A Year in the Lab: Bradford Bacterial Plug Production, PFGE and PCR

Monday 16th June 2014

This week began by inoculating LB broth with single colonies from my Bradford agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. If the sample was above 0.7 abs then chloramphenicol was added to inhibit replication and the sample was left out on the bench. However, if the sample was below 0.7 abs it was returned to the incubator for another hour to allow for further growth to occur. Once reached, each LB broth culture was processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

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Tuesday 17th June 2014

Today started with the washing of my bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC until more restriction enzyme arrives in the lab.

I then prepared and labelled LB broths ready for inoculation tomorrow morning.

Wednesday 18th June 2014

As on Monday today consisted of inoculating LB broth with single colonies from my Bradford agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. Once reached, each LB broth culture was processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

Unfortunately the QIAcube is going to be out of action for a while until an engineer from QIAgen can come to have a look at it, so I decided it would be better for me to extract DNA from my PCR reference strains by hand, through alkaline lysis as I have done before. I therefore inoculated LB broths with single colonies from my previously produced streak plates and incubated these overnight, shaking at 37ºC.

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Thursday 19th June 2014

As on Tuesday today started with the washing of my bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC until tomorrow (Now my restriction enzyme has arrived!).

I then collected my overnight reference strain LB broths from incubation to begin DNA extraction. This was conducted as before, starting by ensuring I had a waterbath set at 60ºC, labelled eppendorfs and 95% ethanol on ice, along with all other prepared reagents and chemicals. I began by pipetting 1ml of incubated sample into an eppendorf, centrifuging it for 5 minutes to form a pellet at the bottom of the eppendorf. I then removed the supernatant and re suspended the pellet in 100μl of TE buffer and 2μg of Lysozyme (100μl TE buffer + 2μl 50mg/ml Lysozyme Stock). This was then incubated for 30 minutes at 37ºC with agitation. 50μl of 10% SDS was added before incubating in a waterbath at 60ºC for 30 minutes. The sample was again centrifuged for 5 minutes, before remove the supernatant to a fresh eppendorf, discarding the old. 100μl of TE buffer was added and then, working in a fume cupboard, 250μl phenol chloroform isoamylalcohol was added. Ensuring the eppendorf is closed securely it was mixed for 1 minute (The mixture will appear white and cloudy). This was centrifuged for 5 minutes, and again in the fume cupboard, the top aqueous layer was carefully removed to a fresh eppendorf. I then added 650μl ice cold 95% ethanol, and incubated on ice overnight.

IMG_4989 Saturday 21st June 2014

I started today by inoculating LB broth with single colonies from my Bradford agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. Once reached, each LB broth culture was processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

I then finished off my DNA extraction from yesterday, by centrifuging for 5 minutes. I removed the supernatant and re suspended the pellet in 70% ethanol. Again I centrifuged for 5 minutes. The supernatent was then removed and discarded, before air drying the eppendorf until all ethanol had evaporated off. Finally 30μl of distilled water was added and allowed to incubate at room temperature for 5 minutes.

In preparation for running my first batch of new plugs tomorrow I cut each the first 14 plugs from the Lincoln collection in half, placed them into a fresh eppendorf and incubated in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.

Sunday 22nd June 2014

As before, today started with the washing of my bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC until they can be restriction digested tomorrow.

I then collected yesterday’s bacterial plugs from incubation, removing the buffer/enzyme before incubating the plugs in 1 x wash buffer for an hour with agitation. This was then removed and replaced with 0.5 x TAE buffer. While they were incubating in TAE I prepared and poured a 1% agarose gel.

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This gel was then loaded with the plugs, PFG and Lambda ladders before running overnight using the following settings:

  • 20 hours
  • 1% agarose gel
  • 0.5x TAE
  • 14ºC
  • 40-100 switch intervals per second
  • 6 V/cm
  • 30 minute stain, 10 minute destain

I ran this gel using the same settings as my last most successful PFGE gel as confirmed last week.

In preparation for running my next batch of new plugs tomorrow I cut each plug in half, placed it into a fresh eppendorf and incubated them in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.

