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Microbiology Concept Inventory
Please note, you must be an educator in higher ed or maybe high school to qualify to recieve the MCI
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|Change in the amount of a cell component
In situations where determining the number of microorganisms is difficult or undesirable for other reasons, the use of indirect methods can be an excellent alternative. These methods measure some quantifiable cell property that increases as a direct result of microbial growth.
The simplest technique of this sort is to measure the weight of cells in a sample. Portions of a culture can be taken at particular intervals and centrifuged at high speed to sediment bacterial cells to the bottom of a vessel. The sedimented cells (called a cell pellet) are then washed to remove contaminating salt, and dried in an oven at 100-105 °C to remove all water, leaving only the mass of components that make up the population of cells. An increase in the dry weight of the cells correlates closely with cell growth. However, this method will count dead as well as living cells. There might also be conditions where the dry weight per cell changes over time or under different conditions. For example, some bacteria that excrete polysaccharides will have a much higher dry weight per cell when growing on high sugar levels (when polysaccharides are produced) than on low. If the species under study forms large clumps of cells such as those that grow filamentously, dry weight is a better measurement of the cell population than is a viable plate count.
It is also possible to follow the change in the amount of a cellular component instead of the entire mass of the cell. This method may be chosen because determining dry weights is difficult or when the total weight of the cell is not giving an accurate picture of the number of individuals in a population. In this case, only one component of the cell is followed such as total protein or total DNA. This has some of the same advantages and disadvantages listed above for dry weight. Additionally, the measurement of a cellular component is more labor-intensive than previously mentioned methods since the component of interest has to be partially purified and then subjected to an analysis designed to measure the desired molecule. The assumption in choosing a single component such as DNA is that that component will be relatively constant per cell. This assumption has a problem when growth rates are different because cells growing at high rates actually have more DNA per cell because of multiple initiations of replication.
Turbidity
A final widely used method for the determination of cell number is a turbidometric measurement or light scattering. This technique depends on the fact that as the number of cells in a solution increases, the solution becomes increasingly turbid (cloudy). The solution looks turbid because light passing through it is scattered by the microorganisms present and the turbidity is proportional to the number of microorganisms in the solution. The turbidity of a culture can be measured using a photometer or a spectrophotometer. The difference between these instruments is the type of light they pass through the sample. Photometers, such as the Klett-Summerson device, use a red, green or blue filter providing a broad spectrum of light. Spectrophotometers use prisms or diffraction gratings supplying a narrow band of wavelengths to the sample. Both instruments measure the amount of transmitted light, the light that makes it from the light source through the sample to the detector.
Figure 4.16. Measuring the turbidity of a culture. A spectrophotometer or photometer quantifies the amount of turbidity of a culture. The amount of light scattered from a solution is proportional to cell number. The instruments measures light that is not scattered by the sample.
When measuring light scattering it is important to consider the wavelength of light used a bacterial culture. Microorganisms may contain numerous macromolecules that will absorb light, including DNA (254 nm), proteins (280 nm), cytochromes (400-500 nm), and possible cell pigments. When measuring bacteria by light scattering it is best to pick a wavelength where absorption is at a minimum and for most bacterial cultures wavelengths around 600 nm are a good choice. However, the exact wavelength chosen is species specific.
The amount of light transmitted through a sample is inversely proportional to cell number and can be expressed in the following equation.
Figure 4.17. The transmittance equation. The ratio of light hitting the sample (I0) to light passing through a sample (I) is the transmittance.
Where T is the light transmitted, I0 is the light entering the sample and I is the light passing through to the detector.
Due to the nature of light scattering, transmittance decreases geometrically as the cell numbers increase. It is more intuitive to think of the units increasing as growth increases and for most bacterial analysis, transmittance is converted into absorbance using the following equation.
Figure 4.18. Absorbance. The absorbance of a sample it the negative log base 10 of the transmittance.
Absorbance increases in a linear fashion as the cell number increases. When measuring growth of a culture the term optical density (OD) is normally used to more correctly represent the light scattering that is occurring; under optimal conditions, little light is actually absorbed by the culture so the term absorbance is misleading. For most unicellular organisms changes in OD are proportional to changes in cell number (within certain limits) and therefore can be used as a method to follow cell growth. If a precise cell number for a given OD is desired, a standard curve can be generated, where viable plate count or cell mass is plotted as a function of OD. It also wise to develop a standard curve to verify that the OD is actually an accurate portrayal of cell growth. After the standard curve is made, it is then possible to simply measure the OD of the culture and read the cell number from the curve.
The turbidity of a culture is dependent upon the shape and internal light-absorbing components of the microorganism and therefore turbidity readings are species-specific and cannot be compared between different microbes or even between different strains of the same species. As above, there are microbes that change cell size or shape at different stages of growth, which introduces some inaccuracy to this method of cell counting. Also both living and dead cells scatter light and are therefore counted. However, the method is very rapid and simple to perform and provides reliable results when used with care, so it is an extremely common method of real time analysis of prokaryotic populations. In fact it is one of the methods we will use for measuring cell number in the experiment on bacterial growth. Turbidometric measurements also do not destroy the sample.