Post by The Cell on Dec 23, 2012 21:34:42 GMT -5
(a re-post from openwetware.org/wiki/DIYbio/FAQ/Equipment )
For Yeast and bacterial work, you'll need some microbiology equipment,
almost all of which is available from www.brouwland.com in the lab
section. Offhand I think you'll just need some gloves, autoclave tape,
and a pressure cooker in addition to the brouwland stuff.
Simply:
1. Go to www.brouwland.com
2. Click "EN" for English site.
3. On the left, click "Measuring/Lab
4. First port of call from here is "Yeast Propogation"
4a: What to buy:
You'll need several of the glass petri dishes, one of the "Inoculation loop
handle + 2 loops", either a bunsen or alcohol burner (I suggest alcohol
unless you know someone familiar with gas burners; huge safety issues
there). You'll also need some agar; you use about 20g/L for agar, so 25g is
enough for 1L of medium, which actually goes a long way when you're starting
out. If you feel you're definitely going to keep biotech on as a hobby, go
for the 100g to save money and later time. You'll get this cheaper on ebay
but it might take some hunting.
5. Go back to "Measuring/Lab" and go to "Glassware for Laboratory"
5a: Go into the Erlenmeyer section and pick up at least one of each 100ml
and 250ml erlenmeyer flasks, they're very useful.
(You don't really need beakers or measuring jugs; you can easily use
cookware equivalents of these that cost less.)
5b: Go into the Test tubes and accessories section and get some test tubes.
You won't be using caps when growing Bacilli, but if you foresee yourself
growing E.coli or yeast you might as well splash out on the capped ones. I
suggest getting at least some uncapped ones; you'll probably be using
tinfoil caps if you can't find tin caps on ebay. The tubes are for growing
small liquid or agar cultures, and tend to retain sterility better than a
petri dish when treated right.
Get a stand for your test tubes, you won't regret it.
You will need one item not available from brouwland (I don't know why), and
that is a test tube brush. Test tubes are otherwise very difficult to keep
clean. You can get a test tube brush from this ebay seller:
stores.ebay.ie/Laboratory-Stuff-and-Fishy-Things
In particular, here's one:
cgi.ebay.ie/8mm-12mm-Test-Tube-Cleaning-Brushes-Glass-Lab-Pk10...
You may need stuff to handle liquids at a smaller level than cookware can
provide, but that's for later. Basic microbiology stuff can be handled with
the above plus some stuff you buy locally.
If you're not buying a bunsen (and I would suggest that you don't,
considering you have probably no experience with propane burners or bunsen
burners and they can kill you dead), you'll need to buy a HEPA-or-better air
purifier and jury-rig it into a boxed arrangement to provide you with a
sterile working area. You can get HEPA air purifiers from eBay, Argos or
some department stores. You want something that'll filter the air until it's
essentially sterile, and HEPA is usually good enough for that. It's
expensive, but it won't kill you if you screw up. You can use an alcohol
burner to sterilise your metal loops while you work under the HEPA
inoculating agar sterilised in a pressure cooker.
You'll need a pressure cooker, and someone who knows how to use one. Modern
pressure cookers aren't as bad as they used to be when they got a bad
reputation, but they are still about as dangerous as a chip-pan, which is to
say they are still dangerous. Find someone who knows how to cook with one,
or can help you learn properly. There are plenty of youtube videos, but half
of them are either wrong or not careful enough; they assume that you're
cooking a casserole for 3 minutes, whereas you're boiling water and glass
for 25 minutes. Big difference.
I suggest also buying this from the Ebay seller above:
cgi.ebay.ie/25mm-Autoclave-Indicator-Tape-NEW-and-UK-/11043099...
This is autoclave indicator tape. If you've used the pressure cooker
correctly to sterilise the stuff inside, the tape will show dark black
stripes. If you did it wrong, it won't change colour sufficiently, and your
agar or broth may become contaminated. This is a nuisance. If you're
disposing of hazardous or legally controlled waste some day, insufficient
sterilisation of waste can get you put in prison; for example, GMOs and
genetically engineered animals/plants etc etc *must* be destroyed completely
under EU law before disposal, and innocuous microbes are no exception. So
get the tape, learn how to sterilise things effectively from the beginning
and don't forget.