Based on my failed first attempt at PCR last week I decided to try again with my new positive control sample. Again I just used my CTX-M group 1 primers, however I changed my positive control to the reference strain DNA from NCTC 13353, using ATCC 25922 DNA for a negative control. I carefully pipetted my required reagents into 50μl PCR tubes, including mastermix Taq2x, primers, plasmid DNA and pure water. I then placed this into the PCR machine and conducted a temperature gradient, ranging from 49.6ºC to 62ºC in order to optimise the most suitable temperature for group 1 forward and reverse primers.

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After 2 hours my PCR finished and I ran the resulting PCR product on a 1.8% agarose gel for 1.5 hours, alongside a 50 bp ladder for reference. I stained the gel for 15 minutes before viewing under UV. The resulting gel was much better than previously, demonstrating clear and accurate banding for both my 50bp ladder and samples. My negative control showed no banding, and my temperature gradient demonstrated that 62ºC was the most appropriate temperature out of those tested. I did notice however that my banding was very thick and dark. This could be as a result of using too concentrated primer and/or DNA.

PCR CTX-M Group 1 NCTC 13353 22.06.14

Next week…

I will be continuing to produce bacterial plugs from my Bradford samples, conducting restriction digests and then running these plugs, along with my previously produced Lincoln plugs, through PFGE to analyse strain. Based on my PCR work this week I plan on conducting both primer and DNA concentration gradients, as well as using the Nanodrop to analyse the purity and amount of DNA present within my extracted NCTC 13353 samples.

A Year in the Lab…

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A Year in the Lab: Lincoln Bacterial Plug Production, PFGE and PCR

Monday 2nd June 2014

This week began with conducting a final PFGE run with both my PFG and lambda ladder, one Escherichia coli sample and reference strain ATCC 25922 to confirm settings before mass plug production and running. As previously the plugs were incubated in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.

I then finished off my preparation for PCR by re-suspending my primers using pure filtered water to produce a 100μM stock, before diluting further to a 10μM working stock. I also diluted my practise bacterial DNA sample in the same way and used to Nanodrop to confirm the presence of DNA before use with PCR.

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Finally I set about calculating the amount of reagents required to produce a large batch of plugs, as I have only ever produced around 10 plugs at a time using 1/2 samples. Once finished I gathered together and prepared the reagents, including: LB broths, 2% agarose, cell suspension buffer, chloramphenicol, lysozyme and proteinase K.

Tuesday 3rd June 2014

I started today by inoculating my prepared LB broth with single colonies from my Lincoln agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. If the sample was above 0.7 abs then chloramphenicol was added to inhibit replication and the sample was left out on the bench. However, if the sample was below 0.7 abs it was returned to the incubator for another hour to allow for further growth to occur. Once reached, each LB broth culture was processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

Once finished I collected my bacterial plugs from incubation, removing the buffer and enzyme before incubating the plugs in 1 x wash buffer for an hour with agitation. This was then removed and replaced with 0.5 x TAE buffer. While they were incubating in TAE I prepared and poured a 1% agarose gel.

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This gel was then loaded with the plugs, PFG and Lambda ladders before running overnight using the following settings:

  • 20 hours
  • 1% agarose gel
  • 0.5x TAE
  • 14ºC
  • 40-100 switch intervals per second
  • 6 V/cm
  • 30 minute stain, 10 minute destain

I ran this gel using the same settings as my last most successful PFGE gel, in order to confirm that these settings are the most appropriate to visulise whole genome banding patters for Escherichia coli. This is in preparation for mass plug production (Of all samples identified as being Escherichia coli, antibiotic resistant and demonstrating plasmid DNA banding when ran through standard gel electrophoresis) and running through PFGE, in order to analyse banding patterns in relation to strain analysis.   

Wednesday 4th June 2014

Today began with the washing of my bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC until more restriction enzyme arrives in the lab.

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Once my PFGE gel had finished I stained it for 30 minutes, destaining for 10 minutes before viewing under UV. The resulting gel was consistent with the previously gel and confirmed that these settings are the most appropriate I have developed for running bacterial plugs containing Escherichia coli samples, alongside PFG and Lambda ladders. Now I have finalised these settings I can continue with mass plug production for all samples within my Lincoln and Bradford collections which fit my desired criteria, before running in batches through PFGE.

