May 14, 2014 colonyforming unit cfu or cfu is a measure of viable bacterial or fungal cells. Only plates between 30 and 300 cfu were used for the calculation of the cell numbers. Finally, multiply the total dilution by the average number of colonies in the plate s and report your answer in cfusml or cfusgram depending upon where the sample came from. Davidsonb a rdecom, edgewood chemical biological center, aberdeen proving ground, md 21010, usa b science and technology corporation, belcamp, md 21017, usa article info abstract article history. Divide the cfu from the dilution 179 by the result from step 1 0. Set up a serial dilution of a bacterial culture and describe why they are used. Dilution factor many laboratory chemicals such as acids are purchased as concentrated solutions stock solutions. Miles and misra technique is employed to calculate cfu. Titer the titer is the smallest amount or concentration that will produce a particular effect or endpoint. This is different than a dilution ratio, which typically refers to a ratio of the parts. Lets say you are the only owner of a company and you own shares. If the ml contains 10ml of the substance, then the 50ml must also have contained 10ml of the substance. For the we need to take log of the values obtained i. Determine the mean cfu ml or cfu per dose for the number of vials tested using the calculation s listed below.
Pdf colony forming unit calculations using spread plate. An easy learning guide dilution multiple dilutions. If no colonies appear on the plate, do not report the count as zero. A simple and fast method for determining colony forming units. We did an experiment where we did 105, 106, 107 dilutions. So colony count x dilution factor the inverse of the dilutions cfu ml of original culture 52 cfu x 106 x 101 5. Calculation answer dilution plate cfus 200 dilution factor of viable plate 1. Chapter six concentrations and dilutions 27 example 6. Calculate the bacterial density, expressed as cfu ml, for the three different samples presented in the table. You are given eight petri dishes of nutrient agar and an abundance of pipettes and 9.
Ezcfutm dilution example when ezcfutm is used according to directions, the following dilutions are conducted to reach a desired concentration of 10100 cfu 0. The logic of the dilution equation you do not need to understand the basis of the dilution equation in order to properly use it. The total dilution is the product of all dilutions. An example of using dilutions to do a cfu calculation could go as follows. Calculate the number of bacteria per cc as follows. Apr 25, 2017 scientists can then use the cfu count to determine roughly how many microbes were in the original sample. For example, when you are making orange juice from a frozen concentrate, you mix 1 can of concentrate with 3 cans of water.
Place in whirltissue pak bag and add 5 ml of sterile homogenization buffer. Then, 1 ml from that mixture is added to 9 ml, and it is mixed together a 102 dilution. In most cases, serial dilutions are employed to reach the final desired dilution. Dilution worksheet and problems biology libretexts.
A dilution scheme to give a total dilution of 108 could be 1. We use two dilutions as inocula because the cells may only be 10% viable. Now we may want to convert the values to log value. Scientists can then use the cfu count to determine roughly how many microbes were in the original sample. Cfu ml number of bacterial colonies counted on plate x dilution factor volume of culture plate. The multiplication factor is determined by dividing the dilution used by the volume plated. Sop for preparation of culture dilutions for analysis. The lab report assistant is simply a summary of the experiments questions, diagrams if needed, and data tables that should be addressed in a formal lab. The cell number as cfu ml was calculated by multiplication of the counted cfu on the petri dish with the dilution factor of 80,000 and multiplication of 20 to correct for the cfu per ml. Serial dilution protocol pdf it is a method of diluting a stock solution where concentration decreases by the same quantity in each successive step. The average cfu counts were comparable and the standard. Detection limit cfu ml 10 sample dilution plate 1 pla7e 2 plate 3 cfu ml 1 103 1816 1698 1885 104 180 159 186 1. For convenience the results are given as cfu ml colonyforming units per.
Back to our example, what is the number of colonyformingunits cfu per ml. Calculate the cfu ml, using the above data and the following equation. Calculations here we give some important calculation tools for the successfull development of membrane processes. For example, a sample size of 1 ml is added to 9 ml of diluent to equal a total of 10 ml. What is a colony forming unit and how is it used to infer microbial. Cfu volume x dilution factor 20,000 cfu ml questions a. For example, if 200 colonies are counted on a plate made with a 1milliliter sample of a solution diluted 1,000 times from its original strength, the original solution contains approximately 200,000 cfus per milliliter. Provide examples of why bacterial growth rates have to be measurable characterized. The purpose can be determination of bacterial, fungal, or viral counts indirectly. Culture dilutions were prepared in 96well plates in 12fold and these were plated using both methods. When these strains are enumerated by conventional cfu counting, typically between 30 and 300 colonies per standard agar petri dish of 8 cm in diameter results in optimal counting.
For example, if a culture contains 1 x 109 cellsml and 0. There is a number of calculations to make before getting your final percentage of dilution. First, from the initial sample your teacher gives you, you take 1 ml and plate it. Dilution and concentration theory sounds ominous but you use dilutions and. This can be mathematically illustrated with the equation d t d 1 x d 2 x d 3 x x d n where d t is the total dilution factor and d n is the dilution ratio. Thanks to tim allen for providing this problem set. Prepare the liquid culture sample you want to plate. You have to prepare 150 ml of the 101 dilution from a water sample.
