In the next few weeks I will be finishing the remaining experiments I need for my PhD thesis and then I will be hiding away in the library writing full-time. As much as I have enjoyed my PhD research I can’t pretend that I haven’t found some of the tasks involved dull, tedious and at times frustrating. Here are a few of the worst:
Most of my experiments require plants to be grown in sterile conditions. Any microbial contamination can ruin an experiment, with fungi the main culprits, so everything must be sterilised and every effort made to keep it so during the experiment. This begins with the sterilisation of the seeds themselves. For my research subject Medicago truncatula the protocol is quick, taking only about 15 minutes to treat the seeds with bleach and wash it off but having done this once a week for 4 years any novelty it may have had wore off a long time ago.
Counting bacterial infection events on Medicago truncatula roots
When nitrogen-fixing bacteria and legumes form mutually beneficial interactions (symbioses) the bacteria infect into the plant root to reach the developing nodules where they are provided sugars in exchange for providing nitrogen for the plant. In many legumes the bacteria infect through the root hair cells on the surface of the root, guided by plant membrane structures called infection threads. These can be viewed under the microscope. In the image on the left the bacteria are stained blue indicating where the infection thread is.
One way to assess whether a plant line has a defect in bacterial infection is to count the number of infection threads formed on each root. This is very time-consuming. However, this experiment has provided some good results for my thesis so I love AND hate it at the same time.
Microinjection of root hair cells
Some of my other experiments have required microinjection of calcium-sensitive fluorescent dyes into root hair cells. The procedure involves using a machine to make a tiny needle by heating and pulling glass capillary tubes, loading the tiny needle with dye, piercing a root hair cell membrane with the needle (without the cell bursting) and injection of the dye by running an electric current through the needle. Oh, and then the needle has to be removed without the cell bursting. All under a microscope. Simple really.
I get a big sense of satisfaction when I successfully microinject a cell but because there are so many things that can go wrong along the way it can also be very frustrating.
Organising my Endnote library
I should start by saying that I am very grateful for reference managers in general. Being able to easily file references on a computer and input them quickly into a piece of writing without having to type the details by hand saves a lot of time. However, when you import references into Endnote (not sure about other reference managers) from a database e.g. PubMed, Web of Knowledge they may be incorrectly formatted (capitals, italics, superscript etc). At some point this should be remedied otherwise these references will be incorrectly formatted in the bibliography of your writing too. Invariably I usually fail to check the formatting of the reference when I import it so occasionally I have to trawl through my library, which contains several hundred entries, correcting as I go…
Repetitive pipetting of tiny volumes of liquid
Moving around tiny (i.e. less than 10 microlitre or µl) volumes of liquid using a micropipette needs to be done carefully to maintain accuracy. Experiments involving DNA/RNA often require mixing of several solutions in tiny volumes and if you have multiple samples of DNA/RNA then you can find yourself setting up many different tubes or wells with various combinations of solutions. It is very easy to miss out a tube/well or add the wrong solution so it is the worst type of dull task: one that requires a lot of concentration.
Despite my grumbles these activities have all been necessary/highly useful for my PhD studies. One of the advantages of the academic research environment is that I have been able to choose what I research and so in general I have been the person making the decisions to do these tasks and I also get to decide when I will do them. So maybe it’s not so bad…