Agar plates are used for culturing microorganisms, and are used in myriad types of experiments. Agar is derived from kelp, and it added to a particular medium (such as Luria-Bertani broth or potato dextrose) to form a solid surface on which one may culture microorganisms such as bacteria, archaea, fungus, terrestrial plants and nematodes (worms). A 1-2% (g/100mL) solution of agar will be solid enough so that your culture will only grow laterally, but it is porous enough to allow the diffusion of nutrients in your medium to your hungry culture.
Reagents
Bunsen burner
In my experience, most Bunsen burners use natural gas, but a propane torch will or an alcohol lamp will work just as well. The drawback of an alcohol lamp is that you have to use a pretty concentrated solution of ethanol (like Everclear) to get a hot enough flame. Concentrated ethanol is heavily regulated in the United States, and therefore quite expensive for laboratory use, which is why I go with a propane torch. autoclaved agar media Latex gloves
You can also use dishwashing gloves; just make sure you sterilize the surface with unflavored vodka, ethanol, Purell or Lysol and make sure they've had time to evaporate before you work around an open flame. sterile petri dishes
Procedure
1. Make sure your agar medium is sterile and liquid. See my section on autoclaving. The best thing to do is pull your media out of the autoclave, let it cool (swirling occasionally to keep it mixed) to the point that you can just barely handle it with latex gloves, and pour. Anything with agar solidifies around 55 degrees Celcius (body temperature is 37), so your medium will need to be warmer than that to pour smoothly. I've also noticed that the hotter I pour it, the less likely my plates are to be contaminated by errant spores. However, if it's too hot to handle it's likely hot enough to melt the plastic in your petri dishes. You can allow your agar to solidify and then microwave it to liquefy it, but (as I'm sure you already know) anything with a metal lid (like a mason jar) can't go in the microwave. Even without a metal lid, I've seen microwaving agar go really wrong (involving paramedics), so I avoid it.
2. While your medium is cooling, make sure you have a clean surface on which to work. Wiping your counter (or lab bench) down with ethanol (unflavored vodka, Everclear) or lysol will minimize the chances of contamination. Set up your plates and Bunsen burner so you can pour your plates between that crucial time when the agar is cooled just enough and when it's cooled to the point of solidifying.
3. Pour. Remove the lid from your vessel of autoclaved media and pass the top of the vessel through your flame. This will heat the air inside the vessel sufficiently to cause it to rise, lifting up (away from the sterile agar) whatever potential contaminants are hovering nearby. A thin layer of media that completely covers the bottom of the plate should be sufficient. Any thicker, and the plate will take longer to dry. If you manage to pour bubbles into your plate, quickly pass the flame of the bubbles. The heat should cause the bubbles to expand and pop, leaving you with a smooth agar surface on which to do your science. Just don't linger with the flame - you can very easily melt the plastic of the petri dish itself.
4. Wait for your agar to solidify. This should take less than an hour.
5. Invert and allow to dry. As the agar cools, water evaporates from it and condenses on the top lid of the petri dish. The water can then drip down onto the surface of your agar, making it very difficult to work. A simple way to avoid this is to simple flip the whole plate over. I usually allow the plates a day or two to dry before I use them.
Autoclaving is a means of heat-sterilizing media and equipment without combustion or oxidation (if you had to sterilize your growth media by burning it, the media wouldn't do you much good). You might ask, "Why not simply sterilize by boiling?" A lot of the time that might work. However, there are several species of bacteria form spores in response to stress, and those spores can often survive temperatures above 100oC.1 To ensure errant spores don’t germinate
in your media, you need to sterilize at even higher temperatures (around 121
oC).
How is this accomplished? Contrary to popular belief, you
cannot simply crank up the heat on your stove and boil the water hotter. Adding
more heat to a liquid will make it boil off faster, but it will not change the boiling
point of a liquid. A phase transition (solid to liquid, liquid to gas, solid to
gas, etc.) happens at exactly one temperature, and that depends on the
substance and the surrounding pressure.
Thus, we can increase the temperature at which water boils by
increasing the pressure (Figure 1). A good way to do that is to start by boiling water.
When the water goes from a liquid to a gas phase, it expands. If the steam is
trapped, it puts pressure on the rest of the container, including the water
that hasn’t yet boiled off. Over time the enclosed system increased in
temperature and pressure.
