A Year in the Lab: Lincoln Collection Bacterial Plugs

Monday 17th March 2014

This week began with preparing overnight cultures of 2 E.coli samples from my Lincoln isolate collection ready for plug preparation. I did this by taking a single colony from the appropriate nutrient agar slope and inoculating into LB broth, incubating overnight at 37ºC with agitation.

Once finished I then continued glycerol stocking my Lincoln bacterial isolate collection by streak plating the rest of the sloped isolates (E 42-.140) onto nutrient agar in order to ensure all samples were purely Escherichia coli and achieve single colonies. These plates were then incubated overnight at 37ºC.

Tuesday 18th March 2014

Today started with removing the overnight streak plates from the incubator and inoculating LB broth with a single colony from each plate. These broths were then incubated overnight at 37ºC.

Once finished I began bacterial plug production by removing the overnight broths and reading the optical density (OD) using a bench top spectrometer at 595nm. Appropriate OD for PFGE plug production is between 0.8-1, as this ensures enough bacterial cells are present to result in a significant amount of DNA after lysis. Each bacterial broth was then 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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Wednesday 19th March 2014

Once plugs had finished incubation in proteinase K, each plug was washed to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs were then stored in 1x wash buffer at 4ºC.

I then removed the overnight LB broth cultures from incubation and continued glycerol stocking. I pipetted 500μl of each overnight LB broth culture into separate tubes before adding 500μl of 10% glycerol LB broth. These were then carefully labelled and stored in the freezer.

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Thursday 20th – Friday 21st March 2014

As I didn’t have enough time left in the week to incubate the new batch of plug in restriction enzyme and then run PFGE I decided to just keep the plugs stored in 1x wash buffer at 4ºC until next week.

I then started preparation for further sample collection next week. As I have been processing my collected water samples they have slowly been whittled down to a much smaller collection than originally started with. Therefore I would really like to collect some more water to ensure I end up with a substantial collection before I begin PCR. I found and autoclaved 2 empty Duran flasks in preparation for water collection, then made, decanted and autoclaved lactose peptone water and tryptone water for E.coli identification.

Next week…

Now I have a new batch of bacterial plugs made from samples in my Lincoln isolate collection I would like to start the week off by running these through PFGE with my best settings to date to see how the banding patterns differ from each other. Therefore, beginning to visulise species differences between Escherichia isolates and allow comparison. I would also like to collect and process more water samples to ensure I have a substantial amount of samples within the Lincoln collection before I began conducting PCR.

A Year in the Lab…

A Bake in the Life: Bakewell Tart

Whenever I go out with friends or family, bakewell tart is one of my favourite pastries choices alongside my usual flavoured latte. I love the mixture of tart raspberry jam, sweet sticky almond filling and crisp pastry especially with a really good cup of coffee! I have never made my own shortcrust pastry for a tart case before so I brought a flan case with a removable base and baking beans ready to give it a go. I decided to use traditional raspberry jam, however I would love to try this recipe out with different jam flavours, particularly one of my mum’s homemade jams!

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Bakewell Tart

You will need:

For the shortcrust pastry

6 oz/175g Plain Flour

2½ oz/75g Chilled Butter

2-3 tbsp Cold Water

For the filling

1 tbsp Raspberry Jam (You can use any jam flavour you like – cherry, apricot or blackcurrant would be delicious too!)

4½ oz/125g Butter

4½ oz/125g Caster Sugar

4½ oz/125g Ground Almonds

1 Beaten Egg

½ tsp Almond Extract

1¾ oz/50g Flaked Almonds

For the icing

2¾ oz/80g Icing Sugar

2½ tsp Cold Water

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Let’s get started!

  • To make the pastry, combine the flour and butter using your fingertips until the mixture resembles fine breadcrumbs. Add 2 tbsp of cold water and begin to pull the mixture together into a ball, if needed add the final tbsp to form a soft dough.

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  • Roll out the dough on a lightly floured work surface large enough to line a 20cm/8in flan tin. Press the pastry gently into the tin ensuring it lays smoothly against the fluted sides.

