bacteria, then swabbed with the polywipe sponges and placed in ringers solution, each plate had 0.1ml pipetted on and there was again 5 repeats for each bone. The same bones were then handled with gloves for 5 minutes and then repeated the swabbing steps to see what grows on bones handled with gloves. Today we made up 60 more agar plates for use tomorrow when we will clean another 5 bones and handle these with bare hands and see how they differ, like the project is called….gloves or no gloves?
We will have to wait and see!!
//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 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 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 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 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 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 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.