Therefore, the amount of drug in the diluted product is the same. All materials checked by dr scott wildman, dr cleopatra. If the concentration is 35,000 cfu ml 104, and 35 cfu ml is the target concentration, the following serial dilutions can be performed. For example, if an average of 65 colonies were counted on plates inoculated with 0. So now i have calculated the cfu ml for each dilution i. Version 4203060002 lab report assistant this document is not meant to be a substitute for a formal laboratory report. For calculation purposes, use the dilution that resulted in 30300 cfu plate or 20200 cfu filter. Protocol for using serial dilutions in the microbiology lab. Counting with colonyforming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all. Bacterial dilutions and a foolproof way to figure them out. Received 9 may 2014 received in revised form 29 august 2014. Cfuml number of c olonies x d ilution f actor volume of c ulture p late. Bacterial enumerationdilutions and plate counts completed. For the first tube of bacteria i grew 400 colonies on a plate after pipetting 100 ul onto the plate with a 105 dilution, 40 for the 106 dilution, and 8 for the 107.
Understanding the basic concepts of dilution and concentration uncomplicates these problems. Calculate the number of bacteria cfu per milliliter or gram of sample by dividing the number of colonies by the dilution factor the number of. Multiply the average number of colonies by the dilution factor and divide by the amount aliquoted. Chapter 10 dilution and concentration 211 critical thinking 10. This is different than a dilution ratio, which typically refers to a ratio of the parts of solute to the parts of solvent, for example, a 1. This will drop the concentration two logs from 103 to 101 cfu ml. Us environmental protection agency office of pesticide. For example, if i added 1g of sample into 9ml of broth 101, and transfer 1ml from 101 to second tube 9ml as well. Whirl pak bag technique o murine tissues in sterile fashion, dissect for lung, do not include heart tissues and weigh them in petri dish. Estimation method for serial dilution experiments avishai bendavid a. Bacterial enumerationdilutions and plate counts handson labs, inc. As shown in the illustration below, it begins when 1 ml of the bacterial sample is added to 9 ml, and it is mixed together creating a 101 dilution. The total dilution ratio can be determined by multiplying the dilution factor of each step leading up to the final step. The dilution factor of the large square 1,250,000 x.
A colonyforming unit cfu, cfu, cfu is a unit used in microbiology to estimate the number of viable bacteria or fungal cells in a sample. Lets look at the information that has been provided and is critical to solving the calculation. However, if the organism normally forms multiple cell arrangements, such as chains, the colonyforming unit may consist of a chain of bacteria rather than a single bacterium. The dilutions used and the number of cfu obtained in each case are also presented in the table. Cfu counting protocols for industrial microbes including lactic acid bacteria, escherichia coli and saccharomyces cerevisiae. Serial dilution method protocol step wise explanation duration. Viable is defined as the ability to multiply via binary fission under the controlled conditions. Calculate the final dilution ratio in a serial dilution. Counting with colonyforming units requires culturing the microbes and counts only viable cells, in contrast. I am confused regarding the calculation of dilution factor. Two pellets are placed in 2 ml of hydrating fluid 1,000 10,000 cfu ml.
To calculate cfu ml or cfu g, average the number of counted colonies from three plates. Dilution photo count and viability viable, tftc, or tntc table 2. You must do this to find the dilution factor which yielded your cfu count. The number of bacteria per cc the average number of bacteria per large square x. Dilutions part 3 of 4 calculating colony forming unitsml youtube. Average between spread plates for a given dilution if using, then average results from the three tubes per treatment.
A set of serial dilutions is made, a sample of each is placed into a liquefied agar. An easy way to calculate this is to convert the fraction into its reciprocal. How can i calculate colony forming unit cfu for bacteria. For example, if 50 colonies were counted in the 102 dilution, the cfuml is 50. How to calculate the concentration of bacteria in your original sample from plated serial dilutions.
Hello all i have already finished analysing all my samples for different types of bacteria. Even though a 100 ml portion was filtered, the colony count for the 10 ml sample dilution is closer to the ideal range and thus, must be used as the reportable result. After incubation, count plates from each series that contains 30 to 300 cfus. This protocol is specific for bacterial counts colonyforming units, cfus, but can be modified for fungi cfus and viruses plaqueforming units, pfus for viral counts. Further, if you do dilution calculations only occasionally, it may. Plating dilution factor pdf when the sample is plated, a dilution factor must also be calculated for this transfer. Dilution is the reduction in the ownership percentage in a certain company as an effect of the issuance of shares. Counting rules for membrane filtration methods for coliform. Determination of inoculum colonyforming units cfus for mic. For determining and verifying the effectiveness of the neutralizer, ensure that. The dilution factor of any dilutions made prior to placing the sample in the counting chamber, such as mixing it with dye 2 in this case c. Student learning advisory service at a glance pharmacy. A serial dilution is the dilution of a sample, in 10fold dilutions. Many foods require a dilution step before plating and the following is a guide to follow when preparing dilutions.
From this, two serial 1100 dilutions are made, and 0. Several 10fold dilutions of the sample are commonly used. Make appropriate dilution of thawed stock culture in broth to yield 104105 cfuml for inoculation into the mic system. Calculate the cfu ml from colonies counted on an agar plate, given the dilution factor and volume correction factor. Dilutions part 3 of 4 calculating colony forming unitsml. A calculation of the total number of cfus in the original milk sample. Since the object of these calculations is to determine cfu ml, the amount plated for the countable plate is divided by 1 ml to get the pdf. To solve type two problems, simply rearrange the formula above to solve for the total dilution factor. Because we generally have no idea of how many bacteria are in a sample, it is almost always necessary to prepare a dilution series to ensure that we obtain a dilution containing a reasonable number of bacteria to count. Cfu ml number of c olonies x d ilution f actor volume of c ulture p late. Determination of inoculum colonyforming units cfus for. The dilution factor df can be used alone or as the denominator of the fraction, for example, a df of 10 means a 1.
348 1144 718 658 904 1208 573 916 988 1578 1599 763 715 227 360 1134 668 1227 1155 1088 766 1376 264 1191 1262 1247 851 17 539 818 869 56 1270 532 584 662 494 699