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  • Chill in the fridge for 30 minutes.
  • Preheat the oven to 200ºC/400ºF/Gas Mark 6.
  • Line the chilled pastry case with foil and fill with baking beans (If you don’t have any you can always use rice, beans or pulses – as long as they are dry and heavy enough to prevent the pastry from puffing up!). Blind bake for 15 minutes, then remove the beans/foil and continue to bake for 5 minute to dry out the centre of the base.

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  • Once baked spoon your chosen jam into the pastry case and smooth out evenly using the back of the spoon.

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  • Melt the butter over a low heat, remove and then add sugar, ground almonds, egg and almond extract – stirring well to combine.
  • Pour into the flan tin and top with flaked almonds.

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  • Bake for around 35 minutes, until golden brown (If the almonds appear to be browning too quickly, cover with foil and continue to bake).
  • Once baked, remove from the oven and allow to cool for in the tin before gently removing and placing on a serving plate.
  • To make the icing, combine icing sugar and water until liquid enough to pipe.
  • You can either drizzle the icing over the cooled tart, or spoon into a pipetting bag and pipe over the tart (As I don’t have a re usable pipetting bag I just use a plastic food bag and cut off a small part of the corner to create a nozzle).
  • You are all done! 🙂

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I’m really happy with how my first attempt at shortcrust pastry turned out. The pastry was crisp and cooked all the way through, no soggy bottom thankfully! I thought that making a homemade Bakewell tart might take a lot of time and effort, however this recipe was really simple to follow and only took around an hour from start to finish. If you are nervous about making your own pastry you could always buy a ready made pastry case, but honestly I found it really easy. As long as you remember to cover the entire base of the case with baking beans, rice, beans or pulses to prevent the pastry from puffing up, and bake for a little longer after to ensure the base dries out completely.

This recipe has opened up a whole new world of tarts and pastry bakes! I would really like to try and make my own quiche or savoury tart, maybe using one of my favourite combinations – caramalised onions and goat’s cheese 🙂

A Bake in the Life…

A Year in the Lab: PFGE and Glycerol Stocking

Monday 10th March 2014

This week started off by completing plasmid isolation on my remaining Lincoln sample collection. I inoculated LB + CTX broth with a single colony from my collection of Escherichia coli nutrient agar slopes (E50-.140). These were then incubated overnight with agitation at 37ºC.

Once finished I then prepared more plugs ready to continue perfecting PFGE settings. 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.

Tuesday 11th March 2014

Today began by removing my inoculated LB + CTX broths and isolating plasmid DNA through alkaline lysis. Once completed, the plasmid DNA was stored at 4ºC until tomorrow when it will be run through gel electrophoresis to visulise banding patterns. I also inoculated tryptone water (TW) and lactose peptone water (LPW) with a small amount of the overnight LB + CTX broths before incubating overnight at 37ºC.

I collected the bacterial plugs from incubation, removed the buffer and enzyme then incubated 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. This gel was then loaded with the plugs and an appropriate PFGE ladder 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 decided to keep all the settings the same as for my last gel, except for the switch intervals which I reduced slightly in order to clean and tighten up the bands that are have become more visible on my previous PFGE gels.

I then began glycerol stocking my Lincoln bacterial isolate collection by initially streak plating sloped isolates (E.1-41) onto nutrient agar in order to ensure all samples are purely Escherichia coli and to achieve single colonies. These plates were then incubated overnight at 37ºC. Fortunately I already have some LB broth and 10% glycerol which was given to me by another student in the lab, so I have enough to continue glycerol stocking tomorrow. Instead of using flip top eppendorfs I will be using screw top, therefore I also prepared and autoclaved screw top eppendorf tubes separately.

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Finally, I made and autoclaved more nutrient agar slopes, before finishing re sloping my bacterial collection.

Wednesday 12th March 2014

I started off today by running the plasmid DNA samples which I isolated the day before on gel electrophoresis in order to visulise the banding patterns present. The resulting gel demonstrated plasmid banding patterns between 1.0 and 1.5 kb for samples: 53, .53, .92 and .126. This therefore indicated that these Escherichia coli samples contained isolated plasmids which can then be run through PCR to look for CTX-M specific Bla genes.

