Serial dilution illustrated
When working with venoms, it is necessary to work with minute quantities. It may
be desirable to work with single drops or quantities as low as one thousandth of
one drop. It would be pretty difficult to try and divide a single drop into one
thousand parts. It is quite easy, however, to add one drop of venom to 999 drops
and then take a single drop from that diluted liquid as being one one-thousandth
of one drop of venom. To inject one one-hundredth of one drop, it would be
impossible to read a syringe and accurately administer a quantity so small.
There is an easier way.
Some of the equipment I use in my immunization program came from Lake
Charles Manufacturing. They can be found online as www.testtubesonline.com
I use 15ml polystyrene centrifuge tubes for sterile water. I boil the water and fill
the tubes myself. This is the water I will use to dilute the venom and inject. You
could use any sterile or bacteriostatic water. Usually the commercially prepared
bacteriostatic water will have .9% benzyl alchohol added to it. A 30ml vial is
usually about $7.00. I feel comfortable using my own sterilized water.
I use a plastic standard fine tip transfer pipette to measure all liquids in exact
drops. It is a clear plastic bulb with a tube and a fine tip at the end, basically like
an eye-dropper. I use these one time and throw them away.
I use 0.5ml sample analyzer cups for dilutions. These are very small, sturdy
plastic cups. They come in a bag of 1000, so I use them once and throw them
away. I put nine drops of sterile water in these and add one drop so I am always
working with ten drops in all my calculations and dilutions. I draw from one of
these cups right into the syringe I am using for dosages.
The syringes I use are from TNB Services. They are Terumo Sterile 1/2cc Insulin
Syringes with 28g x 1/2" Needle. The syringes are marked in “units”. I keep track
of my dosage in units at certain dilution levels. I may be using, for example, a
one hundredth drop solution and inject five units.
centrifuge tube
analyzer cup
pipette
Typically, I set up my boiled sterile water and two or three analyzer cups. I have
made a wooden platform with holes drilled to support these items. I use the
pipette to transfer the sterile water into the analyzer cups, very carefully
dispensing nine drops in each cup I will be using to make dilutions. Then I use
the pipette to transfer the freshly milked venom, dispensing one pure drop of raw
venom into the first analyzer cup. That makes a total of ten drops in analyzer cup
number one, one drop of venom in the cup and nine drops of water.
Each drop in cup number one now represents one-tenth of a drop of venom. If
we were to suck up all the contents of cup number one into a syringe and inject
half of it, we would be injecting one half a drop of venom.
Usually I will further dilute the venom from cup number one in a process known
as serial dilution.
If we use our pipette and take up the one to nine mix from cup one, and carefully
drop one single drop into the nine drops of pure water we put into cup number
two, we will have ten total drops in cup number two. We have now diluted the
original drop of raw venom by one hundred. If we inject only one tenth of cup
number two, we would be getting one hundredth of one drop of the original drop
of raw venom.
This can be further diluted with each successive dilution by ten. By taking one
drop from cup number two, and putting it into nine drops of pure water in cup
number three, we have diluted our one drop of venom by one thousand. Now one
tenth of cup number three represents one thousandth of a drop of raw venom.
It is always important to remember that the dilution cup contains the same
amount of venom that you added to it. It is the division of the cup that dilutes the
venom. For this example, using the contents of the third cup, if you were to suck
up all ten drops into a syringe, you would inject one-tenth of the contents of the
syringe to get one thousandth of one drop of venom. You could inject half the
amount in the syringe and get five-hundredth of one drop. If you injected all of the
syringe, you would be getting one hundredth of one drop. That is how much
venom you put in cup number three.
Just be careful with the dilutions, it is
easy to miscalculate by ten times. Not
good when you are using a potent
venom, or just starting out. The venom
should be considered potentially diluted,
depending on how you use it.
The consistency comes from using the exact pipette so that the drops are equal.
You can experiment with different droppers. The drops will be bigger as the
opening gets bigger. A fine tip pipette will have different sized drops than a
medium tip. But the drops from the same instrument are actually quite consistent.
The specific gravity and surface tension will make each drop nearly exactly the
same.
By using this method, it is fairly simple to start using one thousandth of a drop of
venom and progressively inject stronger doses until you are taking all of the
contents (all ten drops) and then move up to the stronger dilution in your second
cup and on to the first. By using a very small syringe, you will have fairly precise
control of dispensing even one drop in an injection.