Learn techniques for serial dilution and cell counting. For example, in tenfold serial dilutions each successive dilution is one tenth of the previous dilution yielding dilution ratios of 110, 1100. The basic idea behind the dilution effect hypothesis is that biodiversity typically measured by species richness, or. Spectrophotometry page 7 of 10 single dilutions background stock solutions are constantly diluted in biological lab settings. If the technique is done correctly, then the living viable cells will be counted as a colony forming unit cfu that will decrease following the increasing of total dilution and the original sample cell could be calculated based on the result. This is a one millionfold dilution clearly too big to do at once. Estimation method for serial dilution experiments sciencedirect. Serial dilution is the stepwise dilution of a substance in solution. The total dilution factor for a serial dilution is equal to the product of the individual dilution factors.
A serial dilution is the dilution of a sample, in 10fold dilutions. A series of five dilutions is made of this first dilution by diluting it 110, rediluting 110, and then three times more, each resulting solution then being a 110 dilution of the previous one in the series. It should be noted that some level of dilution was achieved as demonstrated with colony counts of 175 and 50 on plates 1a and 2a, respectively. Serial dilutions a serial dilution is any dilution where the concentration decreases by the same quantity in each successive step.
If it is helpful, you can find a microtiter plate image blank in pdf format on the courses server. This methodology utilizes agar as a medium for bacterial growth, a process termed, culturable technology. Introduction to the spectrophotometer and pipetting skills section a. Spread plate technique during this experimentation, we used. After observing dense cultures of tetrahymena last week, to better observe the organisms we must dilute the sample into more easily observable number of organisms. Pdf estimation method for serial dilution experiments. The method maintains this accuracy even in the presence of dilution errors of up. How could your lab technique or the experimental design be improved in order to to produce more accurate data. The independent variable is the concentration of the serial. The objectives of this experiment are to learn the process of enumeration to determine the. For example, when you are making orange juice from a frozen concentrate, you mix 1 can of concentrate with 3 cans of water. They also prepare serial dilutions of phage and use them for a plaque assay. Pdf lab report of microbiology akbar haqi academia. Learn techniques for serial dilution and cell counting understand how to set up experimental design and hypothesis background.
Lab 4 serial dilution and cell counting cili cure spring 2017. What does this mean if a solution has a 110 dilution the number represents 1 part of the patient sample added to 9 parts of diluent. Typically, the dilution factor remains constant for each dilution, resulting in an exponential decrease in concentration. During the first laboratory period students hypothesize the outcomes of.
Introduction microorganism is an organism that is microscopic or submicroscopic, which is too small to be seen under naked eyes. Intro to the spectrophotometer a commonly used instrument in the analysis of cellular extracts is the spectrophotometer. Conclusion we can determine the number of microorganisms in a sample by enumeration. A culture of microbes can be diluted in the same fashion. The independent variable is the concentration of the serial dilutions. Ld50 lab ap environmental science lab report enotebook. An undergraduate laboratory activity demonstrating bacteriophage.
Serial dilutions involve the preparation of successive dilutions that vary the concentration of the solute by a constant factor, so that each successive dilution is a multiple of the previous dilution. The independent variable is the concentration of the. First, the absorption spectrum obtained from methylene blue should peak at an intermediate range approximately equal to 668 nm optical absorption of methylene blue. Serial dilutionagar plate procedure to quantitate viable cells. In the experiment lab students will be diluting environmental toxins to see their effect on fertilization and development. The diluted sample is then used as the base solution to make an additional dilution. Probably the most common application in biology of this technique is in the measurement of the concentration of a compound in solution. You will now create a serial dilution series starting from the 200 pgml wells, resulting in the following dilutions in columns. Prelab questions show work and include in your lab report 1. Upon completion of this lab, the student should be able to proficiently calculate molarities for solutions.
Your microbiology lab instructor taught you the basics of serial dilution and how to use it to determine the number of cells in an original bacterial culture. Calculate the final dilution ratio in a serial dilution. A scientific lab report is simply a paper that explains to an audience an experiment that was done in order to support a hypothesis or null hypothesis. Once an organism has been diluted out and allowed proper incubation time, this is. Serial dilutionagar plate procedure to quantitate viable. Dilution and concentration theory dilution and concentration theory sounds ominous but you use dilutions and concentrations fairly frequently outside of your scientific life. Add cona to the wells at a final concentration of 10 gml. Introduction for unicellular microorganisms, such as bacteria, the reproduction of the cell. This exercise is intended to familiarize us with using scientific notation to express big and small number easily and the process of using serial dilutions to make concentrations of known values.
Hopfully when going into the lab, you are able to know the following skills. The results were off from the hypothesis, the hypothesis was that the ld50 would be at 12. Absorption spectra and the use of light absorption to measure concentration spectrophotometry is a procedure that is frequently utilized in biological laboratories. Two hypotheses may be made prior to performing this experiment. Following one weeks experiment could incubation at 25c, the colonies of the plate were change these and counted manually. Lab 9 serial dilution, pour plates, and enumeration of bacteria introduction the number of bacteria in a small sample can be startling. A series of five dilutions is made of this first dilution by diluting it 110, rediluting 110, and then three times more, each resulting solution then. Standard bacterial plate count uncontrolled copy 3. Many laboratory protocols require the serial dilution of reagents or. To perform a serial dilution, a small amount of a wellmixed solution is transferred into a new container, and additional water or other solvent is added to dilute the original solution. A dilution in chemistry is a process that reduces the concentration of a substance in a solution. A new paper written by dan salkeld formerly of stanford, kerry padgett ca department of public health, and myself just came out in the journal ecology letters this week one of the most important ideas in disease ecology is a hypothesis known as the dilution effect. Use an appropriate dilution of the mixture with regard to the number of cells to be counted. The solutions with the higher concentration of serial dilutions will result in higher conductivity and absorption because of the higher amount of the salts in the solution.
