Download VCLab 9d Spectrophotometer

LAB 28
SPECTROPHOTOMETER
This is another method that is used to count bacteria.
Within this machine, light comes out from a source and goes through a dispersion devise (a
prism), which adjusts the wavelength by spreading all the light out into the full spectrum of the
rainbow colors. This spread-out light then passes through the sample liquid in a special test tube
called a cuvette, which is high quality glass with no imperfections that would distort the light.
Whatever light can pass through the sample is detected on the other side of the machine. Cells or
other causes of turbidity will refract the rest of the light away so it does not get detected.
You can adjust the prism in the machine so it will only put out a red wavelength light. This is
useful in measuring the amount of green chlorophyll in an algae sample. A spectrophotometer
measures the turbidity and absorbance in percent transmission (%T). It also measures the optical
density (OD).
The spectrum of colors ranges:
700nm (red) – orange – yellow – green – blue – indigo – violet (350nm)
Since you can set the prism to measure certain wavelengths, we can measure DNA at 260 nm to
determine the concentration of DNA in a sample.
The spectrophotometer measures both absorbance and turbidity, but we only want to measure the
turbidity in our E. coli sample. How do we eliminate absorbance?
We will use sterile nutrient broth as the diluent, and then pick a wavelength which is the same
amber color as NB. The proper wavelength for this amber is 686nm.
Set up a dilution scheme (standard curve for the spectrophotometer).
Start with the batch of E. coli.
Transfer 2ml into a tube of 2ml sterile water.
Transfer 2ml into another tube of 2ml sterile water.
Transfer another 2ml into a third tube of 2ml sterile water.
Transfer another 2ml into a fourth tube of 2ml sterile water.
The dilution of tube 1 = ½
The dilution of tube 2 = ¼
The dilution of tube 3 = 1/8
The dilution of tube 4 = 1/16
Now make a table to fill out from the spectrophotometer readings:
Dilution
½
1/4
1/8
% Transmission
Optical Density
1/16
For test questions:
Know the name of this instrument.
Why is it used? To measure absorbance and transmission.
Why measure absorbance? To find concentration of a colored substance such as chlorophyll.
When measuring absorbance, use wavelength to differentiate from the color of your liquid.
A special unit called an ultraviolet spectrophotometer can measure proteins and nucleic acids.
DNA is measured at 260 nm
Proteins are measured at 270 nm.
Our spectrophotometer just uses the visible spectrum of light.
How do you change the wavelength? Adjust the prism.
Dial in 686 (color of NB) to eliminate absorbance.
Now it will only measure turbidity (cloudiness).
SPECROPHOTOMETER GRAPH
Use the graph paper on page 557 of your lab manual.
Plot the dilution of the E. coli culture from the spectrophotometer readings on the x-axis.
Plot the optical density on the y-axis.
On the x-axis, plot the Dilutions
40 squares to the right, mark the graph for ½ dilution.
On the 20th square to the right, mark for ¼ dilution.
On the 10th square, mark the 1/8 dilution
On the 5th square, mark the 1/18 dilution
On the y-axis, plot the Optical Density
10 squares up = 0.1
20 squares up = 0.2
30 squares up = 0.3
Now plot the coordinates as the spectrophotometer indicated:
½ = 0.29
¼ = 0.15
1/8 = 0.1
1/16 = 0.03
Draw a straight line that fits the points best. Note that one point falls outside of the
straight line. This can be due to error in pipetting or the cuvette was dirty.
QUESTIONS
1. What does the resulting standard curve indicate? The OD is directly
proportional to cell density and cell density is directly proportional to
dilution. Direct proportionality means that a ½ dilution has twice as many
cells as a ¼ dilution.
Problem
If the OCD was 4.4 x 108, what is the cell density (cfu/ml) of the ½ dilution?
Solution
½ of 4.4 x 108 is 2.2 x 108
Now we see a correlation (relationship) between optical density and cell density.
2. How can you use OD data to correlate with the cell density? Use a ratio
proportion equations to solve for x.
Problem
Since we know from our graph that 2.2 x 108 has an optical density of 0.3, what is the cell
density of an unknown sample of E. coli with an optical density of 0.2?
Solution
0.2
0.3
=
x___
3.3 x 108
0.3x = (0.2)(3.3 x 108)
x = (0.2)(3.3 x 108)/ 0.3
x = 2.2 x 108 cfu/ml
Note that we got this answer without having to do a Standard Plate Count again. That
means we can use the OD data to find the cell density on any E. coli sample by only
doing one SPC.
3. When does the OD data NOT correlate with the cell density? The
spectrophotometer will count dead cells as well as live ones. Therefore, in an old
colony, the SPC numbers will decrease (dead cells don’t grow colonies), but the
spectrophotometer will show and increase. That means you can only use the OD
data on young colonies (16-18 hours old).