Whatever the role of microorganisms at your institution, it is essential to know their growth parameters, especially the specific growth rate under the relevant conditions. Over the last decade microtiter plate readers have been introduced as a high-throughput tool for simultaneously measuring growth in 96 or 384 cultures. In doing so they have tremendously reduced the time required for those measurements, allowing an investigator to spend 2-3 hours setting up an experiment and then allowing the plate reader to automatically monitor growth over a period of many hours, recording the culture density at specified intervals then exporting the results as a computer file.
The output of a high-throughput growth experiment is a file of 96 or 384 time vs OD readings; i.e. growth curves. Turning those growth curves into specific growth rates and other important growth parameters can be done manually using a spreadsheet such as Excel, but it requires at least 5 minutes per well. For a 96 well plate that is 8 person-hours of intense work, for a 384 well plate it is 4 working days of labor. The program GrowthRates, distributed by the Bellingham Research Institute http://www.bellinghamresearch.com/, reduces that time to less than 30 seconds for even a 384 well plate; automatically identifying the optimum time points to use and providing the specific growth rate, lag time, maximum OD and correlation coefficient as a measure of the reliability of the growth rate determination.
Typically a research project will demand numerous growth experiments for a period, and then few or no such experiments for another period. Microtiter plate readers are thus used intensively episodically, but often sit idle much of the time. Plate readers are not inexpensive, so it is advantageous if the plate reader can be part of a core facility where it is available to many investigators on a shared basis without requiring each investigator to invest in the purchase of an individual instrument. Because each experiment typically ties up an instrument for 24 or more hours most instruments do not easily lend themselves to such a multi-user arrangement. An exception is the GrowthProfiler 960 from EnzyScreen http://www.enzyscreen.com/
The GrowthProfiler 960 is unusual among plate readers in several ways. First, it is the only plate reader designed for and dedicated to measuring culture growth. Second it utilizes a unique method to monitor and quantify the population density of each culture. Third, it is perfectly suited for core facility use since it permits up to 10 different experiments to be initiated at separate times and monitored independently.
Most plate readers function as spectrophotometers, recording the OD of each well at user-determined intervals. The plate readers must be capable of regulating the temperature and of shaking the plates both to provide aeration and to maintain the cells in a uniform suspension, but that aside, most function very similarly. The sensitivity and accuracy of spectrophotometers is limited to an OD between 0.05 and about 0.6. Below OD 0.05 the accuracy is unreliable and above 0.6 or so the linear relationship between OD and culture density falls off; culture density increases faster than does OD. As a result, maximum OD does not really reflect maximum population density, and the practical exponential growth phase is limited to that 0.05 to 0.6 OD range.
The GrowthProfiler 960 monitors population density by an entirely different method, photographing each well at the specific time intervals and using image analysis to measure the culture density. Proprietary software converts the image analysis to OD equivalents and permits culture density quantification up to OD = 60; two orders of magnitude greater than allowed by spectrophotometer based plate readers. This is especially valuable when it is important to know the duration of the exponential growth phase or the maximum population density that can be obtained under the employed conditions. The GrowthProfiler 960 also is designed to maximize oxygen transfer rates, thus increasing maximum population densities to those that can be achieved in industrial fermenters.
The GrowthProfiler can accommodate up to ten 96-well plates, hence the 960 designation. The plates use an opaque lid and clamping system that prevents cross contamination between wells, evaporation, and contamination between plates.
Plates can be added and removed during a run after pausing the machine, thus allowing individual users to initiate and terminate experiments independently. This feature makes the 960 particularly well suited for a core facility with multiple users.
Manual calculation of growth parameters almost completely eliminates the “high-throughput” aspect of plate-reader based growth measurements. The program GrowthRates eliminates that time bottleneck, making growth experiments truly “high-throughput”.
There are other programs that estimate growth parameters, but they have two drawbacks: (1) They require familiarity with a programming language such as R, MATLAB or Python, (2) they require manually reformatting the output of the plate reader to match the input format of the program.
GrowthRates requires no familiarity with any programming language.
While plate readers all export their output as Excel files, the format of that file varies enormously from instrument model to model. GrowthRates takes as its input file the tab-delimited text file exported by Excel and translates that format into the GrowthRates input format. All the user needs to do is to choose the instrument make/model from a menu (14 different format so far) and GrowthRates calculates the growth parameters for an entire plate in less than 30 seconds.
GrowthRates writes two output files:
The first gives, in detail, all of the growth parameters for each culture. Those include the growth rate and its standard error, the optimum time points that were used to calculate the growth rate, the lag time, the maximum OD, and the correlation coefficient R of the line of best fit to those time points. Growth rates are expressed as the first order rate constant, both in reciprocal minutes and reciprocal hours. Because most microbiologists are more familiar with growth rates expressed as doubling times, the rates are also given as doubling times in minutes.
R is the best measure of the reliability of the growth rate. An R of <0.99 suggests that the reported rate may be unreliable. When GrowthRates is used according to the User Guide cultures with R < 0.99 are rare.
The second output file is a list that reports the most important growth parameters, with one line per well or culture. That permits quickly scanning down to list to identify any wells with low R values and wells with unusual growth rates.
The mixed growth rate problem
In setting up a growth experiment it is important to choose a reading interval that is appropriate to the growth rates of the cultures in a plate. There should be at least 3 readings per doubling in order to have enough points within the exponential phase of growth. On the other hand, there should not be more than 12 reading per doubling. If readings are too frequent there will be little change in OD between readings, leading to significant scatter and unreliable readings. Usually the investigator has a rough idea of the growth rates and can choose a suitable reading interval with relative ease. A problem arises, however, when some cultures grow rapidly and others grow very slowly. That can happen when inhibitors, such as antibiotics, are present for some cultures or when some cultures are nutrient poor and other nutrient rich. In such circumstances it can be almost impossible to choose a reading interval that is suitable for all cultures.
The GrowthRates package includes a program, EditReadingIntervals, that solves that problem. The investigator chooses a plate reader reading interval that is appropriate to the fastest growing culture, then runs GrowthRates. Many of the slow-growing cultures are likely to have R values that make the growth rates unreliable. Running EditReadingIntervals on the input file allow the investigator to choose to consider, for instance, only every third time point. EditReadingIntervals then re-writes the input file using only those time points. When GrowthRates is then run on the re-written file it is almost always the case that the R values for the slowly-growing cultures are > 0.99 and the associate growth rates can be trusted.
GrowthRates is an appropriate analytical tool for the output from any plate reader, but it is especially suitable for the GrowthProfiler 960 where manual analysis of 10 plates would require 10 working days.
A free 14-day trial of GrowthRates is available from http://www.bellinghamresearch.com/,
Used together the GrowthProfiler 960 plate reader and the GrowthRates program are suitable for a core facility that serves multiple investigators who need to characterize the growth properties of their organisms.
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