Density Gradient Centrifugation for the Separation of Sporulating Forms of Bacteria

Abstract

Urografin has been shown to be suitable as a supporting medium for isopycnic density centrifugation of Bacillus megaterium spores. The complete separation of vegetative cells and spores can be achieved through the use of such gradients. Vegetative cells were found to have an average density of 1.135 g per cm3, while the average spore density was 1.305 g per cm3. Under normal sporulating conditions the spores formed consisted of a heterogeneous population with a wide spectrum of densities. These density differences could be correlated with such properties as resistance to thermal death and ability to be heat-activated. Spores formed in the absence of Ca++ are less dense than those obtained under the usual conditions. The pattern of cell distribution obtained on the Urografin gradient can serve as a gauge of the efficiency of sporulation and degree of synchrony of a sporulating culture.