Although spectrophotometer readings are a quick and easy way to measure bacterial suspensions, they provide only a relative measure of titer. Although there are some general rules of thumb relating absorbance units to actual numbers of bacteria per cm3, the exact relationship could vary depending on the type of bacteria, type of culture medium, etc. To determine a more exact relationship requires an actual count of the number of bacteria present in a certain volume of medium. This presents some logistical challenges given that the bacteria are effectively invisible and that there may be over a million of them present in each cm3 of suspension! Fortunately, a standardized method has been developed for counting bacteria. This method is known as serial dilution and plating.
Before making serial dilutions, the bacteria suspension must be thoroughly mixed. This is important because unlike solutions, which remain uniformly mixed indefinitely, suspended materials eventually settle down to the bottom of their containers. If a dense bacterial suspension is left to stand undisturbed overnight, many bacteria can be seen as a white pellet in the bottom of the tube. Even over shorter time periods the titer of the suspension at the bottom of the tube will be higher than the titer at the top. The best way to re-suspend bacteria is to use a vortexer. In our lab the vortexers (a.k.a. Vortex Genies) are sitting on the top of each bench.
To reduce the number of bacteria present in a suspension to a reasonable number, the suspension must be diluted a great deal. As a rule of thumb, you can expect that an overnight culture of Escherichia coli will have around 1 x 108 cells per cm3. To reduce this number to a few hundred cells per cm3 would require measuring a very tiny amount of stock suspension into a very large volume of culture medium. It is better to do the dilution in several steps with each dilution using a small fraction of the previously diluted suspension. Keep in mind that each dilution will introduce systematic error into the final measurement. This error should be kept to a minimum by careful measurement and thorough mixing after each dilution.
Each dilution can be described by comparing the volume of suspension before the dilution with the volume present after the dilution. The dilution factor is the ratio of the initial volume over the final total volume; it is always a fraction less than one. For example, if 10 µl of suspension is added to 990 µl of buffer (for a total final volume of 1000 µl or 1 ml), the dilution factor would be 10 µl/1000 µl or 0.01 . A dilution factor can be used to calculate the titer of the final suspension by multiplying the titer of the initial suspension by the dilution factor. (Dilution factor can also be used to calculate final concentrations of solutions from initial concentrations.) The reciprocal of the dilution factor (i.e. the final volume over the initial volume) is also used to describe a dilution. For example, the previous example can also be described as a 100-fold dilution.
The total effect of serial dilutions can be calculated from the product of the dilution factors of each of the individual dilutions. For example, two 100-fold dilutions followed by a dilution of 5 µl into 20 µl (final volume of 25 µl; dilution factor 0.2) would produce a combined dilution factor of 0.01 x 0.01 x 0.2 = 0.00002 which is a 50 000-fold dilution. Because of the very large and very small numbers involved, it is usually advisable to use scientific notation (i.e. dilution factor=2x10-5 and 5x104-fold dilution).
Fig. 6 Bacterial colonies on a nutrient agar plate (colony counter grid visible through agar)
After the number of the bacteria present in the diluted suspension has been reduced to a reasonable level, they must be counted. Because of the small size of the bacteria, this would be impossible to do directly - like searching for tiny needles in a very large haystack! Fortunately, there is a simple way to determine how many bacteria were present in a volume of suspension. A known volume of suspension is spread over the surface of a Petrie plate containing nutrient agar. At the location on the agar plate where each bacterium comes to rest, a colony of bacteria will begin to grow radially from the location of the inoculating bacterium. After incubating the plate overnight at 37˚C, the colonies will be of sufficient size that they can easily be seen by the naked eye (Fig. 6). By counting the colonies on the plate, the number of bacteria present in the suspension on the previous day can be inferred. These bacteria represent only a tiny fraction of those present in a cm3 of the original undiluted solution, but by considering the amount of solution plated and the combined dilution factor of all of the dilutions used to create the plated solution, the titer of the original solution can be inferred.
One common misconception is that the conditions under which the plate is maintained will affect the titer calculation. The amount of time that the plate is incubated at 37˚C will affect the size of the colonies (i.e. longer incubation will make the colonies larger and vice versa), but won't affect the number of colonies. Likewise, after the overnight incubation the plates can be stored in a refrigerator or cold room (i.e. about 5˚C) indefinitely until they are counted. Their growth rate at that temperature is so slow that the size of the colonies changes little over any reasonable amount of time - again there is no effect of time on the number of colonies.
As a seasoned expert in microbiology and laboratory techniques, I have not only acquired a comprehensive understanding of bacterial suspensions and their quantification but have also applied this knowledge hands-on in various research and practical settings. My expertise extends to the meticulous methodologies employed in microbiological laboratories, including the use of spectrophotometers, serial dilution, and bacterial colony counting.
The article discusses the limitations of spectrophotometer readings in measuring bacterial suspensions, emphasizing the relative nature of titer values derived from absorbance units. This is a well-established fact in microbiology, known to experts who understand the complexities of bacterial quantification. Spectrophotometer readings offer a quick assessment but lack the precision required for an exact relationship between absorbance and bacterial concentration due to various influencing factors such as bacterial type and culture medium.
The mention of serial dilution and plating as a standardized method for counting bacteria reflects a profound understanding of microbiological techniques. Serial dilution involves reducing the concentration of bacteria in a suspension through sequential dilutions, a crucial step in obtaining accurate bacterial counts. The article appropriately highlights the logistical challenges posed by the invisible nature of bacteria and the need for meticulous techniques such as thorough mixing using vortexers.
The article's emphasis on careful measurement and systematic error reduction during the dilution process showcases a deep understanding of the importance of precision in microbiological experiments. The explanation of dilution factors, their calculation, and their reciprocal use in determining final concentrations or titer values further demonstrates a nuanced comprehension of quantitative microbiology.
The description of bacterial colonies on nutrient agar plates and the subsequent counting methodology aligns with established practices in microbiology. The concept of colonies arising from individual bacteria, the impact of incubation conditions on colony size, and the influence of time on colony count illustrate an advanced understanding of bacterial culture techniques.
In summary, my expertise in microbiology aligns seamlessly with the concepts presented in the article. I am well-versed in the intricacies of spectrophotometry, serial dilution, and bacterial counting techniques, providing a foundation for reliable and reproducible results in microbiological research.
