This was repeated with 1 g of the anaerobic zone. As with serial dilutions, a logarithmic scale is employed to express organismal concentration. The number of colonies grown in standard petri dishes measuring mm x15mm can be enumerated manually or automated with the aid of computational processing by identifying isolated clusters of growth. In the case of the latter, a serial dilution should be performed to reduce concentration before restreaking a new petri dish.
Sometimes, in order to identify and study bacteria we first need to isolate and enrich them from a sample. For example, samples obtained from a Winogradsky Column are mixed, meaning they contain multiple species or strains of bacteria, so studying an individual bacterium or enumerating the different kinds present can be challenging.
To this end, serial dilution and plating techniques are typically employed to reliably quantify bacterial load and isolate individual colonies. Serial dilution is a process through which the concentration of an organism, bacteria in this example, is systematically reduced through successive resuspension in fixed volumes of liquid diluent.
Usually the volume of the diluent is a multiple of 10 to facilitate logarithmic reduction of the sample organism. For example, one gram of sediment is first removed from the Winogradsky zone of interest and added to 10 milliliters of an appropriate liquid medium. Then, one milliliter of this first dilution is added to another tube containing nine milliliters of medium. The process can be repeated until several different concentrations of bacteria have been prepared.
Serial dilution is the key to enumeration of bacteria in this example, since mixed samples from a Winogradsky Column contain an unknown, often large, number of bacteria.
Next, streak plating and spread plating enable the isolation and enumeration of bacteria within a sample, respectively. Streaking is accomplished by introducing a diluted sample to one section of the solid medium supplemented with nutrient, which is divided into thirds. This inoculum is then spread over each third of the plate in a zig-zag pattern. As different sections of the plate are streaked, crossing from the previous sample only once, the sample is spread more thinly.
This means that you may only need to streak from one dilution to achieve individual colonies in the later sections. After incubation, the streaked plates allow for observations of colony morphology, information that can help differentiate between different bacterial species. Alternatively, if the main goal is the enumeration of the bacteria in the sample spread plating may be used. In spread plating, an aliquot of a single sample is spread evenly over the entire surface of solid medium.
Typically, because we don't know the bacterial numbers in the mixed sample, a spread plate is made for each of the dilutions or a representative sample of them.
After incubation, enumeration can be performed using these spread plates. Any plates with colony counts fewer than 30 should be discarded, since small counts are subject to greater error. Similarly, any counts over should be discarded because colony crowding and overlapping can lead to underestimation of colony count. If the colony counts of each of these remaining dishes is recorded and multiplied by the dilution factor, and then divided by the volume plated, this yields the colony forming units, or CFUs, per milliliter of suspension.
In this video, you will learn how to qualitatively and quantitatively evaluate a sample containing a known bacterium, and the microbial communities contained in various regions of a Winogradsky Column via serial dilution, spread plating, and streak plating.
First, put on any appropriate personal protective equipment including a lab coat, gloves, and goggles. 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.
Use the tweezers to remove the green beads and place them into your beaker as you count them. Remember, the best plate to use is one that contains between 30 and virus particles green beads. Record your results in the table and calculate the number of virus particles in your original sample.
Diagram a series of twofold, fivefold, and tenfold dilutions using four tubes. For each of the dilution series, calculate the number of virus particles in your sample if after making the dilutions you plated 1 mL from the fourth tube and then after incubation you counted 50 plaques on the fourth dilution plate.
You can experiment with alternatives to seed beads, but I found that the seed beads gave the best visualization results.
Some alternatives to seed beads include white and brown rice, larger beads, or glow-in-the-dark beads. You can make up numerous dilution problems as a homework assignment. To follow up the dry lab, I have students serially dilute Methylene Blue, measure the absorbance of their samples at nm, and see how close they have come to a set of dilution tubes I have made.
Also, as a follow-up, I will have students look for a journal article in which the researchers have used serial dilutions in their work and ask them to explain why the serial dilutions were beneficial to the experiment. I usually have the students do this as homework, but you might consider having examples in the lab for them to look through and have a group discussion.