Once my PFGE gel had finished I stained it for 30 minutes, destaining for 10 minutes before viewing under UV. The resulting gel demonstrated good clear individual bands for 2 of my bacterial isolate samples (52 and 53).

PFGE

For sample 52 – 2 very clear bands approximately 680kb and 610kb, and 2 blurred bands approximately 356-450kb and 285-365kb. For sample 53 – 2 very clear bands approximately 945kb and 610kb, and 2 blurred bands approximately 356-450kb and 285-365kb. The next step I would like to pursue is producing a further batch of bacterial plugs using samples from my new Lincoln collection, in order to perfect my PFGE running procedure.

I then removed my streak plates from the incubator and inoculated LB broth with a single colony from each plate. These broths were incubated overnight at 37ºC.

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Finally I removed the overnight LPW and TW inoculated with E50 -.140. I added Kovac’s reagent to the TWs then observed and noted down the results to both tests. A colour change from red to yellow in LPW and the presence of a cherry red ring in TW, indicate Escherichia coli.

Thursday 13th – Friday 14th March 2014

Today began with the removal of my overnight LB broth cultures and continuation of glycerol stocking. I pipetted 500μl of each overnight LB broth culture into separate tubes before adding 500μl of 10% glycerol LB broth. These were then carefully labelled and stored in the freezer.

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Next week…

Now I have perfected PFGE settings for the current batch of bacterial plugs I have been using, I would really like to move onto running further PFGE gels using E.coli samples from my new Lincoln collection. I would also like to finish off glycerol stocking my collection to ensure I have continued access to my isolates over the next few months. As I have selectively identified CTX resistant/intermediate E.coli, isolated and visulised plasmids I have significantly reduced my number of original isolates. Therefore I would like to collect another water sample in order to increase my number of samples a bit more before starting to conduct PCR.

A Year in the Lab…

A Bake in the Life: Raspberry, White Chocolate Crumble Muffins

This recipe comes from my favourite muffin recipe book, which is pretty much the first place I look when I want to try out something new. I am a massive fan of white chocolate and raspberry together, so when I spotted this recipe I knew I had to make it! I have seen muffins in coffee shops/bakeries that have this style of crumble topping and I really enjoy both the look and texture it gives, particularly on top of fruit based muffins. I have made Apple and Blackberry Crumble before so I know how to make a good crumble, however this will be my first time sprinkling it over muffins 🙂

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Raspberry, White Chocolate Crumble Muffins

You will need:

For the Muffins

10 oz/280g Plain Flour

1 tbsp Baking Powder

½ tsp Bicarbonate of Soda

Pinch of Salt

4 oz/115g Caster Sugar

2 Eggs

9 fl oz/250ml Natural Yoghurt

3 oz/85g Melted and Cooled Butter

1 tsp Vanilla Flavouring/Extract

5½ oz/150g Frozen Raspberries

3½ oz/100g White Chocolate Chips/Chunks

For the Crumble Topping

1¾ oz/50g Plain Flour

1¼ oz/35g Butter

1 oz/25g Caster Sugar

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Let’s get started!

  • Preheat your oven to 200ºC/400ºF/Gas Mark 6.
  • Begin by greasing or lining a 12 cup muffin tray with paper cases.
  • To make the crumble topping, combine flour and butter then rub between your fingertips to create a fine breadcrumb like texture.
  • Stir in the sugar and set aside until later.

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  • In a large bowl sift together flour, baking powder, bicarbonate of soda and salt, then stir to combine.
  • Beat together the eggs, butter, yoghurt and vanilla in another bowl/measuring jug.

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  • Add the liquid ingredients to the dry along with the chocolate chips/chunks and gently stir to combine.

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  • Spoon the mixture into the prepared muffin tin then top each with 2-3 raspberries (I decided to do this just before baking to prevent the raspberries from all sinking to the bottom, but you can combine them when you add the chocolate).

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  • Finally, sprinkle your prepared crumble mixture over the top of each muffin and bake for 15-20 minutes until golden brown and well risen.

