Dilution can apply to a number of various things depending on the situation. It may refer to a decrease in the ownership proportion of a stock in a company or it may relate to the process of dilution, which we learned about in elementary school science. The term "dilution" in this text refers to the process of lowering the pH of a chemical (gas, vapour, or solution), which is further defined as the process of lowering the concentration of a solute in solution, typically by mixing with more solvent. There are many types of dilution in microbiology namely; homeopathic dilution which is a process in which a substance is diluted with alcohol or distilled water and then vigorously shaken in a process called “succession” and the commonly known dilution which is serial dilution. Serial dilution is a series of sequential dilutions used to reduce a dense culture of cells to a more stable concentration where each dilution will reduce the concentration of bacteria by a specific amount. Serial dilution enables one to determine the amount of bacteria one started with by calculating the total dilution over the entire series. In this lab report, we will discuss the importance of serial dilution, the procedure on carrying out the lab experiment and its background information. The objectives of this experiment are to learn the process of enumeration to determine the number of microorganisms in a sample and utilize two methods in enumeration that are pour plate method and spread plate method.
Introduction
In most cases, we encounter rivers or small water catchment areas that have been closed or more so swimming pools, this is due to contamination of them by different types of microorganism like Escherichia coli. It is known that microorganisms are small to be seen under the naked eyes and they need to be discovered in certain aspects as the dairy industries or treating of rivers or disease investigation, and this has led to the development of a variety of methods like serial dilution that have enabled the enumeration of the microorganisms (Jacquelyn, 2014).
Many areas of science use serial dilutions in preparations for different experiments. Serial dilutions are usually made in increments of 1000, 100, or 10. The concentration of the original solution and the desired concentration will determine how great the dilutions need to be and how many dilutions are required. Important also is the total volume of solution needed. If only small quantities of solutions are needed then greater numbers of dilutions are necessary. Serial dilution method is cost-effective and it allows small aliquots to be diluted instead of unnecessary large quantities of materials (Jacquelyn, 2014). This technique involves the removal of some of the original solution and then adding it to another container which contains a known amount of the same buffer as the original solution. This type of dilution describes the ratio of the solute to the final volume of the diluted solution.
Microbiologists have come up with indirect methods that give precise measures of population density, one of them being viable count. In viable plate counts, we are measuring the number of viable cells, unlike the microscopic counts that cannot distinguish live from dead cells. Before doing plate counts, serial dilutions are required. This is because it is hard to count more than 300 colonies on an agar plate if we inoculated directly from the original bacterial suspension or sample without serial dilutions (Kathleen and Talaro, 2015). Either to complete plate counts, we could use pour plate method or spread plate method and each of them has their advantages and limitations. The pour plate, like other viable plate count methods, involves adding a sample to a solid medium that will support microbial growth incubating the plates so that each bacterial cell multiplies to form a colony and counting the number of colonies that develop. However, spread plate consists of serial dilutions of a sample spread onto the surfaces of agar plates. The spread plate technique is faster than the pour plate, but can result in colonies close to each other making it harder to count CFUs. All the visible colonies are calculated and represented as colony forming units (CFU). Then, the CFU is multiplied with the corresponding dilution factor. As a result, the population of the original sample is known.
Procedure
Pour Plate Method
Six nutrient agar pours are melted in a boiling water bath. After they liquefy, they are mixed and placed in a 50°C water bath until they are ready to use. Six petri plates are labeled 10-2 through 10-7.The unknown sample is shaken to ensure an even distribution of microorganisms; 1 mL of sample is removed aseptically with a sterile pipette and transferred to the 10-2 dilution blank.The dilution blank is shaken vigorously to distribute the bacteria evenly. Using a new sterile pipette, 0.1 mL and 1.0 mL are transferred aseptically from the 10-2 dilution to the agar plates labeled 10-3 (0.1 mL) and 10-2 (1 mL) respectively. With the same pipette, an additional 1.0 mL is removed from the 10-2 dilution blank and transferred to the 10-4 dilution blank. The original 1 mL of the sample has now been diluted 1 part in a total of 10,000 parts. The procedure is repeated for the 10-4 and 10-6 dilution blank.A tube of melted agar (50°C) is poured aseptically into each Petri plate to which already added a dilution of the sample. The plate is swirled to mix the sample with the agar. The agar is made sure does not run over the edges of the plate. The lid is replaced. The agar is allowed to cool and solidity. The inverted plates are incubated at 30°C. After incubation, the colonies are counted on each plate, and the number of OCD determined.
Spread Plate Method
The bacterial sample is transferred to the given dilutions and four TSA plates are taken and labeled.From the 10-4 test tube, we used 1 plate and added 0.1 mL to the plate. A bent glass spreader was used to spread the plate, the bent glass spreader was dipped in alcohol and passed through a flame of Bunsen burner and with an angle of 45° used the spread.The bent glass spreader was sterilized and the inoculums were left to dry for about 10 min before inverting. They were later incubated at 35°C for 24-48 hours; the FDF is indicated as 10-5. This procedure is carried out for the rest of the three test tubes.The number of colonies was counted and the OCD (original cell density determined).
References
Jacquelyn G. Black, 2014, Microbiology: Principles and Explorations 4th Edition, Prentice-Hall, Inc., USA.
Kathleen Talaro, Arthur Talaro, 2015, Foundation in Microbiology, Wm. C. Brown Publishers, Kerper Boulevard.
