After spending the last few weeks with family back home in Oxfordshire, and it is good to be back in Lincoln ready to start back in the lab. Now I have phenotypically identified all the organisms within the bacterial collection, I am moving onto working to genotypically analyse the organisms.
I am planning on using two specific techniques:
- Agarose gel electrophoresis, specifically pulsed field gel electrophoresis (PFGE)
- Isoeletric focusing (IEF)
I have worked with agarose gel electrophoresis and PFGE before, however IEF is a new technique to me. Standard electrophoresis works on the principle that one electrical field in one direction causes the separation of DNA and RNA, based on the sample’s size and charge. Therefore smaller and more negatively charged molecules move faster, while larger and less negatively charged molecules move slower through the gel. As a result separation occurs due to individual electrophoretic mobility, and bands produced can be compared to known sized ladders.
On the left – PFGE gel demonstrating bands created by DNA fragments moving through gel based on size and charge. On the right – Appropriately sized ladder, which is used as a comparison and allows calculation of unknown band fragment size.
In comparison, PFGE uses several electrical fields from multiple directions at interchangeable switch intervals. DNA or RNA present within a sample form a standard state, which when applied with electrical fields elongates and begins to travel through the gel according to charge and size.
Time lapsed microscopic images of DNA in sample during PFGE, showing standard state, elongation and travel through gel (Birren and Lai 1995)
This change of direction and electrical field allows DNA that would previously have been too large, able to be separated. This is because DNA within the samples actually moves in a zigzag manner, due to the continuous change in electrical field direction, decreasing the length of agarose gel required for effective separation. As a result of this, PFGE is particularly useful for my research as it allows the separation of DNA over 10Mb/10,000Kb, separation of entire genomes and therefore analysis of individual bacterial strains.
PFGE equipment, including: CHEF-DR II unit, cooling system, buffer pump and power pack
IEF allows the separation of soluble proteins within a sample based on charge, which I will use to investigate bacterial enzyme production.
I also hope to develop an antibiotic resistance measurement protocol from techniques I have used before. Starting by collecting a water sample from the Brayford, running through established sample preparation and bacterial isolation. Then incoulating mueller hinton agar and conducting antibiotic resistance testing with antibiotic discs.
Monday 13th January 2014
I began the week by preparing stocks and plates. As I want to conduct antibiotic resistance testing on isolated organisms from the Brayford using antibiotic discs I will need Mueller Hinton agar plates. I produced 20 plates of MacConkey, Nutrient and Mueller Hinton agar by combining appropriate agar powder with distilled water in a conical flask, gently mixing, sealing with cotton wool and grease proof paper before autoclaving. Once finished I allowed the agar to cool before pouring aspectically and allowing to set.
As I will be running PFGE, I decided to start by reacquainting myself with the technique by practising running a standard electrophoresis gel. For this I require TAE buffer, so I produced a 1L 50x TAE stock that can be diluted to use during both standard electrophoresis and PFGE.
How to produce 1L 50x TAE buffer stock
- Begin by preparing an ethylenediaminetetraacetic acid (EDTA) solution.
- For a 500ml 0.5M stock solution, add 93.05g of EDTA disodium salt powder to 400ml of distilled water and allow to dissolve on a magnetic hotplate with magnetic stirrer.
- The EDTA disodium salt powder won’t dissolve entirely until it reaches pH 8, therefore sodium hydroxide (NaOH) must be added to adjust the pH.
- A 100ml 0.5M stock solution of NaOH was made by combining 2g of powder in 100ml of distilled water. The pH was measured using a pH probe and NaOH added until the pH reached 8 and all EDTA disodium salt powder dissolved.
- The solution was then topped up to 500ml using distilled water.
- 242g of Tris-Base was then weighed out and dissolved in 750ml of distilled water.
- 57.1ml of Glacial acetic acid was then added, followed by 100ml 0f the prepared 0.5M EDTA solution.
- Once completely combined, the solution was made up to 1L in a volumetric flask with distilled water.
- This solution was then separated into two brown Winchester bottles (50ml each) and autoclaved.
One finished I spent some time checking over and re setting up the PFGE equipment, as it was cleaned and packed away once we finished using it during the UROS project. I also did the same for the spiral plater and plate reader due to both being moved for lab fumigation.
