Passive permeabilities of the cell wall and protoplast of Bacillus megaterium strain KM were characterized by use of 50 hydrophilic probing molecules (tritiated water, sugars, dextrans, glycols, and polyglycols) which varied widely in size. Weight per cent uptake values (RW) were measured at diffusional equilibrium under conditions that negated the influences of adsorption or active transport. Plots of Rw for intact cells as a function of number-average molecular weight (Ma) or Einstein-Stokes hydrodynamic radius (iES) of the solutes showed three phases: a protoplast uptake phase with a polydisperse exclusion threshold of M1, = 0.6 x 103 to 1.1 x 103, tES = 0.6 to 1.1 nm; a cell wall uptake phase with a polydisperse exclusion threshold of MR = 0.7 x 105 to 1.2 x 105, rES 8.3 nm; and a total exclusion phase. Isolated cell walls showed only the latter two phases. However, it became evident that the cell wall selectively passed only the smallest molecules in a heterodisperse polymer sample. When the molecular-weight distributions of polyglycol samples (M1, = 1,000, 1,450, and 3,350) were determined by analytical gel chromatography before and after uptake by intact cells or isolated cell walls, a quasi-monodisperse exclusion threshold was obtained corresponding to M" = 1,200, rEs = 1.1 nm. The permeability of isolated protoplasts was assessed by the relative ability of solutes to effect osmotic stabilization. An indefinite exclusion threshold, evident even with monodisperse sugars, was attributed to lengthwise orientation of the penetrating rod-shaped molecules. Altogether, the best estimate of the limiting equivalent porosity of the protoplast was 0.4 to 0.6 nm in radius and of the cell wall, 1.1 nm.

The limiting sizes of molecules that can permeate the intact cell wall and protoplast membrane of Saccharomyces cerevisiae were determined from the inflection points in a triphasic pattern of passive equilibrium uptake values obtained with a series of inert probing molecules varying in molecular size. In the phase identified with the yeast protoplast, the uptake-exclusion threshold corresponded to a monodisperse ethylene glycol of molecular weight = 110 and Einstein-Stokes hydrodynamic radius (rEs) = 0.42 nm. In the cell wall phase, the threshold corresponded to a polydisperse polyethylene glycol of number-average molecular weight (Mn) = 620 and average radius (rEs) = 0.81 nm. The third phase corresponded to complete exclusion of larger molecules. The assessment of cell wall porosity was confirmed by use of a second method involving analytical gel chromatographic analyses of the molecular weight distribution for a single polydisperse polyglycol before and after uptake by the cells, which indicated a quasi-monodisperse threshold for the cell wall of Mn = 760 and rEs = 0.89 nm. The results were reconciled with two situations in which much larger protein molecules previously have been reported able to penetrate the yeast cell wall.

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The threshold surface porosity in the dormant spore of Bacillus cereus strain T was assessed by measuring passive permeabilities to a series of polydisperse polyethylene glycol samples which increased in average molecular size. The apparent exclusion threshold at diffusional equilibrium corresponded to a polymer of number-average molecular weight (MU) = 150,000 and equivalent hydrodynamic radius (PEs) = 16 nm, which confirmed a previous report. However, analytical gel chromatography before and after uptake by the spores revealed that only the low molecular weight fractions in a polymer sample distribution were taken up. From graphical analyses of the changes in molecular weight distributions, a quasi-monodisperse exclusion threshold was determined corresponding to MU = 8,000 and rES = 3.2 nm. Thus, the equivalent porosity in the limiting outer integument appeared much more restrictive than heretofore shown for spores, although still more open than the monodisperse equivalent for the cell wall of vegetative bacilli.

The thin-sectioned spore of Bacillus thuringiensis resembles that of Bacillus cereus in fine structure. Planar inclusions occur between the exosporium and spore coat and are structured differently from the parasporal crystal outside the exosporium.