If and when you get all this stuff together and ordered, get B.subtilis.
You don't actually need B.subtilis though, because you can
get baker's or brewer's yeast in most shops near the
breadmaking supplies; wake it up with some water and you can
teach yourself microbiology with yeast right away.
B.subtilis is only different in that it's a bacteria rather
than a yeast, and that it cannot be easily grown without air
(you can seal yeast, you can't seal B.subtilis or it'll
choke).
The loop handle is a separate item. Petri dishes are on the
same list, but just come as a glass petri dish sold singly.
The loop handle is for holding metal loops that are used for
inoculating and spreading cells, which can be sterilised
quickly between uses in a flame. The loop handles sold on
www.Brouwland.com come with two loops, but treated well you
probably won't need to replace the first loop anytime soon.
For green points, just recycle jars of pasta sauces or other
heat-sterilized foods: those *are* canning jars, which are
normally discarded.
Look for ones with the freshness dimple on the lid, because
it'll work again.
Just make sure you don't real the jars before sterilizing-
leave the lids loose, then seal them while still
uncomfortably-but-dangerously hot. The dimples will pop in
as they cool.
Then, trust in the dimples. If they pop without you opening
the can, it implies unwanted fermentation is taking place
and your sterilisation failed. Or perhaps that lid is
faulty and lets air leak in, though so far jars have worked
well for me.
PDA is great as an all purpose recipe. I, too, recommend
this venerable medium (see below).
Yes, you'll also need pipettors. I suggest for larger volumes
(5mls - 25mls) to buy a rubber-bulb pippettor and glass
pippettes from a good brewing supplier such as
www.brouwland.com/ Don't forget to get a pippette
cleaning brush! You'll need it. To sterilise glass pippettes
of this size/type, it's probably easiest to wrap them
individually in tin-foil and bake them in dry heat for a long
time. Google dry heat sterilisation to find out how long and
at what temperature.
For smaller pipette volumes, consider using glass-droppers
sterilised with bleach for normal routine stuff, presterilised
pasteur plastic pipettes for important stuff, and for under
1ml you'll need to get a micro-pipette set and tips to match.
Those guys can unfortunately be expensive.
For working in a sterile environment, you'll want a bunsen
burner probably, and/or a new HEPA-or-better air purifier. A
Bunsen creates a zone of sterile circulating air surrounding
the flame when it is blue, as long as you're in a room with
still, relatively clean air. Don't lock yourself anywhere
airtight though or it might suffocate you...
HEPA air
purifiers filter air of spores and microbes, giving you an
airstream of sterile air (in theory at least). Arranging
things so this airflow hits your working area from above can
mean that contamination can't reach your work. It's also less
flammable than a bunsen, but you don't have a flame handy when
you want to sterilise metal surfaces, such as the little metal
inoculating loops normally used to transfer bacteria about the
place.
For growth media for bacteria, you can use
potato-dextrose-broth and agar for growing a lot of the cool
bugs, including Bacillus subtilis. It's really easy to make.
So easy I put it on the back of my business cards!
Here's a
recipe for Potato-Dextrose Broth/Agar:
1. Boil 200g sliced potato in 1L bottled or deionised water (to avoid
chloramine. If the water authorities use chlorine rather than chloramine,or
if you have a well, you can use tapwater! Ring and ask, it'll save you loads
of money). Boil for 30m.
2. Filter through a J-cloth or Cheesecloth, then leave to settle in a
tall vessel so it can clarify. When clear, carefully pour off the clear
liquid into another container, and add 20g glucose/dextrose (same thing).
This is Potato-Dextrose Broth, when you bring it back to 1L with water. If
you want Agar, add 20g of Agar before bringing to 1L.