Tuesday 14th January 2014
In order to conduct antibiotic resistance testing and establish a protocol I started the day by collecting a fresh water sample from the Brayford, using the same method as previously. On returning to the lab I inoculated nutrient broths with 50μl, 100μl, 500μl and 1000μl of water sample. Incubating these overnight at 37ºC.
I spend the rest of the day writing and preparing a restriction digestion and gel electrophoresis procedure, based on previous work and research papers. Once finished I collected together and prepared any equipment, reagents and chemicals required. Including loading buffer diluted with 50% glycerol.
Wednesday 15th January 2014
I began the day by observing the overnight nutrient broths, as expected universals were progressively cloudier from 50μl, 100μl, 500μl through to 1000μl. I then inoculated each sample onto MacConkey agar using the spiral plater and incubated overnight at 37ºC.
I spent the rest of the day reviewing the DNA extraction, restriction digest and plug production for PFGE, as well as calculating reagent and chemical stocks.
Thursday 16th January 2014
I observed the incubated spiral plated MacConkey plates, isolating single colonies and inoculating each into separate nutrient broths. These were then incubated overnight at 37ºC. The isolated colonies were preliminary identified as:
- Lactose positive violet – Enterobacter spp., Citrobacter spp. or Escherichia coli
- Lactose negative pink/cream – Proteus spp. or Pseudomonas spp.
- Lactose negative brown – Salmonella spp.
- Lactose negative light pink – Serratia spp.
Friday 17th January 2014
Overnight isolated colonies in nutrient broth were observed and streak plated onto nutrient agar, then stored on the lab bench over the weekend to allow slow growth ready for Monday. In order to work continuously with isolated organisms agar slopes can be used to allow storage for an extended period of time. I decided to produce 30 nutrient agar slopes.
How to make nutrient agar slopes
- Begin by combining nutrient agar powder with distilled water in a conical flask (The amount you use will depend on how many slopes you want to make).
- Dissolve and heat on a magnetic hotplate with magnetic stirrer.
- Once the mixture reaches boiling point, remove and allow to cool slightly before pouring 10ml into each glass universal.
- When all universals are filled loosen the lids and autoclave.
- Once finished remove and place into a metal basket, leaning the basket at an angle to create a slope once set.
- Leave slopes to set.
I then spent the rest of the day writing and preparing a DNA extraction procedure, based on previous work and research papers. Once finished I collected together and prepared any equipment, reagents and chemicals required. Including: Tris-EDTA (TE) buffer, lysozyme, SDS and ethanol.
How to make TE buffer
- Begin by producing a 1M 500ml stock of Tris-Cl, by adding 60.57g of tris in 500ml of distilled water, allowing to dissolve on a magnetic hotplate with magnetic stirrer.
- Tris powder won’t dissolve entirely until it reaches pH 8, therefore concentrated hydrochloric acid (HCL) must be added to adjust the pH 8.
- As I already had a stock of 0.5M EDTA prepared, I then combined 2ml of this with 10ml of prepared 1M Tris-Cl and 998ml of distilled water in a 1L volumetric flask.
How to make 50mg/ml lysozyme
- Combine 0.5g of lysozyme with 5ml of TE buffer.
- Gently mix to combine.
How to make 10% SDS
- Combine 10g of SDS with 80ml of distilled water.
- Gently mix to combine (SDS is a detergent and will create a lot of bubbles when mixed – so leave for a few minutes to settle before use).
- Make up to 100ml with distilled water.
How to make 95% and 70% ethanol
- Combine 9.5ml of 100% ethanol with 0.5ml distilled water.
- Gently mix to combine.
- Combine 7ml of 100% ethanol with 3ml distilled water.
- Gently mix to combine.
As I expected this week was mostly stock and reagent preparation, as well as procedure writing. I have four organisms isolated and streak plated ready to be inoculated on nutrient agar slopes next week. As well as mueller hinton agar ready to be inoculated and antibiotic resistance tested. I also have all reagents and equipment prepared to run a refresher standard gel using identified Escherichia coli samples from the bacterial collection. I hope that next week I will also be able to use these identified Escherichia coli samples to start producing plugs for use with PFGE.
Birren and Lai, E. (1995) Pulsed field gel electrophoresis – A practical guide, Academic Press Inc., CA, USA.
A Year in the Lab…