It was found that the raw cotton used for the cotton plugging of test tubes contained numerous growth factors for bacteria and such action was tested by a bioassay. It was thereby found that such factors were contained in approximately 3.5γ/g. as pantothenic acid, approx. 7γ/g. as biotin, approx. 4γ/g. as nicotinamide, approx. 0.15γ/g. as riboflavine, and approx. 0.8-5mγ/g. as vitamin B₁₂. Same tests were carried out on absorbent cotton and glass wool but such activities were not found in them. It is therefore concluded that a strict caution is necessary in using cotton plugging for test tubes in microbiological assay.