3. Stir for broth to dissolve, and simmer for agar to dissolve the agar.
Before it solidifies, put it into glass test tubes or glass petri dishes (
www.brouwland.com/) as needed, and then pressure cook it for at
least 20m at full pressure and steam. This is needed to sterilise it
effectively, as spores from the potatoes themselves can often grow in the
agar if not done correctly. You're best off searching for someone who knows
how to use a pressure cooker, or doing a lot of reading. They are honestly
quite safe these days, as safe as any cooking implement (chip pans and
untended frying pans are much much worse!), but if you don't do it right,
you might damage the pot, burn yourself with steam, or at least not
sterilise things correctly.
4. When they are fully sterilised, leave them to cool within the pressure
cooker, and try to remove them only when you plan to use them so they can't
get contaminated. Using masking tape around the rim of petri dishes once
inoculated can help prevent contaminating air circulating in and out, but
use low-stickiness tape or you'll send dishes flying trying to get it off.
If they are masking-taped securely, you can dry them a bit by putting them
upside-down somewhere warm; this will help prevent bacteria from washing all
over a plate when you inoculate them due to surface water.
So you've got incubation, sterility and a cheap and easy
medium to grow at least yeast/bacillus in. Advanced
manipulating will also require a centrifuge: I recommend for
low-speed, high safety that you consider the NCBE
microcentrifuge:
www.ncbe.reading.ac.uk/ncbe/materials/dna/microcentrifuge.html
Please note though that, so far as I can tell, they
overestimate the G-forces it can reach, because they measure
the centrifuge's rotary radius from the centre to the edge of
the tubes. Convention suggests instead that you measure to
half-way down the tube, and re-calculating the results gives
you a lower range. Still, it's enough to centrifuge bacteria
out of suspension easily.
If you need more power without any
endorsement of safety, you can get a Dremelfuge here:
www.shapeways.com/model/77306/Dremelfuge.html You'll
need a Dremel to go with it, I use the Dremel 300 plug-in
tool. You'll also need safety googles at the very least! Bolt
it to something so the rotor is horizontal if you plan to use
it, and be careful to load samples equally on all axes, and
securely. This one reaches some pretty obscene speeds and
forces, and isn't necessary for microbiology at all, although
for spinning down DNA and protein it'll work quite nicely.
Mgmt
For Yeast and bacterial work, you'll need some microbiology equipment,
almost all of which is available from www.brouwland.com in the lab
section. Offhand I think you'll just need some gloves, autoclave tape,
and a pressure cooker in addition to the brouwland stuff.
Simply:
1. Go to www.brouwland.com
2. Click "EN" for English site.
3. On the left, click "Measuring/Lab
4. First port of call from here is "Yeast Propogation"
4a: What to buy:
You'll need several of the glass petri dishes, one of the "Inoculation loop
handle + 2 loops", either a bunsen or alcohol burner (I suggest alcohol
unless you know someone familiar with gas burners; huge safety issues
there). You'll also need some agar; you use about 20g/L for agar, so 25g is
enough for 1L of medium, which actually goes a long way when you're starting
out. If you feel you're definitely going to keep biotech on as a hobby, go
for the 100g to save money and later time. You'll get this cheaper on ebay
but it might take some hunting.
5. Go back to "Measuring/Lab" and go to "Glassware for Laboratory"
5a: Go into the Erlenmeyer section and pick up at least one of each 100ml
and 250ml erlenmeyer flasks, they're very useful.
(You don't really need beakers or measuring jugs; you can easily use
cookware equivalents of these that cost less.)
5b: Go into the Test tubes and accessories section and get some test tubes.
You won't be using caps when growing Bacilli, but if you foresee yourself
growing E.coli or yeast you might as well splash out on the capped ones. I
suggest getting at least some uncapped ones; you'll probably be using
tinfoil caps if you can't find tin caps on ebay. The tubes are for growing
small liquid or agar cultures, and tend to retain sterility better than a
petri dish when treated right.
Get a stand for your test tubes, you won't regret it.
You will need one item not available from brouwland (I don't know why), and
that is a test tube brush. Test tubes are otherwise very difficult to keep
clean. You can get a test tube brush from this ebay seller:
stores.ebay.ie/Laboratory-Stuff-and-Fishy-Things
In particular, here's one:
cgi.ebay.ie/8mm-12mm-Test-Tube-Cleaning-Brushes-Glass-Lab-Pk10...
