- To introduce the principles behind optical density measurements.
- To relate optical density to cell density and colony forming units (CFUs).
- Optical Density
Hi everybody! Welcome back to Synthetic Biology One. Today I want to talk about how we measure bacterial growth. If there’s really ten billion bacteria in your test tube, how do you count them all?
Well, one way to do it is by plating. You take a bunch of bacteria. You dilute them repeatedly until you get to very very high dilution factors of like 10 million. And then you spread them out evenly on Petri dishes to produce colonies. Each single bacterium in the original culture forms one colony, so you can count the colonies to count the bacteria.
But plating is hard. You have to do all that diluting and diluting. You have to make all of those plates. And then you have to wait for like 12 hours or more for the bacteria to grow into colonies and everything is the worst. You don’t want to wait 12 hours. You need that growth measurement now! Well I can solve that problem with this one simple trick.
Here is what a culture of bacteria looks like in LB medium when it is freshly inoculated. And here is what that same culture looks like after shaking it overnight in a 37 degree incubator. The difference is pretty clear. By which I mean that the stationary phase culture is not clear anymore. As bacteria grow, they gradually make the media more and more cloudy. The scientific name for this cloudiness is optical density and we can use it to get a fast, simple estimate of how many bacteria we have.
First, let’s take our observation that bacteria are cloudy and turn it into something that we can precisely measure. In physics, optical density is also known as absorbance. It is a way of measuring the amount of light transmitted by a given material. If we shine a light across a culture of E. coli, not all of it will pass though. Some of the light will hit the E. coli cells and become scattered.
The amount of light scattering depends on two main factors. First, the thickness of the material. Obviously, the longer the light has to travel the more likely it is to be scattered. But we’re not really interested in measuring thickness, so instead we control for it by doing all of our measurements in a cuvette that is exactly 1 cm across. A cuvette, by the way, is just a test tube with clear, flat sides that we can shine a light through.
The second factor that determines how much light can get through the sample is the absorbance. We measure the absorbance in the following way. First, we take a blank cuvette with just pure media. We turn on the light and measure how much comes through. This is the baseline measurement of light received by the material. Then we add the media with cells, turn the light on again and take a second measurement that represents the light transmitted by the material.
The ratio of the light received to the light transmitted is called the transmittance. And the negative logarithm of the transmittance is the absorbance.
You should notice that this equipment is nothing fancy. All we are doing is measuring how much light comes through the cuvette. The measurement doesn’t tell us anything specific about what is blocking the light or what happens to the light that is blocked. The light could be absorbed by a pigment, either in the cells or in the media. Or it could be scattered by the cells, reflecting off their tiny little bodies and away from the detector.
We know from experience that light with a wavelength around 600 nm, in the green part of the visible spectrum, is easily scattered by particles the size of bacteria. Commonly used bacteria don’t produce pigments that absorb in that part of the spectrum, so our measurement reflects mostly the number of bacteria, not their color. You will often see the wavelength written out as a little subscript when describing the measurement: OD600.
Another important thing to keep in mind when you are doing an absorbance measurement is that the readings will only be accurate if at least some light gets through to the detector. If your absorbance is 1 or less, it means 10% or more of the light is passing through and you get an accurate reading.
So let’s say I’ve done my absorbance measurement and I get a reading of 0.3. Now what? What does absorbance 0.3 mean? What I really wanted to know is the number of bacteria. Well, unfortunately you’ll have to calibrate it. The only surefire way to know how many bacteria correspond to an OD of 0.3 is to count them the old fashioned way. You have to dilute them, then plate them, then wait 12 hours, then count the colonies and blah blah blah. But the good news is that you do it once, you never have to do it again. And the other good news is that OD is linear with cell density, meaning that twice the OD means twice the cells, so you can estimate the cell counts over a good range of ODs.
And the other other good news is that for most common bacteria, the relationship between cell counts and OD is already basically known. If you want really precise data, you still have to do the calibration yourself. It has to be your cells and your media and your equipment etc. But if you only need an estimate, you can just dig up some numbers from the internet. Or listen to some guy in a YouTube video. For E. coli, an OD of 1 means about 109 cells/mL. For a yeast like S. cerevisiae OD 1 is more like 108 cells/mL.
OK so I hope that clears up optical density. Until next time, stay shiny!