Bacterial factors 1. Bacterial culture time The typical morphology and staining of bacteria are in the logarithmic growth phase. Gram staining of bacteria at this time can reflect the true staining of the bacteria. If the bacterial culture time is too long and it becomes old-age bacteria, the stainability will change, and Gram-positive bacteria may be stained as Gram-negative bacteria. 2, the composition of the medium is generally believed that Gram-positive bacteria contain a special nuclear protein magnesium salt and polysaccharide complexes, it is a combination of iodine and crystal violet compound is very strong, not easy to decolorize. However, if there is a lack of nuclear protein magnesium salt in the medium, it will not be able to synthesize the nuclear protein magnesium salt of the bacterial component. The combination of bacteria and iodine and crystal violet is not strong, easy to decolorize, and may cause Gram-positive bacteria to become Gram-negative Negative bacteria. Dyeing agent 1, iodine liquid Iodine as a mordant can increase the affinity of the dye and the dyed material. If the iodine solution is left for a long time, or fails to be irradiated by sunlight, the effect of mordant will be lost, and the combination of crystal violet and bacteria may be weak, and it may be easily decolorized, resulting in the Gram-positive bacteria being stained as Gram-negative bacteria. 2, crystal violet Crystal violet concentration is too high, it will make the color of the dye is not easy to be bleached by alcohol, may make Gram-negative bacteria stained Gram-positive bacteria. 3, alcohol as a decolorizing agent, decolorized alcohol concentration of 95% Gram stain, at this concentration of Gram-positive bacteria stained on the crystal violet can not easily be removed, keep the purple, Gram-negative bacteria stained with crystal violet easy It was taken off and finally it was red. However, when the concentration of alcohol is 70%, its decolorization ability is the strongest and it may cause the crystal violet dyed by the gram-positive bacteria to be removed, so that Gram-positive bacteria can be stained as Gram-negative bacteria.