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This was not the first time that microbiologists experienced problems with agar. A phenomenon called “The Great Plate Count Anomaly” baffled researchers in the early 20th century when they observed that the number of cells seen under a microscope didn’t match the actual number of colonies growing on an agar plate. Investigating this discrepancy, researchers found agar itself to be the culprit: when nutrient broths are heated with agar during boiling, harmful byproducts (hydroperoxide) can form due to the reaction of agar with phosphate minerals contained in the media. Researchers can avoid this by autoclaving agar separately from the nutrient broth, or by reducing the amount of agar used.
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Source: Computational Materials Science, Volume 267