A Year in the Lab: Getting Started in the Lab!

After the initial period of supervisor meetings, filling in what seemed like every form under the sun, plenty of literature research and getting together a good starting plan I finally got to start working in the lab! As a postgraduate research student in the field of Biomedical and Medical Science, I am based in the University of Lincoln‘s microbiology and molecular biology postgraduate research lab. I have been in this lab before, but only ever under supervision, so it was really fun if not slightly scary to be able to work there unsupervised!

After my initial literature research and discussion with my supervisor, my research work begins with further classification of a collection of bacterial samples that were collected and stored by a MScRes student last year. These samples were collected in Lincoln from waste water, post treatment and analysed for antibiotic resistance along with genotypic analysis by PCR. The collection remains phenotypically unclassified and therefore is the starting point of my research.

IMG_2722University of Lincoln postgraduate research lab

Wednesday 16th October

I began my work in the lab by observing the collection of samples (Currently stored in the freezer as glycerol stocks), describing their appearance, taking photos and making a note of the amount of samples in the collection. The previous work conducted on the collection should mean that I am working with a collection of samples that contain a variety of Gram negative Enterobacteriace.

IMG_2731Bacterial sample collection in glycerolIMG_2739Individual samples in the collection

In order to observe the bacterial samples, obtain single colonies and begin classification of the collection of samples, I began by making up MacConkey agar plates. I am using MacConkey agar as it allows the growth of Gram negative organisms and the differentiation of lactose fermenting bacteria from non lactose fermenting bacteria.

How to make up MacConkey agar

  • To begin with, weigh out an appropriate amount of MacConkey agar powder into an appropriate sized conical flask (You can calculate this based on the production information on the side of the container, and on how many agar plates you require).

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  • Dissolve the powder gradually in distilled water.

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  • Using cotton wool, plug the top of the flask, cover with grease-proof paper and seal this with autoclave tape. Make sure to label with your initials and the content of the flask, for example MAC (For MacConkey agar).

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  • Autoclave the agar for 45 minutes at 121ºC.

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  • When it is finished, remove the flask carefully and allow it to cool until comfortable to the touch.

 

How to pour agar plates

  • Begin by setting up an aseptic work space.

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  • Ensure you have empty plates, a bunsen burner flame and flask of molten agar within reach.
  • Remove the paper and cotton wool, and briefly flame the neck of the flask.
  • Begin to pour the plates in stacks according to your preference, gently swirling between pours to ensure even coverage and prevent bubbles (How many plates you pour at a time is up to you, I have small hands and therefore pour 5 at a time!).
  • Allow them to cool completely before inverting.
  • Clean the flask out with hot water to prevent any remaining agar from solidifying.
  • I allowed the plates to dry overnight at 18ºC.

Although I haven’t ever used MacConkey agar before and have limited experience in pouring plates I found the process fairly simple and problem free. However, during this first plate production I learnt several very important tips. Firstly, remember to dissolve the powder slowly and carefully in order to prevent clumping and burning during autoclave. When removing the cotton wool remember to twist as you remove, as this prevents cotton wool from sticking to the rim of the flask and burning when being flamed before use. Make sure the plates are inverted to prevent excessive moisture forming on the surface of the agar, as this can interfere with bacterial growth.

IMG_2726MacConkey plates are a really nice colour when poured 🙂

Thursday 17th October

I began by removing my MacConkey agar plates and selected bacterial samples from storage, allowing them to reach room temperature before use. I also vortexed the samples individually, to ensure they were as homogeneous as possible before spread plating. After labeling my plates with my initials, the date and sample identification number, I spread plated 50µl of each sample onto individual agar plates. Once spread I allowed the plates to absorb the sample for around 15 minutes before inverting and incubating at 30ºC for 24 hours.

