Hi there! I'm Jayme Dyer. If i want to measure my
height, I'll use a measuring stick. But this cool device, it measures things
using light. It's called a spectrophotometer. It
measures the intensity of the light of different wavelengths in the visible
spectrum. Spectrophotometers can be used to do
lots of different things. Let me give you one example: in this
2017 paper, scientists characterized a strain of bacteria that can do
bioremediation, which is when living organisms are used
to break down or remove toxic chemicals in the environment.
It's a way to use living things to clean up pollution.
In this paper, the scientists identified a type of bacteria that can do
bioremediation of a toxic weed-killing herbicide called quinclorac.
It's used to kill weeds in rice fields and lawns but it's toxic to many types
of crops and it's slow to break down in the
environment. The scientists isolated a strain of bacteria from a field that was
contaminated with high levels of Quinclorac.
But they needed to show that as the bacteria grow the bacteria actually
degrade the toxic chemical. To do that the scientists showed that, in
the lab over several days, the bacteria grew in number.
And higher numbers of bacteria correlated with higher degradation of
quincloac. How they measured bacterial growth was
with a spectrophotometer. iIhave here two glasses of kool-aid. One
of them is more concentrated than the other.
Can you tell which one? This one it's darker.
What you just did with your eyes is essentially what a spectrophotometer
does. When you looked at the glasses, the
kool-aid particles in the darker glass absorbed more light.
That's why it looked darker. Spectrophotometers do a similar thing,
they shine light through a sample and measure how much light goes through.
When a sample has more light-absorbing particles, less light goes through the
sample; Which the spectrophotometer reads out as
absorbance. But there's one thing that makes a
spectrophotometer a little more complicated than just you looking
through a glass of kool-aid. Spectrophotometers don't use plain old
light. They use specific wavelengths of light. White light is composed of all
the colors of the rainbow, which can be separated by a prism.
Each color is a specific wavelength of light.
By using specific wavelengths of light, the spectrophotometer is more accurate.
When measuring the absorbance of a sample,
the scientists were able to measure the growth of the bacteria using a
spectrophotometer because more bacteria cells in the
liquid meant higher absorbents. So using the spectrophotometer was a
quick and easy way to measure how many bacteria were in the culture.
You need to use a specific wavelength of light that depends on what your sample
is. Make sure the spectrophotometer is set
to that wavelength.
Failing to set the spectrophotometer to the correct wavelength will give you an
absorbance value that is totally
meaningless.
Absorbance is never negative. If you get a negative number, something
went wrong. So you should start over. Almost everything absorbs a little bit
of light. So if what you want to measure is the absorbance of bacteria, then you
don't want to include in your measurement the absorbance of the media
they're growing in. To avoid doing that, before you measure
your sample, you must set the blank. Your blank is a cuvette with the same
kind of liquid that your sample is in but without the sample in it.
if you forget to set the blank or if your blank is a different kind of liquid
than what your sample is in, you will get an absorbance value that's
totally meaningless. Make sure you set the blank using the
same kind of liquid that the sample is
in. So let's say you have multiple samples
that are in the same kind of liquid that you're measuring with the same
wavelength. You only have to set the blank once in
the beginning.
Measuring the sample is pretty easy pipette it into the cuvette,
insert the cuvette into the spectrophotometer, and close the lid.
Note that the cuvette is designed for light waves to pass through it in a
specific direction. Make sure you orient it in the
spectrophotometer so that the light beam goes through it the right
way.
Don't touch the part of the cuvette that the light goes through because your
fingerprints can interfere with the absorbance reading.
iI fact it's good practice to wipe the cuvette –
just to make sure you remove any potential fingerprints or dust before
putting it into the spectrophotometer. On many spectrophotometers, once you put
the sample in, it will automatically display the absorbance without even
needing to push a button. That's it! Now you know how to use a
spectrophotometer and maybe one day you'll be the one using a
spectrophotometer to do research on bioremediation or something else really
cool!