You may need stuff to handle liquids at a smaller level than cookware can
provide, but that's for later. Basic microbiology stuff can be handled with
the above plus some stuff you buy locally.
If you're not buying a bunsen (and I would suggest that you don't,
considering you have probably no experience with propane burners or bunsen
burners and they can kill you dead), you'll need to buy a HEPA-or-better air
purifier and jury-rig it into a boxed arrangement to provide you with a
sterile working area. You can get HEPA air purifiers from eBay, Argos or
some department stores. You want something that'll filter the air until it's
essentially sterile, and HEPA is usually good enough for that. It's
expensive, but it won't kill you if you screw up. You can use an alcohol
burner to sterilise your metal loops while you work under the HEPA
inoculating agar sterilised in a pressure cooker.
You'll need a pressure cooker, and someone who knows how to use one. Modern
pressure cookers aren't as bad as they used to be when they got a bad
reputation, but they are still about as dangerous as a chip-pan, which is to
say they are still dangerous. Find someone who knows how to cook with one,
or can help you learn properly. There are plenty of youtube videos, but half
of them are either wrong or not careful enough; they assume that you're
cooking a casserole for 3 minutes, whereas you're boiling water and glass
for 25 minutes. Big difference.
I suggest also buying this from the Ebay seller above:
cgi.ebay.ie/25mm-Autoclave-Indicator-Tape-NEW-and-UK-/11043099...
This is autoclave indicator tape. If you've used the pressure cooker
correctly to sterilise the stuff inside, the tape will show dark black
stripes. If you did it wrong, it won't change colour sufficiently, and your
agar or broth may become contaminated. This is a nuisance. If you're
disposing of hazardous or legally controlled waste some day, insufficient
sterilisation of waste can get you put in prison; for example, GMOs and
genetically engineered animals/plants etc etc *must* be destroyed completely
under EU law before disposal, and innocuous microbes are no exception. So
get the tape, learn how to sterilise things effectively from the beginning
and don't forget.
If and when you get all this stuff together and ordered, get B.subtilis.
You don't actually need B.subtilis though, because you can
get baker's or brewer's yeast in most shops near the
breadmaking supplies; wake it up with some water and you can
teach yourself microbiology with yeast right away.
B.subtilis is only different in that it's a bacteria rather
than a yeast, and that it cannot be easily grown without air
(you can seal yeast, you can't seal B.subtilis or it'll
choke).
The loop handle is a separate item. Petri dishes are on the
same list, but just come as a glass petri dish sold singly.
The loop handle is for holding metal loops that are used for
inoculating and spreading cells, which can be sterilised
quickly between uses in a flame. The loop handles sold on
www.Brouwland.com come with two loops, but treated well you
probably won't need to replace the first loop anytime soon.
For green points, just recycle jars of pasta sauces or other
heat-sterilized foods: those *are* canning jars, which are
normally discarded.
Look for ones with the freshness dimple on the lid, because
it'll work again.
Just make sure you don't real the jars before sterilizing-
leave the lids loose, then seal them while still
uncomfortably-but-dangerously hot. The dimples will pop in
as they cool.
Then, trust in the dimples. If they pop without you opening
the can, it implies unwanted fermentation is taking place
and your sterilisation failed. Or perhaps that lid is
faulty and lets air leak in, though so far jars have worked
well for me.
PDA is great as an all purpose recipe. I, too, recommend
this venerable medium (see below).
Yes, you'll also need pipettors. I suggest for larger volumes
(5mls - 25mls) to buy a rubber-bulb pippettor and glass
pippettes from a good brewing supplier such as
www.brouwland.com/ Don't forget to get a pippette
cleaning brush! You'll need it. To sterilise glass pippettes
of this size/type, it's probably easiest to wrap them
individually in tin-foil and bake them in dry heat for a long
time. Google dry heat sterilisation to find out how long and
at what temperature.
