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In the field of microbiology, the counting of bacterial colonies is fundamental and mandatory. This is done to estimate the number of bacterial cells in every 1 milliliter or gram of sample. The counting takes a long time and is tedious, so it requires an accurate and fast counting method. The image quality used is very low and contains noise. Therefore, a preprocessing method is needed to reduce the noise. The Perona-Malik filter method is known to be able to remove noise well. However, it is difficult to determine the appropriate gradient threshold parameter ( ) for each different image. To find the appropriate value of , the original Sobel Mask method and Sobel Mask Fractional-Order are used to estimate the value of . The experimental results show the results of noise reduction using PMD with a value of from the original Sobel Mask and Sobel Mask Fractional-Order. The results of the accuracy of determining the value of k with the Sobel Mask Fractional-Order (α=1.0) show higher results based on the F-Measure values for samples 1, 2, and 3 respectively 97%, 98%, and 90%.