This is the idea behind a pressure cooker, and why for
generations people have used canning as a very successful method of preserving
food. And while you can purchase an autoclave for several hundred to several
thousand dollars, a decent
substitute can be procured for around $20.
Procedure
1. Put your media/instruments in an autoclave-safe container. To autoclave media, mix up the reagents in a jar and cap it loosely so the jar doesn't over-pressurize (if your media has agar - if you're pouring plates, for example - the agar won't completely dissolve until autoclaving anyway). To autoclave instruments, you can either put them in a dry jar and cap loosely, or put them in disposable autoclave bags. Just make sure to put the sealed autoclave bags on top of something dry (like an uncapped glass jar) to keep it out of the water.
2. Put water in the pressure cooker. You'll need the water level to be high enough so that the water doesn't boil off before you've been sterilizing for at least 15 minutes. You also don't want the level of the water to go above the pressure cooker's limit (there will be a line inside the vessel). It may take some experimentation to hone in on the ideal water volume; I fill the pressure cooker about 5 centimeters from the bottom.
3. Seal the pressure cooker and heat it on the stove top. As pure your pressure cooker's instructions, the water will need a few minutes of boiling before there's enough steam to build up pressure. When the pressure is high enough to be "autoclaving," the top piece will bob up and down to off-gas excess steam (it usually takes 10 minutes before my pressure cooker enters "sterilization phase"). Once it does, let the pressure cooker continue to cook for at least another 15 minutes (the minimum sterilization time I've seen for any autoclave cycle).
4. Cool. Once you're sterilization phase is complete, simply turn off the stove's heat and allow the whole pressure cooker to cool. If you're sterilizing media with agar (e. g., for plates), you'll want to crack the pressure cooker lid (be careful not to get a faceful of steam!). Media with agar needs some tending; with oven mitts, gently swirl the jar so the agar remains well mixed. This will also help you gauge when the jar of agar is cool enough to handle without an oven mitt. For more, see the section on pouring plates.
References
1. Abraham, G.; Debray, E.; Candau, Y.; Piar, G. 1990. Mathematical model of thermal destruction of Bacillus stearothermophilus spores. Appl Environ Microbiol. 56(10):3073-80
A typical analytic balance is quite expensive (hundreds of dollars at a minimum). For the level of precision and scale (pun intended) of a kitchen lab, something like this is more economical.
Make sure your balance is clean before you turn it on. For convenience, you'll probably want to put a small piece of wax paper or a plastic container on the balance (if you feel like treating yourself, you can used disposable weigh paper or weigh boats), and measure your reagents on that.
After you place your paper/dish on the balance, but before you measure your reagents, hit the TARE button. This will zero your balance, and automatically subtract the weight of your paper/dish from the overall weight. Thus you can directly measure the weight of your reagent.
If you're weighing multiple reagents, make sure to tare in between reagents.
Today I celebrate the man to whom I owe much of my
professional existence. It’s July 20th, and on this day Gregor
Mendel, the Father of Modern Genetics, was born.
Like many Ice Dynamo favorites, Mendel’s story has
rural beginnings; in 1822, Johann Mendel was born to a family who had been
working their farm for 130 years. And like Ice Dynamo favorite Charles Brush,
young Mendel was a bit of an iconoclast. Though he gardened and kept bees from
an early age, it soon became clear that his agricultural interest was not the
result of tradition. He wanted to understand nature, and his family shouldered
the financial burden of sending him to secondary school.
His parents hoped he would return and take over the
farm, but Mendel was determined to continue his pursuit of knowledge. He
studied physics and math Philosophical Institute of the
University of Olmütz (studies which ultimately led to his greatest
discoveries). Though he tutored privately, he was unable to finance his own
education; his sister gave her dowry to cover tuition.
Mendel’s finances
continued to strain, so he became a friar, changed his name to Gregor and persisted
with his education free of charge. Twice he tried to become a certified
teacher, and both times he failed. Nevertheless, when he returned to his monastery,
he threw himself into the investigation of the natural world. During that
decade (roughly 1854-1864), he researched phenomena that would become the
foundation of modern genetics.