I spent the rest of the day preparing, running and visulising my first attempt at PCR. I decided to begin with just my CTX-M group 1 primers, using my previously extracted plasmid DNA as a positive control (Confirming DNA presence using the Nanodrop) and replacing this for water in a negative control. I carefully pipetted my required reagents into 50μl PCR tubes, including mastermix Taq2x, primers, plasmid DNA and pure water. I then placed this into the PCR machine and conducted a temperature gradient, ranging from 50ºC to 63.8ºC in order to optimise the most suitable temperature for group 1 forward and reverse primers.

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After 2 hours my PCR finished and I ran the resulting PCR product on a 1.8% agarose gel for 1.5 hours, alongside a 50 bp ladder for reference. I stained the gel for 15 minutes before viewing under UV. The resulting gel was disappointing but not surprising, my ladder was present and demonstrated clear bands however my plasmid DNA was not suitable as a positive control. I had previously expected that this may be an issue. so for my next attempt at PCR I will be using the reference strain Escherichia coli NCTC 13353 instead of using plasmid DNA. NCTC 13353 is recommended for use as a positive control for CTX-M group 1 ESBL’s, and it is currently glycerol stocked in one of the lab’s freezers.

Thursday 5th June 2014

As on Tuesday, today started by inoculating LB broth with single colonies from my Lincoln agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. Once reached, each LB broth culture was processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

I spent some time looking through the reference strains that we have in the lab’s freezers, and found 5 that could be useful for my particular research area. NCTC 11560, which is a TEM-1 Beta lactamase producer. NCTC 13353 (that I mentioned previously), which is a CTX-M-15 (Cefotaxime) ESBL producer and is often used as a control strain for group 1 bla CTX-M multiplex PCR assays (Like mine!). ATCC 23355, which produces cephalosporinase beta-lactamase II and can act as a bacteriophage host. ATCC 7006003A/B, which are SHV-18 producers. In order to check whether these glycerol stocks were still viable and visulise single colonies I decided to streak and spread plate them onto nutrient agar, along with ATCC 25922 which I use as a reference for identification of Escherichia coli. These plates were then incubated overnight at 37ºC.

Friday 6th June 2014

As on Wednesday, I spent today washing the bacterial plugs, in order to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC.

Out of the 6 reference strain nutrient agar plates NCTC 11560 was the only sample that didn’t grow. All others showed good growth on the spread plate and clean single colonies on the streak plate. In order to perform DNA extraction of the reference strains I then inoculated each into separate LB broths. These broths were then incubated shaken overnight at 37ºC.

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Saturday 7th June 2014

I started today by inoculating LB broth with single colonies from the last of my Lincoln agar slope collection. These broths were incubated shaken at 37ºC for 3 hours then the optical density (OD) was measured at 590nm. Once reached, each LB broth culture was processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

I had hoped to use my overnight LB broth cultures in the QIAcube, however when I arrived and tried to turn it on the screen displayed an error which indicated that a software issue was preventing me from accessing any of the programmes. I emailed our laboratory technician to let him know, but as it was a Saturday I knew there was no way I was going to be able to do an extraction today. So, I decided to centrifuge the samples anyway, remove the supernatent and freeze the pellet for use in the future.

Sunday 8th June

I finished off this week with bacterial plug washing! The washing procedure and times were kept the same as previously, and plugs stored in 1x wash buffer at 4ºC.

Next week…

Monday afternoon I will be traveling back home to Oxfordshire to attend job interviews for when I finish up my masters in September! Depending on what happens at the interviews I will be away for most of next week, and so lab work will be on hold until I’m back. When I get back I will be carrying on with bacterial plug production, beginning the Bradford part of my collection now all my Lincoln plugs are made. Hopefully more restriction enzyme will have been delivered and I can get started on restriction digesting the new plugs ready for running through PFGE. Lastly if the QIAcube is not up and running again I will be manually extracting DNA from my reference strains and having another go at PCR!

A Year in the Lab…

A Day in the Life: Woolsthorpe Manor, Woolsthorpe by Colsterworth, near Grantham, Lincolnshire, UK

Last week I traveled back home to Oxfordshire to attend job interviews for positions starting this coming September, once my masters has finished. After a successful but nerve wrecking week, I spent Father’s Day driving back to Lincolnshire with my parents. As the journey back takes around 3 hours, we decided to take a break around lunchtime and visit Woolsthorpe Manor near Grantham. My parents have visited nearby Belton House before and really enjoyed it, so I thought it would be good to explore another National Trust property in a similar area.

