We Need to Stop Pigeon-Holing Science - Crossing the boundries between disciplines and addressing the interesting questions, as opposed to getting caught up in what we think a *insert field here*-ist should focus on, is the way to go.

Postdocs always overestimate their intellectual contributions - In a repost, the topic of thinking too highly of oneself is discussed.

Are We Training Too Many Scientists? - With competition in academia for funding is sky-high, this post asks a good question.

Gene Patents and Genetic Testing Should patenting of genes be permitted?

During my PhD I saw others around me working extremely long hours in the lab and not really having much of a personal life and quite early on I made the decision that this was not for me.

Although I enjoy my work, having a good life outside the is also very important. Also, I found that if I worked very long hours then I tended to be far less efficient overall.

But, I still wanted to get through as many experiments as possible. So I also made the decision that my approach would be to work a regular 8 hour day and be as efficient as possible during that time. My basic recipe for an efficient working day was:

1. Good time planning

2. Multi-tasking

3. Efficient and accurate record keeping

4. Short breaks (!)

Most of this worked quite well, but in retrospect the multi-tasking part was not a good idea, at least to the extent I took it to.

My aim in multi-tasking was to fill every part of the working day with something useful. So I would normally have several experiments going on at once and flit between them in a packed schedule. Gaps in the schedule were filled with the other necessaries: writing up, reading, making solutions.

I definitely got through a lot of experiments that way. But that was the only good thing that such a packed schedule delivered.

Multi-tasking with few gaps in the day made me stressed, error-prone and took the enjoyment out of the job. That’s not surprising looking at it now, but at the time I thought it was the best way to work.

A quick Google search on multi-tasking brings up a whole host of articles highlighting the inefficiency of multi-tasking (see here and here for example - both contain links to other useful articles including primary literature). Apparently the brain takes a certain amount of time to switch between tasks, so constant switching means constantly losing time and train-of-thought.

These days I still multi-task - although your brain is not designed for it, there’s no way around it. But I pay close attention to the number of tasks I am juggling. Where possible I perform only one or two experiments in parallel, and guess what? I have a much higher rate of success, and much less stress than before.

What do you think of multi-tasking?

In this article I’ll add the possibility of using a microwave to sterilize or decontaminate growth media. From the outset I’d like to say that I am not too sure about this, but I’ll make the case and you can tell me what you think.

Normally, growth medium is sterilized or decontaminated using an autoclave. Autoclaves are generally expensive, energy-hungry beasts that (in my experience) break down a lot so I would be very happy to use them less if I could.

Decontamination using microwaves.

The case for using microwave ovens for decontamination of cultures or materials was made back in 1977 by Latimer and Matsen. They showed that 1-5 minutes in a conventional microwave was sufficient to decontaminate 5mL cultures or petri dishes of common clinical pathogens including E. coli, S. aureus and K. pneumoniae. B. subtilis spores proved a bit more subborn, requiring more than 10 minutes of microwaves to wipe them out.

Border and Rice-Spearman backed this up with a 1999 study that showed materials contaminated with various bacteria and yeast strains were completely decontaminated by one minute in the microwave (I guess their microwave was better). And in 2006, Silva et al, investigating the decontamination of dentures, showed that 6 minutes in the microwave sterilised S. aureus and C. albicans but only partially disinfected P. aeruginosa and B. subtilis.

Sterilisation using microwaves

A 2001 Biotechniques paper by Weiss and Galande showed that LB plates made from microwave-sterilised LB-agar were apparently sterile (control plates were no detectably contaminated by microorganisms), had a similar shelf-life to autoclaved plates and supported bacterial growth as normal. The plates were prepared from dry powders dissolved in distilled water and aliquoted into 50mL tubes.

This is a very fast way to make plates and has the added advantage that the antibiotics can be added in from the start as they are not destroyed by microwaves. Invitrogen have a product that takes advantage of this. ImMedia is LB medium provided as sachets of dried, weighed power containing all of the required media components (including antibiotics). It is designed so that you can just add the sachet contents to water, microwave and your media is ready. But Weiss and Galande’s method is just as good and much cheaper.

My view

My take-home from this is that microwaves are are reasonably good at decontamination but more stubborn microorganisms (e.g. the spore-forming B subtilis) are not effectively disinfected. So the method does not sound too reliable to me. Also, filling the lab with smelly fumes from contaminated stocks does not seem to be a good idea. For easy liquid culture decontamination I think I will stick with Virkon, and for solid media, autoclaving seems to be the only good option.