From time immemorial,
people have performed selective breeding: pairing together two individuals with
a desirable characteristic in the hopes of producing offspring with that same
characteristic (e. g., breeding grass-like maize over generations to produce
modern-day corn). However, it was thought that breeding produced a general
blending of physical characteristics. Observationally, that wasn’t a stupid
idea: children usually grow to a height that falls somewhere between their
parents’ height.
Some of Mendel’s
earliest experiments yielded a surprising result: if you bred plants with
yellow peas to plants with green peas, the offspring’s peas weren’t
greenish-yellow. All of them were yellow. What’s more, this wasn’t just the
case for an isolated characteristic like pea color; it was true of flower
position, plant height and four other characteristics.
Mendel persisted. He
took the yellow pea offspring and bred them together. One might expect that the
offspring of yellow pea plants would produce yellow peas – that the green color
had been bred or “blended” out of organisms. The “grandchild” generation showed
no yellowish-green peas, no greenish-yellow peas, no mix of yellow and green
peas on the same organism . . . but some of them had green peas. In fact, for
every three yellow-pea “grandchildren,” there was one green-pea “grandchild.”
Here’s where Mendel’s
background in statistics enabled modern genetics: he recognized that 3:1 was a
very special ratio. The “children” of the original pair had inherited one “character”
(one copy of their genes) from each parent. Furthermore, one “version” (allele)
of that heritable unit (gene) was dominant in the presence of the other. In the
presence of a yellow pea allele, the peas will be yellow rather than a blended
color.
Astonishingly, these “characters”
(genes) didn’t blend together into one indivisible unit – they separated when
the “children” made gametes (sperm and eggs). Thus, the “children” had one
green pea allele and one yellow pea allele, and could give either to the “grandchildren.”
Half of the time, the “grandchildren” would receive a green pea allele from one
parent and a yellow pea allele from the other parent. Because yellow is
dominant, these “grandchildren” had yellow peas. A quarter of the time the “grandchildren”
would get the yellow allele from both parents, but another quarter would get
two green alleles. In the latter case, no yellow allele was present to
overshadow (dominate) the green allele, so the peas would be green.
The Law of Segregation –
the idea that heritable units are indivisible, and behave in statistically
predictable ways – was an historic discovery, but Mendel went still further. He
followed two traits; he bred tall yellow-pea plants to short green-pea plants.
Unsurprisingly, all the offspring were tall with yellow peas. The question was:
if he bred these tall yellow-pea hybrids together, would he get three tall
yellow peas for every short green pea?
The short answer was
no; the results were a whole lot weirder. He got tall plants with yellow peas, tall
plants with green peas, short plants with yellow peas and short plants with
green peas. At this point, another person might have thrown up their hands and
quit science, but once again, statistics saved the day. Mendel realized the
ratio of these four different plants was 9:3:3:1. This meant that the alleles
for peas color and plant height still segregated and yielded 3:1 ratios, but
pea color alleles segregated independently from plant height alleles. He dubbed
this phenomenon the “Law of Independent Assortment.”
As is so often the
case, these great ideas were relegated to obscurity. After he died, his
monastery’s new Abbott burned his research; the only evidence of his work that
survived was a little-known paper published in the Natural History Society of
Brno. What is even more tragic is the fact that Charles Darwin published his
magnum opus, On the Origin of Species,
just six years earlier. Darwin’s central thesis was that individuals had
natural variation, and that these variations were heritable. Mendel’s research
identified that variation, and showed how it was inherited, yet Charles Darwin
never heard of Mendel. Indeed, it wasn’t until the next century that Mendel’s
work was rediscovered and properly appreciated.
Mendel’s story is one
of tenacity. Despite the disapproval of his family, frequent illness, financial
hardship, repeated failures and a dearth of recognition, Mendel was as
persistent as he was diligent. Gregor Mendel made me realize why I love science
so much: it’s the novelty. When I discover some scientific phenomenon, I open a
brand new door into human understanding. I get to be the first to walk through –
the first to know something – and I
get to usher everyone else in. Nothing else – not fortune, not approbation, not
notoriety – offers that kind of exhilaration.
Music swirls around you, and delectable aromas hurl
distant memories into the immediacy of the present. Woven between the sounds
and scents are dancing bodies; lights illuminate them to reveal the faces of
your loved ones.
You remember the sensory overload of previous years’
River Town Days in Bainbridge, Georgia, and it seems only natural that this
year’s festival immediately precedes the birthday of a man without whom River
Town Days – indeed, our modern civilization – might not have been possible.