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  • Allow to cool for 5 minutes in the tin then cool completely on a wire rack.
  • You are all done! 🙂

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Placing the raspberries on top of each muffin just before baking worked really well, ensuring that they didn’t all sink to the bottom and instead were visible once they had baked. In combination with the golden brown crumble topping the bright pink colour of the oozing raspberries looks great! Once you break them apart you get the sweet taste of the white chocolate chips as well as a subtle hint of vanilla. The crumble was really simple to make, as were the muffins and all together only took around 30 minutes. I decided not to top these with an icing, but instead just sieved some icing sugar over each. Definitely try these out if you love the taste of sweet tart raspberries and milky white chocolate 🙂

A Bake in the Life…

A Walk in the Life: Nocton, Lincolnshire, UK

Now that the weather is definitely starting to feel more like Spring and Summer we decided to head out for a walk in the nearby village of Nocton, east of Lincoln. This walk is one part of two neighbouring walks which explore both the villages of Nocton and Dunston, as well as the surrounding woodland, fens and heaths. As with many of the walks I have tried out this is another walk from the Visit Lincolnshire Stepping Out series, which I would definitely recommend!

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Nocton Village Walk

This walk starts from the Village Hall car park, Main Street, Lincolnshire, LN4 2BH, accessed via the B1188 Lincoln Road. There is plenty of free parking behind the village hall, which is quick to find off the main road through the village. From the centre of Lincoln it took around 15 minutes to get there by car.

Off we go…

1. From the car park behind Nocton Village Hall turn right and head towards the centre of the village.
2. Opposite the post office, follow the way markers along the restricted byway (Look out on the left hand wall for the Nocton cow and a small snail plaque!).

March 2014 015Smiling Nocton Cow – made from old pieces of painted metal

March 2014 017Mosaic tile snail 

3. Continue along this path until you reach a crossroad. Take the bridleway on the left, keeping the Nocton Cricket Ground on the right.

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4. Follow this path as it bends to the right (Look out for views over an orchard with the village church behind on your left!) and continue on through Burton Plantation.

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5. As the path bends left continue to follow it through Grotto Holt (Look out for buzzards flying overhead and smaller birds in the hedgerows – great tits, chaffinches and blackbirds).

6. When you reach a crossroad beside the driveway up to a large house, head straight over and continue up a gentle slope to a metal gate (Make sure to stop here and look out over the surrounding fields and listen to the birds in the nearby fir trees).

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7. Turn right here, through an area of trees and continue until you reach a crossroads of grassy paths.

8. Take the left path and follow this, keeping Nocton Wood on your right.

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9. As you reach the end of this path, head off left along a grassy path between young trees.

10. At the end of this path, carry straight on towards a group of houses known as Wasps Nest (Definitely stop and look out over the surrounding fields towards Whisby Nature Park).

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11. At Wasps Nest turn left and follow the ancient Roman Car Dyke until you reach a sharp right bend in the road.

12. Leave the road and follow the way markers left onto a grassy path heading back towards Nocton.

13. Continue along this path as it runs alongside a river and then into Nocton.

14. Follow the way markers through the houses and back to the car park (Make sure to stop and look around the old houses in the village, especially the post office).

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15. You are all done! 🙂

This walk is 4 miles/6.4km and takes around 2 hours to complete at a leisurely pace, however the walk does have several shortcuts/extensions which you can take to decrease/increase the original walk. When you walk through the village look out for several mosaics, carvings, cast metal panels and photography pieces which are part of a village trail. As we left the car park there were also loads of beautiful spring flowers (Crocuses and snowdrops) which were great to see!

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I really enjoyed this walk, as it not only allowed you to explore the beautiful village of Nocton, but also nearby woodland, surrounding fields and farm land. On our visit we saw plenty of interesting wildlife, including a variety of hedgerow birds (Great/Blue tits, chaffinches, gold finches, blackbirds and thrushes), Buzzards and mallards swimming along in the river. Nocton village itself is full of beautiful old houses and buildings, in particular the old post office built in 1833 and row of connected houses nearby. I would definitely recommend this walk, and I can’t wait to try out the Dunston walk in the next village!

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A Walk in the Life…