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Woolsthorpe Manor

Woolsthorpe Manor is the 17th century family farmhouse home of Sir Issac Newton. Newton was born in the house on Christmas Day, 25th December 1642, to Hannah Ayscough 3 months after his father (Also named Issac Newton) died. He was born prematurely and was not expected to live very long, especially in the harsh world of Medieval England. Three years later his mother remarried and decided to leave Woolsthorpe Manor with her new husband, Reverend Barnabus Smith. Newton remained at Woolsthorpe under the care of his maternal grandmother, Margery Ayscough until his mother returned in 1659 when she was widowed again. Newton attended The King’s School in Grantham from the ages of 12 to 17, leaving briefly to help his mother with farming at the family home before finishing off his education and being accepted at Trinity College in Cambridge. In 1665 Newton returned to Woolsthorpe when the Great Plague struck England and the university was closed as a precaution against further infection. During his time back home at Woolsthorpe he continued to develop his theories and conduct experiments regarding gravity, optics and calculus, until his return to Cambridge in 1667.

Gardens and orchard

As it was lunchtime when we arrived, we decided to head straight to the little cafe which is situated inside one of the many farm barns. We enjoyed our homemade carrot and coriander soup while looking at the Newton family timeline which ran around the top of the barn and the large solar system hanging from the ceiling. Once finished we headed out to explore the surrounding gardens and orchard containing Newton’s famous apple tree! Part of the orchard contained a wildflower garden and a mosaic tile human sundial which was really beautifully made and designed. Unfortunately as it wasn’t very sunny we couldn’t test it out 😦

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Following the path around to the front of the farmhouse we reached the site of the famous 400 year old apple tree, which was named 1 of 50 Great British trees in 2002. Naturally over 400 years the tree has been subjected to quite a lot of damage, falling over during a storm but thankfully re rooting as can be seen today.

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Farmhouse

We began our exploration of the manor house by ducking through the small main front door which would originally only have been used for weddings and funerals. Above the door you can see the Newton family coat of arms. Once inside we headed left into the parlour which was covered with reed matting to simulate the original flooring that would have been there before the current Victorian wooden floor was laid. I really liked an old English map on the wall, which showed the ‘countrie and citie of Lyncolne’ and how the county was once divided. Moving on across the hall we headed into the dining room and kitchen, at the back of the ground floor. Throughout the house there are many drawings and carvings on the walls, made by Newton himself, which have been framed so that they can be viewed easily. It was also great to see traditional food on display in the kitchen, including pork pies (From nearby Melton Mowbry), gingerbread and knot biscuits. Speaking to one of the volunteers we also found out that the original staircase was between these two rooms, concealing a priest’s hole underneath.

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Heading back into the hallway we went upstairs starting off in the left bedroom, in which Newton was born. Here we also found the house’s resident cat, sleeping next to the beautiful four poster bed. Hannah Ayscough’s spinning wheel is situated next to the fire place, which has a plaque above documenting Newton’s birth. Moving along the hall the next room is full of more information about the Newton family, life in medieval times and on the farm. The final room upstairs is another bedroom, containing several pieces of Newton’s equipment and notes from his experiments. My favourite part of this room were the papers displayed on the main desk, written on Sundays by Newton himself when he visited Woolsthorpe, confessing his weekly sins. I particularly liked one which confessed to ‘using another’s towel at university’!

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The site at Woolsthorpe is much smaller than other National Trust properties I have visited, however there is still plenty to see and do, especially in the science discovery centre. From Lincoln it would take around 45 minutes to get here, heading through Newark-on-Trent and then to Water Lane, Woolsthorpe by Colsterworth, near Grantham, Lincolnshire, NG33 5PD. The manor house, grounds and farm buildings are open everyday 11-5pm, except on Tuesdays when the manor is closed. As my family are members of the National Trust we got in for free, however at £6.04 per adult and £3.04 per child to visit the whole property I think it’s fairly inexpensive. I think it would be especially interesting for children who are just learning about Sir Issac Newton 🙂

A Day in the Life…

A Bake in the Life: Double Orange Cake

Whenever I am browsing through cookery books I always seem to end up choosing a recipe that feeds my love for the taste of citrus! This cake caught my eye as I was looking through a Mary Berry recipe book while back home for a week in Oxfordshire. There is nothing quite like a traditional sponge cake, but I do like to try and liven it up a bit with a fresh taste that stops it from becoming too heavy and sickly. Originally this recipe was a one layer sponge with a simple orange juice icing sugar topping, however I decided to split the batter into two tins to make a double layered cake and top it with a cream cheese frosting instead 🙂

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Double Orange Cake

You will need:

For a double layered cake

6 oz/175g Butter

6 oz/175g Caster Sugar

6 oz/175g Self Raising Flour

3 Eggs

1½ tsp Baking Powder

Grated zest and juice of 1 orange

Cream cheese icing

10½ oz/ 300g Icing Sugar

2oz/ 50g Butter

4½oz/ 125g Cream Cheese

Grated zest of 1 orange

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Let’s get started!

