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The 1953 paper The isolation of protoplasts from Bacillus megaterium by controlled treatment with lysozyme by Claes Weibull (pp. 688–690, Figures 2–3) tested Bacillus megaterium strain KM. Apparatus included a Klett-Summerson colorimeter with a red filter and a Spencer no. 8 phase-contrast microscope. Lysozyme in phosphate buffer produced empty residual bodies; adding $0.2\mathrm{M}$ sucrose preserved granular spheres. Diluting their surroundings caused rapid osmotic lysis.

The same study compared untreated, boiled, lysozyme-exposed and sucrose-protected samples. Filled, stoppered tubes reduced damage from air exposure. After centrifugation, a Beckman DU spectrophotometer measured ultraviolet wave absorption of the separated liquid. Protected samples resembled untreated controls in releasing little ultraviolet-absorbing material; boiled and unprotected samples released much more. Later-growth cultures reduced spontaneous breakdown as a confounder.^[ref]

Together, the shape and release readouts support removal of mechanical protection while a permeability barrier persists. They do not establish unchanged selective permeability to every solute, long-term viability, or universal susceptibility.

An instructive extension of this experimental logic is a matched comparison: lysozyme present or absent, crossed with osmotic support present or absent. Keep starting material, buffer, observation interval and handling comparable; include a blank to distinguish optical effects of reagents from changes in the biological cells. Then dilute already formed protoplasts as a separate intervention. This separates access and wall removal from failure after removal. These are proposed controls for interpreting the mechanism, not additional reported results from the historical paper. A fall in optical signal alone cannot establish rupture: shape changes also alter scattering of light.

Awarded to Frits Zernike for the phase-contrast microscopy method, particularly the phase-contrast microscope.^[ref]

Phase-contrast microscopy converts specimen-dependent phase differences into visible light intensity differences. It can reveal structures in living, unstained biological cells, allowing their changes to be followed without first making an absorbing preparation.^[ref] The key question is how an optical microscope can read a phase when an ordinary detector records light intensity.

Consider a transparent specimen with complex amplitude transmission $t(x,y)=e^{i\varphi (x,y)}$. In ideal in-focus coherent imaging with all relevant spatial frequencies transmitted, its image has $I=|t{|}^2=1$: varying $\varphi$ alone produces no brightness detail. This is a particular model, not a claim that all transparent specimens are invisible. Diffraction, defocus, absorption and a finite optical pupil can already change a real image.

A phase-contrast microscope images an illuminating annulus onto a phase plate ring near the objective's rear focal plane. The aligned ring attenuates and shifts direct light relative to most diffracted light; their interference changes image brightness.^[ref]

The method is beautiful because the bright direct beam becomes its own reference: a small specimen-dependent complex amplitude can interfere with it and change the image at first order. Merely magnifying an invisible phase pattern would leave $|t{|}^2$ unchanged.