Objectives. Understand how to quantify bacterial cells. Learn how to solve a dilution problem.It is a common practice to determine microbial counts for both liquid and solid specimens-suspensions of E. Coli in nutrient broth all the way to soil samples and hamburger meat.
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Most specimens have high enough numbers of microorganisms that the specimen has to be serially diluted to quantitate effectively. The following is a step-by-step procedure to working dilution problems, and includes some practice problems at the end.The purpose can be determination of bacterial, fungal, or viral counts (indirectly). This protocol is specific for bacterial counts (colony-forming units, CFUs), but can be modified for fungi (CFUs) and viruses (plaque-forming units, PFUs for viral counts). A set of serial dilutions is made, a sample of each is placed into a liquefied agar medium, and the medium poured into a petri dish.
The agar solidifies, with the bacterial cells locked inside of the agar. Colonies grow within the agar, as well as on top of the agar and below the agar (between the agar and the lower dish). The procedure described above produces a set of pour plates from many dilutions, but spread plates (sample spread on top of solidified agar) can be used also. The agar plate allows accurate counting of the microorganisms, resulting from the equal distribution across the agar plate. This cannot be done with a fluid solution since 1) one cannot identify purity of the specimen, and 2) there is no way to enumerate the cells in a liquid. STEP 1:Determine the appropriate plate for countingLook at all plates and find the one with 30-300 colonies (or plaques), preferably.
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Greater than 300 and less than 30 is a high degree of error. Air contaminants can contribute significantly to a really low count and a high count can be confounded by error in counting too many small colonies. Use the total dilution for the tube from where the plate count was obtained. If duplicate plates (with same amount plated) have been made from one dilution, average the counts together. The LibreTexts libraries are and are supported by the Department of Education Open Textbook Pilot Project, the UC Davis Office of the Provost, the UC Davis Library, the California State University Affordable Learning Solutions Program, and Merlot.
Aug 22, 2010 Serial dilutions & 'spread plate technique'? If you were to directly take a given serial dilution and do a count under a microscope what would be the advantages and disadvantages of this method versus carrying out the serial dilution-agar plate procedure to count the number of 'cells'? What the advantages and disadvantages of the serial dilution-agar plate procedure? What are the advantages/disadvantages of Iran's 'peaceful' power. Published: 23rd March, 2015 Last Edited: 11th May, 2015. Cell Counting of Live Bacteria, S. What Are The Advantages And Disadvantages Of The Serial Dilution?
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. A variety of techniques has been developed for the isolation of microorganism, mainly the bacteria, from the specimen or from the sample cultures. The spread plate method is a technique to plate a liquid sample containing bacteria so that the bacteria are easy to count and isolate.
A successful spread plate will have a countable number of isolated bacterial colonies evenly distributed on the plate. Spread plate culture technique is among the most widely used culture technique for isolating the bacteria.Principle. In this technique, a serially diluted specimen containing 2 or more bacteria or microbe (Mixed culture) is used which is spread over the solidified agar media plates as a thin layer with the help of a sterile L-shape glass rod (Spreader) while the media plate is spun on a turntable.The principle behind this method is that when the Media plate is spun, at some stage, single cells will be deposited with the bent glass rod (Spreader) onto the surface of the Agar media. Some of the cells present in the specimen / diluted specimen will be separated from each other by a distance sufficient to allow the colonies that develop to be free from each other. Make a dilution series from a sample.
Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate. Dip the L-shaped glass spreader into alcohol. Flame the glass spreader (hockey stick) over a Bunsen burner.
Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petri dish underneath at the same time. Incubate the plate at 37°C for 24 hours. Calculate the CFU value of the sample. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/mL in the original sample.Applications. The Spread Plate Technique, in conjunction with serial dilutions, is a valuable research tool. To demonstrate the cultural characteristics of the bacteria (e.g.
Color, texture, size, elevation, etc.). To isolate the bacteria in discrete colonies from the specimen containing more than 1 bacterium. For determining the Sensitivity and/or Resistance of bacterium towards the particular Drug/Antibiotics or Test substances. To obtain the sufficient of the bacterium for various biochemical and other tests. To estimate the viable counts of the bacteria in the specimen. To maintain the stock cultures.
To transport or short-term storage of the specimen (e.g. Stab culture).Advantages. Hartman D.
Perfecting your spread plate technique. Journal of microbiology & biology education, 12(2), 204-5. Doi:10.1128/jmbe.v12i2.324.
Plate Technique.
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