  • Pre heat your oven to 180ºC/350ºF/Gas Mark 4, and grease/line two 6in/15cm cake tins.
  • Beat together the butter and sugar until light and fluffy.

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  • Sift in the self raising flour and baking powder, stir to combine fully.
  • Beat together the eggs, then add the orange rind and juice.

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  • Add to the dry ingredients and beat well until completely blended.
  • Divide the mixture evenly between the two tins, spread flat and bake in the pre heated oven for 30-35 minutes until well risen and golden brown.
  • Turn out onto a cooling rack and leave to cool completely.
  • In a separate bowl beat together the butter and cream cheese until smooth.

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  • Sift in the icing sugar and beat together for around 5 minutes until the icing becomes thick and fluffy.
  • Once the cakes are cool, fill and top with the cream cheese icing then decorate with grated orange zest or a slice of orange.
  • You are all done!

I really like that the orange zest gives this sponge cake tons of citrus flavour, while the juice keeps the cake moist. You could of course substitute the orange for another citrus fruit, but I love the colour orange zest/juice gives to this cake and the icing. The cream cheese icing complimented the cake well, and I would definitely recommend trying it out! Although I decided to make a double layered sponge cake I think this recipe would work well as cupcakes, topped with cream cheese icing and one of my favourite jelly oranges/lemons 🙂

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A Bake in the Life…

A Year in the Lab: Preparing for PFGE and PCR of Lincoln and Bradford Collections

Tuesday 20th May

In order to confirm that all my isolated water samples are purely Escherichia coli and that my nutrient agar slopes haven’t become contaminated over time I decided to inoculate nutrient agar plates. Instead of streak plating completely I decided to just streak once across the whole plate as I am only concerned with viewing colony growth/morphology. I began with all my isolated Lincoln Escherichia coli samples as well as a reference strain for Escherichia coli ATCC 25922 to allow comparison. These plates were then incubated overnight at 44ºC.

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Wednesday 21st May

Today started with removing my streaked agar plates from incubation and comparing them to my reference strain in order to confirm purity. I used a piece of black cloth as a background in order to examine the colony growth/morphology carefully. The samples which were positively confirmed as being Escherichia coli will be made into bacterial plugs for use in PFGE, as well as for PCR.

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Wednesday 28th May

I began today by preparing, autoclaving and pouring more nutrient agar plates.

I spent the rest of the day writing up a plan for lab work for next month, after my exam facilitation work is finished. I also showed one of our laboratory technicians, who will be completing some research work, how to use the Spiral Plater. I prepared some MLSA plates as well as some Escherichia coli overnight broths, which we diluted before spiral plating and incubating overnight.

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Thursday 29th May

I started today by streaking the nutrient plates I made yesterday with all my isolated Bradford Escherichia coli samples as well as a reference strain of Escherichia coli ATCC 25922 to allow comparison. These plates were then incubated overnight at 44ºC.

Following on from yesterday’s spiral plating work, we viewed the incubated spiral plates and I showed him how to use the automatic plate reader.

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Friday 30th May

I removed my streaked agar plates from incubation and compared them to my reference strain in order to confirm purity. Again I used a piece of black cloth as a background in order to examine the colony growth/morphology carefully. The samples which were positively confirmed as being Escherichia coli will be made into bacterial plugs for use in PFGE, as well as PCR.

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Next week…

Now my exam facilitation work is all finished I will be able to get back into the lab and start producing bacterial plugs from all the samples which were positively confirmed as being Escherichia coli (Both Lincoln and Bradford collections). While I am producing these I will simultaneously be running my PCR primer temperature gradients, as well as beginning to run the newly produced bacterial plugs through PFGE.

A Year in the Lab…