Flow Cytometry In A Nutshell

FACS was used in the trial treatments of leukemia last Saturday's post.

Fluorescence-activated cell sorting analysis, or FACS, uses a flow cytometer to separate individual cells in a heterogenous suspension based on epitope type. Fluorochrome-labeled antibodies are added to the cell sample. The antibodies bind to specific epitopes on or within the cell. The fluidics system delivers a stream of cells or particles one at a time through an interrogation point, where it passes through a laser. A cell traveling through the laser beam scatters the beam’s light forwards and sideways as a function of its size and granularity, respectively. When the laser beam strikes cells labeled with fluorescent-labeled antibodies, the fluorescent dye becomes excited and fluoresces at a unique wavelength. The intensity of the scattered and fluorescent light is collected and filtered by the optics system, recorded by the detector which translates the light into a quantifiable electrical impulse that can be represented graphically as a dot plot or a histogram.

We can use different fluorochromes at the same time, as long as their emission peaks are far enough apart for us to easily distinguish. A peripheral computer can instantaneously analyze the forward and side scatter light and fluorescence to identify the characteristics of individual cells and separate them into different subpopulations by charging each droplet with either a negative or positive charge, depending on the intensity and wavelength of fluorescence, as they leave the stream. The droplet is deflected either to the right or left by charged electrodes into one of three sample tubes. Intensity and wavelength of fluorescence and be plotted in a two dimensional box plot, where subpopulations in the sample can be distinguished by looking at two parameters. 

2D results with two different parameters makes visualizing cell populations so easy!

A histogram measuring the frequency of celled labeled with antibody A is plotted on the y-axis of the two-parameter box plot, and another histogram measuring the frequency of cells labeled with antibody B is plotted on the x-axis. The box plot in this example shows two subpopulations, distinguished by the intensity of fluorescence f protein expression.

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Enumerating Bacteria In Lab

Serial dilutions allow us to do viable cell counts or total cell counts.

Serial dilution and plating can determine the amount of viable cells in a culture. Serial dilutions allow a discrete number of colonies of bacteria to grow, whereas concentrated cultures may contain billions of bacteria per milliliter. In serial dilutions, smaller dilutions are repeated in succession, and the dilutions can be multiplied to obtain the total dilution. Thus, serial dilutions are more practical than doing the total dilution in a single time. For example, if I have a 100 ml bacterial culture, I can add 1 ml of it to 99 ml of water, add 1 ml of the first dilution to 99 ml of water, and then add 1 ml of the second dilution to another 99 ml of water. I end up with a 10-2 X 10-2 X 10-2 = 10-6 dilution of the original bacteria culture. I can then plate .1 ml of the final dilution on growth medium. The goal is to dilute the culture so that, when plated, the number of bacterial colonies is discrete and each colony arises from one viable bacterial cell. We can use the number of viable cells in the undiluted culture by dividing apparent colony-forming units with the product of milliliters used and the dilution factor. For example, if 150 colony-forming units were counted on the plate that was streaked with .1 ml of the 10-6 dilution, there is about 150 / (.1 ml X 10-6) = 1.5 x 109 bacteria/ml in the original culture. This method is useful because I am using only a small portion of the original culture, and large volumes of solution are not required for many-fold dilutions.

The Petroff-Hausser Counting Chamber can also be used to validate cell counts. The cell suspension is vortexed and a drop is applied to the chamber with a Pasteur pipette. Etched squares on the surface of the chamber representing specific areas and volumes are then examined under high magnification. Count the number of bacterial cells per chamber cell and multiply to obtain the concentration of cells per milliliter.

The turbidimetric method indirectly determines the quantity of insoluble particles in a liquid by comparing light transmittance in reference to a standard solution. A spectrophotometer shines a specific wavelength of light at the sample. Insoluble particles suspended in the sample will absorb and the incidental light, decreasing the amount of light transmitted to the photocell. Optical density is the measure of the turbidity of a solution, and it increases as the concentration and size of the particles increase. For example, as the concentration of bacteria reaches about 107 cells per ml, the liquid medium will appear cloudy or turbid.

Aλ= log10(Io/I) = εbc

The absorption of light is described by the Beer-Lambert Law, where A is absorbance, Io is the intensity of light incident on the sample, and I is the intensity of light transmitted through the sample. Beer’s Law states the optical density is proportional to the concentration of the compound in the solution, c, and the light’s path length, b. Thus, the concentration of bacteria in a pure culture can be determined if the molar absorbtivity, ε, and the path length, c, are known. OD600 refers to the optical density of a sample when the incident light has a wavelength of 600 nanometers. 

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