Charles Francis Brush was born March 17, 1849 on his
family’s farm – a farm not so different from those sprinkled across Bainbridge.
You can imagine the bemusement of his parents – both farmers – when seemingly
from infancy Charles showed an insatiable interest in electricity. He was a
mere twelve when he built his first static electric machine.
He graduated college when he was twenty, and
immediately went to work repaying his student loan, granted to him by his
uncle. Charles spent his days selling iron ore and his nights devising a new
dynamo – an early version of the electric generator. He was twenty-eight when
his tireless efforts earned him his first patent.
As abundant and reliable as electricity is for us
today, it’s hard to imagine what Brush’s dynamo meant for nineteenth century
Americans. At the time, electricity was so inefficient and uneconomical that it
little more than a novelty; what lighting existed was almost exclusively in the
form of kerosene lamps.
The dynamo was a great achievement, but for Charles
Brush, it was just a stepping stone. He envisioned a world lit by arc lights (a
technology similar to light bulbs). That vision required not only economical
electricity, but efficient and reliable arc lights. Once he’d completed his
dynamo, he turned his focus to arc lights, and received his first of four
patents in 1878.
Charles Brush loved his own life too much to
relegate himself to thankless toil in an obscure lab. He was eager for the
world to benefit from his genius, and wanted to be remunerated for his effort.
Thus, in 1880 he established the Brush Electric Company. It was a herculean
undertaking; he competed directly with Thomas Edison’s titan of a company,
General Electric. Nevertheless, in a few short years Brush’s arc lights
illuminated the streets of cities such as San Francisco, Montreal, Boston and
New York. His hydroelectric power plant in Minneapolis was one of the first in
the United States to generate electricity from water.
When Brush was 42, he merged his company with
General Electric and retired to the mansion he’d built in Cleveland. His home
included a private laboratory in the basement and the world’s first automatic
wind turbine generator. Even in retirement, he never stopped investigating
scientific phenomena.
Charles Brush’s inventions – such as his dynamo –
were incredible machines, but they were so much more. Those inventions were the
product of a child who was born with a singular purpose, and never let being an
iconoclast stop him from pursuing that purpose. They are the result of a young
man’s inexhaustible dedication to his work, and an industrialist’s fearless
determination to bring light to the world.
Which brings us to the article’s title. My favorite
author described machines as “the frozen form of a living intelligence.” When
you’re at the River Town Days Festival, among friends and family, you’re
surrounded by more life than you may have realized. All the machines are ideas
frozen into physical form. Thaw them out, and you’ll see the incredible living
intelligences that made those ideas – those “ice dynamos” – real.
It turns
out that scientists are not immune to hard economic times. I was trying to find
ways to use my science to improve my and my family’s situation.
My first
thought was wine. There are lots of health benefits to a daily glass of wine:
the antioxidants in wine can help prevent heart disease.1
Additionally, there is evidence that certain polyphenols (such as resveratrol
in red wines) retard the effects of aging.2,3 There’s also a great
deal to be said for having a relaxation routine - a moment to yourself in which
you kick off your shoes and sip a glass - to combat the immune-suppressing
effects of stress.4
However,
even with a modest $10 bottle of wine amounts to $2.50 a day - almost $1,000 of
your annual budget.
That’s
where science - and cider - come in. While brewing beer can be time-consuming
and investment in ingredients can be costly, you can fill eight to ten 12-oz
bottles for about $5 in a relaxed half hour.
Alternatively,
the airlocks we use fit quite nicely in a 4L (slightly more than 1 gallon) wine
jug, such as those made by Livingston. If you’re going to pay for a glass jug,
you might consider one with wine in it.
If you
can’t get your hands on fresh-pressed cider, make sure you get cider that isn’t
fortified with vitamin C. Ascorbic acid (vitamin C) acts as a preservative,
which means it discourages microbial growth. This is exactly what you don’t
want if you’re trying to culture yeast in your cider. Certain juice brands
(such as Langer’s and White House) do offer juice without vitamin C; we had bad
results with White House and good results with Langer’s, but you may have
different results.
Procedure
Initial/Primary
Fermentation
1. Sterilize everything that’s going to touch
your media.This includes the glass jug, stopper, airlock, funnel and
measuring cup. The specifics of the sterilization procedure will vary depending
on what sterilizer you use, so just follow the directions on the package.