Because of this, a variety of methods have been developed. In homeopathy, serial dilutions called potentisation are often taken so far that by the time the last dilution is completed, no molecules of the original substance are likely to. A simple method is presented to estimate the microbial counts obtained with the serial dilution technique for microorganisms that can grow on bacteriological media and develop into a colony. If it is too dissolved, the sample size will not be.
The power point can be used to reveal instructions to the students during the lab. I have completed the lab report for the solutions and dilutions part of the lab. Serial dilutions and quantification of bacterial viable counts. Note that serial dilution is a special case of series of dilutions. The following formula can easily be used to carry out a simple dilution. Aseptic technique, dilution, streaking, and spread. Hypothesis predict how the serial dilution of your drink solution will affect the colour, flavor, and other properties of the solution. Ic50 assays, commonly used to evaluate drug efficacy, and assay development. Label 2 or 3 refer to the firms outline of production op plates per dilution series for each serial.
It is assumed that the volume of cell suspension placed in the chamber represents a truly random sample. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. Dilution and concentration theory university of minnesota. Serial dilution lab in this unit we will be discussing measurements of concentration that us e both very big and very small numbers.
Dilutions in the range 101 110 to 108 1100,000,000 are generally used, although with particular types of samples the range of dilutions can be restricted. Calculating concentrations based on dilution factors. Culturing and enumerating bacteria from soil samples. It is critical that you master both serial and simple single dilutions. They will need to understand dilution in order to be able to do these experiments in any kind of meaningful way. Prior to the lab, you will also need to complete your prelab report, which will test your understanding of the following concepts.
Serial dilutions are used to accurately create highly diluted solutions as well as solutions for experiments resulting in concentration. Sample lab report introduction to the spectrophotometer. The dependent variables are the conductivity and absorption. In this lesson an attempt has been made to relate dilution to the real world.
Before doing plate counts, serial dilutions are required. After observing dense cultures of tetrahymena last week, to better observe the organisms we must dilute the sample into. Hemocytometer counts are, however, subject to the following sources of error. Mar 30, 2014 serial dilution is a method intended to reduce the numbers of colonies to a range between 30 and 300. Culturing and enumerating bacteria from soil samples protocol. Conclusion form this experiment, the bacteria that found in the porridge are. Titration of microorganisms in infectious or environmental samples is a corner stone of quantitative microbiology. Understand how to set up experimental design and hypothesis. The results from the lab fully support my hypothesis in that the solutions with the higher concentrations have higher conductivity and absorption. This can be mathematically illustrated with the equation dt d1 x d2 x d3 x x dn where dt is the total dilution factor and dn is the dilution ratio. Calculate the mass of sucrose needed to prepare 200 ml of 1. Place the used dilution tubes in the disposal baskets in the hood. In conclusion, lampdlfd proved to be highly specific and sensitive.
As such, microbiologists have employed serial dilution and various plating. Lab 9 serial dilution, pour plates, and enumeration of bacteria introduction. Each student might be required to turn in a separate lab report, but they are. Serial dilution protocol pdf it is a method of diluting a stock solution where concentration decreases by the same quantity in each successive step. Perform a serial dilution of a bacterial sample according to instructions in. One way to enumerate the number of bacteria present in a soil sample is to utilize dilution and plating methodology. In addition, the absorptions of serial dilutions of methylene. This will not be a valid assumption unless the suspension is monodispersable and free of cell clumps. In this lab, a serial dilution will be made of a sample of bacteria, and then those dilutions will be used to. The method maintains this accuracy even in the presence of dilution errors of up to 10% for both the aliquot and diluent volumes, microbial.
Calculating serial dilutions serial dilutions are a way of dividing up a large dilution into a number of successive dilution steps with dilution factors of 100fold or less. Serial dilutions involve diluting a stock or standard solution multiple times in a row. To have more practice in serial dilutions and calculations. To verify this conclusion, the first experiment varying the number of mix.
Yeast suspension 2 mllab counting chamber 1student pipettes, pasteur 1student. Serial dilutions austin community college district. Bio101 lab 3 bacteria enumeration north central college. For this assay, an antibody against il2 is coated onto the microtiter well. Repeated serial dilutions will be necessary to determine accurate concentration. Today you will learn how to use the spectronic 20 spectrophotometer, commonly referred to as the spec 20.
Serial dilution is a series of dilutions, usually twofold or tenfold, used to determine the titer or concentration of a substance in a solution. The teacher may wish to go step by step, making sure that all groups have. Disscussion and conclusions the hypothesis for this lab was that serial from. Serial dilutions reduce the concentration of a sample in small steps or fractions. We will solve this problem by doing a serial dilution. Interested in isolation of soil bacteria lab report. Using a turntable and sterile bent glass rod see fig. The total dilution ratio can be determined by multiplying the dilution factor of each step leading up to the final step. In this experiment, we used serial dilution and spread plate.
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