A wide series of dilutions e. A number of spread plates is needed because the exact number of live bacteria in the sample is usually unknown. Greater accuracy can be achieved by plating duplicates or triplicates of each dilution. Care was taken to avoid spreading to the edges of the plates as it is more difficult to count colonies along the edge of the agar.
These plates have densely packed colonies, are too numerous to count, and most likely more than CFUs. On the other hand, plates 10 -3 , 10 -4 and 10 -5 have a countable number between of CFUs. The most common dilutions are ten-fold and multiples of ten-fold. Alternatively, a fold dilution can be made directly from the original sample by mixing 1 mL of sample and 99 mL of buffer.
These dilutions can be made in successive steps or a series to give a wide range for any given sample. Once the dilution is made, an aliquot can be spread on an agar plate to create a spread plate.
After incubation, the colonies which arise can be counted and the number of cells more precisely the number of colony-forming units or CFUs in the original sample can be calculated.
For example in the image below, you did a serial dilution of a culture of the red pigmented bacterium, Serratia marcescens and made a series of spread plates. In plate 1, this was the 10 -1 dilution, in plate 2 is the 10 -2 dilution, and in plate 3 is the 10 -3 dilution.
After incubation, you count colonies were present on the plate 10 -2 dilution. Next, gather two milliliter Erlenmeyer flasks and label one broth and the other agar.
To prepare LB agar solution, mix approximately 6. Then, prepare LB broth by combining 2. After autoclaving the flasks, use a heat resistant glove to remove the flasks from the autoclave and place them in a 40 to 50 degree Celsius water bath. Once the flasks are 50 degrees Celsius, carefully prepare three milliliter aliquots of the broth solution and label each aliquot solution zero.
Next, gather 10 sterile petri dishes and label them with the date, name, type of media used, and the Winogradsky Column zone that the organisms will be harvested from. Pipette 15 milliliters of agar from the agar flask into each petri dish. Then, use the pipette tip to remove any bubbles, replace the plate lids, and allow them to solidify on the bench top overnight. Next, label 10 20 milliliter test tubes T1 through T10 and place them in a rack. Pipette nine milliliters of.
Now, cover each of the 10 test tubes loosely with their caps and transfer them to an autoclave-compatible test tube rack. After the cycle is complete, remove the saline blanks using heat resistant gloves and allow them to cool. Store the tubes at room temperature until they have reached approximately 22 degrees Celsius. To cultivate a known target organism, E. Then, cover the tube and incubate it over night at 37 degrees Celsius.
To evaluate the regions of a Winogradsky Column, add approximately one gram of material from the aerobic zone to T1 and resuspend by vortexing. Then, repeat this process with one gram of material from the anaerobic zone. Remove the tube containing solution zero inoculated with E. Then, pipette one milliliter of the solution into a T1 test tube and vortex to mix. Remove one milliliter of solution from T1 and transfer it to T2, vortexing to mix.
Repeat this process through tube T To evaluate the aerobic and anaerobic zones of the Winogradsky Column, remove one milliliter of solution from each of the previously prepared T1 tubes and transfer it to the appropriate T2 tubes. Then, continue the serial dilutions through the T10 tubes as previously demonstrated. To spread plate, pipette microliters of the diluted sample from each T3 tube on to the corresponding petri dish.
Then, use a sterile spreading rod to gently distribute the sample on to the petri dish and replace the plate lid. Repeat this process for the T6 and T9 dilutions, as previously demonstrated.
Incubate the plates containing aerobic organisms in a 37 degree Celsius incubator for 24 hours. Incubate the plates containing anaerobic organisms in an anaerobic chamber set to 37 degrees Celsius for 24 hours. The next day remove the T3, T6, and T9 dilution plates from the incubator and the anaerobic chamber and transfer them to the bench top.
Working with one plate at a time, glide a sterile inoculating loop across the top of the media in a zig-zag pattern. Then, replace the petri dish lid. Repeat this streaking method for the remaining plates, as previously shown. Then, place the streaked plates containing aerobic organisms in a 37 degree Celsius incubator overnight and the streaked plates containing anaerobic organisms in an anaerobic chamber set to 37 degrees Celsius overnight.
Cultures were harvested from the aerobic and anaerobic zones of a seven day Winogradsky Column. Then, the cultures were serially diluted prior to streaking and spreading on LB agar plates. Streaking revealed a mixed population from each of the evaluated Winogradsky zones, and the spread plates produced similar results. A plate streaked from a mixed population will result in bacterial colonies of different shapes, sizes, textures, and colors.
In contrast, the streaked and spread plates containing the known organism, E. Generally, it is best to calculate CFUs per milliliter using the average colony count of three plates spread with the same sample and dilution factor. Multiply the average number of colonies by the dilution factor and divide by the amount aliquoted. Finally, isolated colonies chosen from each plate can be used in further enrichment assays to determine species identity. Bacterial enumeration and strain isolation by plating requires manageable concentrations of target organisms.
Successful plating is therefore contingent upon serial dilution. As such, the aforementioned techniques remain the cornerstone of microbiological examination and experimentation. Though simple by design, dilution factors and plating technique can be modified to by the experimenter to bolster outcomes without compromising the integrity of each method. Plotting the four phases of bacterial growth can be helpful when characterizing desired microbes.
These phases, lag, log, stationary, and death, are marked by changes in bacterial replication. The lag phase features slow growth due to physiological adaptation, the log phase is the period of maximum proliferation featuring an exponential rise in viable cells, stationary phase is then reached due to environmental limitations and accumulations of toxins, before the death phase where cell counts begin to fall.
This can be accomplished by serially diluting or 1-step diluting to avoid confusion Solution 0 every hour for a total of 8 hours, beginning at Time 0 Solution 0 should be returned to a shaking incubator after each dilution. Repeat until each time Time 1 -Time 8 are plotted on the X-axis. Serial Dilutions and Plating: Microbial Enumeration.
To learn more about our GDPR policies click here. If you want more info regarding data storage, please contact gdpr jove. Your access has now expired. Provide feedback to your librarian. If you have any questions, please do not hesitate to reach out to our customer success team. Then you determine the average number of microbes per chamber by counting some or all under a microscope. Finally you use this average to calculate the number in the original unit.
The major drawback for direct microscopic counts is that it's difficult to distinguish living microbes from dead ones, so this method may not give an accurate viable enumeration. Turbidity tests are forms of indirect enumeration. Turbidity is the cloudiness of a liquid. In turbidimetric measurement you put a sample in solution, measure the new solution's cloudiness by shining light through it with a spectrophotometer, then estimate the number of living microbes it would take to produce the observed cloudiness level.
The drawback here is that someone must have already done numerous standard plate counts of the microbe in question in order to make sample solutions of varying turbidity, so that you have a standard to measure your current sample against.
You must also beware of overly concentrating your sample, because a turbidimetric count is only accurate if no microbes in the sample are blocking any others.
Materials and Methods Bacteria C. Infection of Mice Mice were challenged with an aerosol produced from a ml suspension of C. Statistical Analysis Direct comparisons between groups were made by paired student t -tests, with Bonferroni correction applied for multiple tests. Open in a separate window. Figure 1. Figure 2. Ethics Statement All procedures were conducted under a project license approved by internal ethical review, and in accordance with both the United Kingdom Act of Parliament and the United Kingdom Home Office, Codes of Practice for the Housing and Care of Animals used in Scientific Procedures.
Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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