During my undergraduate degree I had the opportunity to spread plate, so I was familiar with the process and therefore found it pretty easy and problem free. One problem I did have was more to do with the consistency of the samples. Even after vortexing some samples contained very large pellets and this made it very difficult, if not almost impossible to remove 50µl of sample. I made a note of these samples as I went, so that I can refer back if growth is subsequently effected.

How to spread plate

  • Begin by setting up an aseptic work space.
  • Ensure you have spreaders, an appropriate pipette, pipette tips, waste bucket, samples, and labelled plates within reach.
  • Using your pipette, take your required amount of sample and disperse it onto the surface of the plate.
  • Dispose of the tip in the waste.
  • Using a spreader, spread the liquid evenly across the plate.

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  • Dispose of the spreader in the waste (Unless you are using a reusable spreader).
  • Allow the sample to absorb into the plate for around 15 minutes before inverting and incubating appropriately (This will depend on your sample/plate).

IMG_2771Sleep tight plates!

Friday 18th October

I started the day by removing my plates from the incubator, and observing them using the colony counter. Although I wasn’t counting colonies, this gave me a good backing light in order to observe the plates. Working through the plates I made detailed notes, took photos of the plates and began to group similar looking plates/colonies together.

IMG_2815Selection of observed plates

Once finished, I selected a few plates with single colonies and interesting growth to heat fix onto a slide and Gram stain. As the collection should contain Gram negative bacteria, I used the Gram stain as a way to ensure this was the case and to view individual cell morphology.

How to heat fix a slide

  • Begin by setting up an aseptic work space.

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  • Ensure you have blank microscope slides, water, plates and a wire loop within reach.
  • Start by sterlising your wire loop in the blue bunsen flame (Ensuring you sterlise as much as the wire as possible).
  • Allow to cool then place 1/2 drops of water in the centre of the slide.
  • Sterlise the wire loop, before taking a single colony of your chosen sample and spreading it thinly and evenly in the water on the slide.
  • Sterlise your wire loop before laying it on the bench.
  • Keeping the slide close to the bunsen, or holding the slide above the flame (Making sure it doesn’t get too hot!) allow the water to evaporate.

How to Gram stain

  • Make sure you are wearing gloves, as the chemicals used during Gram staining can stain the skin.
  • Place your heat fixed slide on the bars over a sink. If you are using more than one slide, make sure the slides aren’t touching, as this can allow stains to move from slide to slide.
  • Apply crystal violet to the slide for approximately 30 seconds (This allows staining of Gram positive organism’s thick peptidoglycan wall, giving them a purple colour).
  • Rinse well with tap water.
  • Apply iodine to the slide for approximately 30 seconds (This binds crystal violet ensuring it remains within the cell wall in Gram positive organisms – it sometimes appears black on the slide).
  • Rinse well with tap water.
  • Rinse the slide with ethanol until it runs clear, around 10 seconds (This allows rapid decolourisation of the sample, as Gram positive organisms have a thick peptidoglycan wall and have been treated with iodine, the crystal violet is not removed. However as Gram negative organisms have an outer membrane with a thinner peptidoglycan wall beneath, they are decolourised – so don’t be worried if all the staining is washed off!)
  • Rinse well with tap water.
  • Apply safranin to the slide for approximately 2 minutes (This allows staining of Gram negative’s thinner peptidoglycan wall, giving them a pink colour).
  • Dry the slide before use (If you are too heavy handed you can rub your stained sample off the slide – so be careful!).

IMG_2822Gram staining

Microscopy

The slides were then observed under kholer illumination at X1000 oil immersion magnification.

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All the samples I Gram stained were Gram negative, rod shaped organisms, which was consistent with what was expected of the collection. I have heat fixed slides and Gram stained many times before, so I found these processes very simple and easy to complete.

48 - x1000 magGram stained sample at X1000 oil immersion demonstrating the individual Gram negative rod shaped cells

This was taken down the microscope on my phone, so apologies for the quality! I should be getting to use the microscope camera soon, so hopefully better quality photos 🙂

A Year in the Life…

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