The microwave media prep method is certainly interesting. Weiss and Galande’s results seem to be pretty robust and I would consider this method for an emergency media prep - if I need to start an E. coli culture last thing at night and there’s no sterile media available. Although the decontamination results show that microwaves don’t kill everything, E. coli grows so quickly that for routine purposes, a low level of contamination by slower growing organisms can be tolerated.

But I would not use this method for anything other than routine cultures and certainly not for slow growing organisms. Maybe that’s just me being a typical scientist, reluctant to take on new methods as Liam suggested. The microwave method is also limited by the fact that only small volumes (50ml) can be sterilised so it is never going to replace the autoclave for batch media production.

That’s my view - what’s yours?

As Schrödinger observed, the theoretical physicist deals with statistical approximations of matter and energy, but at such massive numbers of electrons and protons that variation and uniqueness among them appears canceled out. Not so with the biologist, who deals with molecules of far, far greater diversity. How do organisms cope with such apparent chaos, and how do they transmit that organization with such high fidelity, from generation to generation?

In What is Life?, Schrödinger introduced the idea of an “aperiodic crystal” that contained genetic information in its configuration of covalent chemical bonds. In the 1950’s this idea both stimulated enthusiasm for discovering the genetic molecule and could be seen (in retrospect) as having been a well-reasoned theoretical prediction of what biologists should have been looking for during their search for the genetic material. Francis Crick attributed his interest in DNA to having read this book.

What is necessary for a reading of What is Life?, and indeed any understanding of molecular biology, is recognition of how biology transcends the physical realm. Yes, biology operates according to the laws of physics, but physics alone cannot recapitulate life. Ernst Mayr, in What Makes Biology Unique describes the autonomy of biology in much the same sense. That is, new phenomenon emerge in living organisms that could never be anticipated by physics alone. Or at least not until we understand life and its emergent properties better.

And no one has better described these aspects of life than Schrödinger, to my knowledge, in the sixty-four years since the book was published.

Whose Genome? -
“What is a genome?” and “whose genome was sequenced?” are legitimate questions, and what follows is an attempt at clarification that is, by necessity, as much philosophical as scientific.

The Human Genome is Old News. Next Stop: the Human Proteome -
A Nature News article describes the initial plans for an ambitious effort to begin mapping the complete human proteome: the set of all human proteins expressed in all of our cells at all points during our development and adult life.

Widdle Biddy Stem Cells -
Very small embryonic-like stem cells pay provide a potential clue as to tissue renewal in adults.

The Individuality of Bacteria -
Larry debunks the common misconceptions about the biological study of life, which is that it promotes a determinism that denies individuality and freedom.

Where the Wild Microbes Are: A New Theory on How Pathogens Survive Food Processing -
Common sense says that washing and proper handling of our food should simply be enough to prevent illness outbreaks, but this isn’t always true.

That’s one of my main gripes with creationist simpletons who imply that scientists are uncritical of their peers, and that criticism is directed solely at those who refuse to take their claims at face value. They have no clue whatsoever what they’re talking about.

Every scientific claim, as it’s actually being formulated, must be paved with meticulous attention to detail. The scientist advancing some newly-considered possibility must endure a constant barrage of critiquing, on both the grant application and results publication stages.

It’s for a darn good reason - people, even scientists, are prone to error.

I’m not saying this because I’m complaining, although venting does feel good. Criticism does make us better.

The scientific method is a winnowing process, and the writing process is ripe with revisions and the repeated phrase “it’s not good enough.” Competition of ideas with the tangible results as the decider is how science inevitably moves forward. For the individual researcher however, it is humbling. It IS frustrating. But it’s what we do. Curiosity and enthusiasm began our careers in science, and a driving need to maintain an income reminds us to stick to it.

The human frailty in me craves nothing more than the pat on the back, the kind appraisal, and the reassurance that I know what I’m doing. It doesn’t matter what career you’re in, science or otherwise, but you simply won’t get that form of condescension AND a competitive place in the job market. The only solution is to pay attention to detail, learn how to be thick-skinned, and learn how to write persuasively to grant foundations and scientific journals.

