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