For smaller pipette volumes, consider using glass-droppers
sterilised with bleach for normal routine stuff, presterilised
pasteur plastic pipettes for important stuff, and for under
1ml you'll need to get a micro-pipette set and tips to match.
Those guys can unfortunately be expensive.
For working in a sterile environment, you'll want a bunsen
burner probably, and/or a new HEPA-or-better air purifier. A
Bunsen creates a zone of sterile circulating air surrounding
the flame when it is blue, as long as you're in a room with
still, relatively clean air. Don't lock yourself anywhere
airtight though or it might suffocate you...
HEPA airpurifiers filter air of spores and microbes, giving you an
airstream of sterile air (in theory at least). Arranging
things so this airflow hits your working area from above can
mean that contamination can't reach your work. It's also less
flammable than a bunsen, but you don't have a flame handy when
you want to sterilise metal surfaces, such as the little metal
inoculating loops normally used to transfer bacteria about the
place.
For growth media for bacteria, you can use
potato-dextrose-broth and agar for growing a lot of the cool
bugs, including Bacillus subtilis. It's really easy to make.
So easy I put it on the back of my business cards!
Here's arecipe for Potato-Dextrose Broth/Agar:
1. Boil 200g sliced potato in 1L bottled or deionised water (to avoid
chloramine. If the water authorities use chlorine rather than chloramine,or
if you have a well, you can use tapwater! Ring and ask, it'll save you loads
of money). Boil for 30m.
2. Filter through a J-cloth or Cheesecloth, then leave to settle in a
tall vessel so it can clarify. When clear, carefully pour off the clear
liquid into another container, and add 20g glucose/dextrose (same thing).
This is Potato-Dextrose Broth, when you bring it back to 1L with water. If
you want Agar, add 20g of Agar before bringing to 1L.
3. Stir for broth to dissolve, and simmer for agar to dissolve the agar.
Before it solidifies, put it into glass test tubes or glass petri dishes (
www.brouwland.com/) as needed, and then pressure cook it for at
least 20m at full pressure and steam. This is needed to sterilise it
effectively, as spores from the potatoes themselves can often grow in the
agar if not done correctly. You're best off searching for someone who knows
how to use a pressure cooker, or doing a lot of reading. They are honestly
quite safe these days, as safe as any cooking implement (chip pans and
untended frying pans are much much worse!), but if you don't do it right,
you might damage the pot, burn yourself with steam, or at least not
sterilise things correctly.
4. When they are fully sterilised, leave them to cool within the pressure
cooker, and try to remove them only when you plan to use them so they can't
get contaminated. Using masking tape around the rim of petri dishes once
inoculated can help prevent contaminating air circulating in and out, but
use low-stickiness tape or you'll send dishes flying trying to get it off.
If they are masking-taped securely, you can dry them a bit by putting them
upside-down somewhere warm; this will help prevent bacteria from washing all
over a plate when you inoculate them due to surface water.
So you've got incubation, sterility and a cheap and easy
medium to grow at least yeast/bacillus in. Advanced
manipulating will also require a centrifuge: I recommend for
low-speed, high safety that you consider the NCBE
microcentrifuge:
www.ncbe.reading.ac.uk/ncbe/materials/dna/microcentrifuge.html
Please note though that, so far as I can tell, they
overestimate the G-forces it can reach, because they measure
the centrifuge's rotary radius from the centre to the edge of
the tubes. Convention suggests instead that you measure to
half-way down the tube, and re-calculating the results gives
you a lower range. Still, it's enough to centrifuge bacteria
out of suspension easily.
If you need more power without any
endorsement of safety, you can get a Dremelfuge here:
www.shapeways.com/model/77306/Dremelfuge.html You'll
need a Dremel to go with it, I use the Dremel 300 plug-in
tool. You'll also need safety googles at the very least! Bolt
it to something so the rotor is horizontal if you plan to use
it, and be careful to load samples equally on all axes, and
securely. This one reaches some pretty obscene speeds and
forces, and isn't necessary for microbiology at all, although
for spinning down DNA and protein it'll work quite nicely.
Mgmt