2. Measure your sugar. We found
that one cup of white sugar plus one cup of brown sugar per gallon works well.
However, the whole point of this blog is to experiment; please try whatever
types and amounts of sugar you wish.
3. Add some of your cider(make
sure it’s either fresh-pressed or not fortified with vitamin C) to your sugar
to partially dissolve it. This will make it easier to pour the sugar into your
fermenter jug.
4. Add yeast.If this is
your first time, you’ll need to reconstitute the yeast according to the
package’s instructions. If you’re using yeast from a previous batch, just make
sure the bottle you’re using didn’t come in direct contact with your mouth. If
it did, it’s certainly contaminated by the bacteria living in your mouth, which
will foul up your fermentation.
5. (Optional) Add yeast nutrient, pectic enzyme,
and/or potassium metabisulphite. Truthfully, I haven’t
noticed much difference between batches with yeast nutrient and batches without
it, but you’re probably the kind of person who wants to see for yourself.
Pectic enzyme (also called pectinase) breaks down some of the more complex
carbohydrates in cider into sugars that are more easily utilized by the yeast.
Again, I haven’t noticed pectic enzyme making much difference. However, the
fresh-pressed cider I get is different from batch to batch - different apple
varieties and different proportions of these varieties go into each batch. You
may find that pectic enzyme helps you control for some of this variation.
Potassium metabisulphite will kill any wild yeast languishing in your
fresh-pressed cider, leaving real estate for your own yeast (you don’t need
potassium metabisulphite if you’re using cider you purchased at a grocery
store).
6. Attach the airlock.Fill the
jug at least up to the neck. Insert the stopper and the three-piece airlock on
to the fermenter. Partially fill the airlock with hard liquor, so that it
covers the slates of the loose piece but not so full that the liquor leaks back
into your media. The liquor will allow the loose piece to bob up and down as it
releases carbon dioxide that the yeast evolves during catabolism (metabolic
breakdown) of sugars. At the same time, any contaminants that enter the airlock
are sterilized by the liquor. Of course, there are other liquid sterilizers you
could put in your airlock (such as bleach), but if you bump the airlock and
some of your liquid sterilizer enters the fermentation, a little bit of liquor
in your hard cider is infinitely preferable to a little bit of bleach.
7. Ferment. A dark, cool basement or
garage is a great place to incubate your fermentation. However, our lab is at
room temperature, and we haven’t noticed any effect on our cider. Whatever your
conditions, let your reaction incubate for at least two weeks.
Initial/Primary
Fermentation
8. Rebottle. After at least two weeks
of initial fermentation, it’s time to kickstart your yeast to make even more
bubbles and ethanol. Of course, rebottling also allows you to repackage your
hard cider into more consumer-friendly bottles.
Once
again, your first step is to sterilize your bottles, and everything that’s
going to touch your medium (bottles, funnel, tablespoon), just as you did for
the initial fermentation. Add sugar (I found that about 3 tablespoons of sugar
total per 12-oz bottle works really well) to each bottle. I found that varying
the types of sugar you use (e. g., one tablespoon each of white sugar, brown
sugar, and agave per bottle) makes a big difference, so experiment widely. Distribute
your initial fermentation into your bottles, seal the bottles and wait at least
two more weeks before sampling your work.
9. Enjoy! To reiterate, open your
bottle carefully and pour it into a glass rather than drinking from the bottle.
If you like what you’ve made, seal the bottle back up and leave it in the
fridge until you’re ready to start your next initial fermentation.
References
1. Opie, L. H.; Lecour, S. 2007. The red
wine hypothesis: from concepts to protective signalling molecules. Eur Heart J. 28(14): 1683-93
2. Sandhya, K.; Venkataraman, K.;
Hollingsworth, A.; Piche, M.; Tai, T. C. 2013. Polyphenols: benefits to the
cardiovascular system in health and aging. Nutrients.
5(10): 3779-3827
3. Marks, S. C.; Mullen, W.; Crozier, A. 2007.
Flavonoid and hydroxycinnamate profiles of english apple ciders. J Agric Food Chem. 55(21): 8723-30
4. Padgett, D. A.; Glaser, G. 2003. How
stress influences the immune response. Trends
Immunol. 24(8): 444-8
