1.Inoculating holding medium kept at refrigeration temperature at about 4-5 C as to increase the shelf life of media. 2.Ferrous sulfate is added as it is Indicator of H2S formation which means the black color of ferrous sulfide is seen. 3.Vitamin K1 -support the growth of fastidious anaerobes, especially Bacteroides, Prevotella, and Porphyromonas when incubated anaerobically,it is used for the isolation of strict anaerobes from clinical specimens and used for susceptibility testing of anaerobes with the E test method. Solution 1.Inoculating holding medium kept at refrigeration temperature at about 4-5 C as to increase the shelf life of media. 2.Ferrous sulfate is added as it is Indicator of H2S formation which means the black color of ferrous sulfide is seen. 3.Vitamin K1 -support the growth of fastidious anaerobes, especially Bacteroides, Prevotella, and Porphyromonas when incubated anaerobically,it is used for the isolation of strict anaerobes from clinical specimens and used for susceptibility testing of anaerobes with the E test method..