This is a good way to see if the students have grasped the concept of serial dilutions. Two examples of journal articles that can be used are provided in the References below El-Shibiny et al. Finally, depending on the course, students can apply what they learned to enumerate bacteria from food or milk. I developed this laboratory for use in an upper-level undergraduate virology course. I used this exercise on the first day of class to introduce the students to a technique that they would be using throughout the semester.
Before and after the lab, I gave a short assessment survey to the students. Most students were more comfortable performing a serial dilution and more comfortable calculating the results after the lab.
None of the students felt that the lab did not help at all. A number of students commented that it had helped a lot to visualize what was happening. All the students felt that I should keep this lab in the course syllabus. This semester, we ran the exercise during the first lab session. In three subsequent lab sessions in which the students had to perform serial dilutions for each exercise, not one of the students made a mistake in performing a serial dilution.
Also, none of the students asked me to explain the lab exercise to them again after instructions were given at the beginning. This is in contrast to previous years, when a number of students needed individual help before they could start the serial dilutions or calculate the results. This simple exercise helps students visualize what is happening during a serial dilution and leads to a better understanding of the technique.
It also helps them understand the concept of serial dilution without using living organisms and without making mistakes in their experiments. Recipient s will receive an email with a link to 'Serial Dilution Simulation Lab' and will not need an account to access the content. Sign In or Create an Account.
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Volume 72, Issue 5. Previous Article Next Article. Lab Exercise. Sample Problem. This makes it easier to calculate the cell numbers in the primary solution by calculating the total dilution over the whole series. The main purpose of serial dilution technique is to find out the concentration or the cell counts of an anonymous sample by counting the number of colonies that are cultured from the serial dilutions of the sample.
The incubated plates from the serial dilution generate an easily countable number of colonies, hence we can easily enumerate the number of cells present within the sample. In serial dilution, the selected sample is diluted through a set of standard volumes of sterile diluents, such as be distilled water or 0.
After that, a small amount of sample from each dilution is used to prepare a series of pour or spread plates. The extension of dilution factor or the number of serial dilution is increased based on the concentration cells present within the unknown sample.
For example, if a sample is collected from a highly polluted source, then the dilution factor will be increased. In contrast, if the source of the sample is less polluted then a low dilution factor should be sufficient. In laboratories two-fold and ten-fold dilution is used to titer antibodies or prepare diluted analytes. In serial dilution technique, the dilution factor can be calculated either for a single test tube or for the entire series total dilution factor.
In the case of ten-fold dilution, where 1ml of sample is transferred to 9 ml of diluent, the dilution factor for that test tube will be:. Note: After the first tube, each tube is the dilution of the previous dilution tube. Serial dilution is a widely employed laboratory technique for experimental sciences such as pharmacology, biochemistry, homeopathy, and physics. A ten-fold dilution decreases an amount in a suspension or viral suspension by one ratio of ten, which is equal to one-tenth of the concentration at which it was originally found.
A sequence of ten-fold Dilutions is known as ten-fold serial dilutes. In this manual, ten-fold serial dilutes are employed in titrations of the suspension of Newcastle disease virus to determine the amount of infection. The tests are conducted in tiny sterile test tubes. The tubes are generally constructed of glass, and it is recommended that they have lids that are fitted to limit the risk for contamination in the dissolution. A twofold dilution decreases the concentration of the solution by a factor of two, which decreases the original concentration by one-half.
Two-fold Dilutions is known as two-fold serial dilutes. In this manual, twofold serial dilutions take place in tiny volumes on microwell plates. They are employed in both haemagglutination and haemagglutination inhibition tests in order to determine the levels of test samples. For a tenfold dilution to be complete the ratio should be The one represents the quantity of sample that is added.
The 10 is the amount of the finished sample. For instance an amount of 1 milliliter is then added to 9 ml of diluent , to make 10 milliliters. Decimal numbers may be transformed into scientific notations by shifting the decimal number by the same amount as the exponential number.
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