Which brings me to an intriguing book that I came across recently - How to Write a Lot: A Practical Guide to Productive Academic Writing. I’m pretty sure that it will help my grant writing skills, but maybe the grad students out there will benefit from reading it before beginning the grant writing beat. After all, it’s something that they don’t teach you well enough in grad school.

Accounts on how amenable electroporation cuvettes are to recycling vary, but I find that as long as you treat them well it is possible to use single cuvette many times.

It’s the metal parts of the cuvette you need to worry about the most - you need to get them clean of DNA and cells and dry again quickly to prevent corrosion.

So the key is to wash and dry as soon as possible after transformation.

My protocol is to wash with water first to clear away most of the residual cells/DNA, then treat briefly with HCl to destroy any remaining DNA - which you obviously don’t want hanging around the next time you do a transformation - then wash with 70% ethanol. The ethanol rinses away the HCl and sterilises the cuvette but because it is volatile it makes it easy to dry the cuvette in air.

So the protocol in detail is:

1. Rinse the cuvette five times with purified water. Ensure, especially with 1-2mm gap cuvettes that the chamber is fully washed out in all washing steps.

2. Fill the cuvette with 0.2M HCl and allow to stand for 10 minutes (but no longer as this will promote corrosion).

3. Rinse the cuvette five times with 70% ethanol, again ensuring the chambers are fully washed.

4. Under a sterile hood if possible, decant the ethanol and use a pipette (or syringe and hypodermic needle for small gaps) to remove as much residual ethanol as possible.

5. Air dry for 60 minutes, then replace the cap.

…and now your cuvettes are ready to go again.

Among the picks are:

Twenty-five years of quantitative PCR for gene expression analysis

Bacterial genetics: past achievements, present state of the field, and future challenges

Over the rainbow: 25 years of confocal imaging

Mass spectrometry: m/z 1983–2008

Kits and their unique role in molecular biology: a brief retrospective

Remember that access to Biotechniques is free, but registration is required.

Do you remember where you were 25 years ago?

I suppose rational scientists often have irrational superstitions.

One example of an old method that could be improved is the growth media used for plasmid preparation.

The majority of us, throughout our university careers, have used either SOC, LB or TB, for recombinant plasmid propagation, typically in E. coli. LB or Luria-Bertani broth has been in use for almost 60 years or thereabouts, while SOC has certainly been in use for 2 decades.

But by adding in a few more ingredients or being more economical on others (especially yeast extract and tryptone) that you could get a higher plasmid yield, quicker and with less money.

To counter the naysayers, nobody wants to make very complex with 15 ingredients requiring filter sterilisation, as this obviously defeats the object of economy of time and budget. Indeed, there are trade-offs between optimising for biomass, plasmid yield, quality, stability and cost with the difference between protein production and plasmid production being that plasmid production requires only cell growth, division, and plasmid stability.

The good news is that Michael Danquah and Gareth Forde from Monash University down-under have devised a stoichiometrically optimised medium for plasmid production. PDM, supposedly yields under the conditions they tested, twice the amount of plasmid in both volumetric and specific yields compared to TB , LB is left in the dust. Better yet, because it uses less tryptone and yeast extract, the cost per mg of DNA is roughly one quarter compared to LB.

The recipes for LB, TB, SOC and PDM are shown below. If you decide to break with tradition and give PDM a go, be sure to tell us how it goes.

Note – Autoclave glucose, KH₂PO₄ and Na₂HPO₄ separately

From one whose dream of tenure track just came true:
In Which the Werewolf is Admitted to Paradise - An amusing tale of motivating students to ask questions during a teaching evaluation. The end result - paradise - the coveted tenure track job.

And from those newly in a tenure track position, a series of posts discussed -
When PIs Do Experiments: Sciencewoman, Dr. Free-Ride, Dr. Jekyll & Mrs. Hyde, PhysioProf, and JuniorProf all weighed in.

The Public and Science

The Personal Genome discussion - Sandra covers a panel discussion with some big names at the University of Washington.

Genes and the Environment - Some thoughts on the eternal “nature vs. nurture” debate, in an era of genomics and PLoS.

Science Itself (titles say it all)

How Can Chromosome Numbers Change
The Definition of Life; and, Taxonomy as We Know It
Reversing dermal aging by inhibiting NF?B
RAS is Regulated by miRNAs