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The structural features observed in freeze-fracprecooled object stage (-150 °C) . After the temperatured bacterial membranes have, in the now ture of the object is raised to -100 °C in vacuo, the numerous reports, generally been interpreted as holders are unlocked . This is carried out by pushing belonging to the external membrane surfaces . the microtome arm against a small protruding part of However, several lines of evidence indicate the the upper holder. Immediately thereafter the fracoccurrence of membrane splitting as contrasted tured surfaces are replicated according to Moor (9) . The replicas are then cleaned and mounted on to cleavage along both surfaces in freeze-fractured Formvar-coated copper grids (Belden Mfg . Co., biological membranes (for a review see Branton Chicago, Ill ., supplied by VECO, Eerbeek, Holland) [1969]) . according to the usual procedure (10) . Because of In earlier observations on the plasma membrane their larger size, yeast cells have been used as markers of Bacillus subtilis we have assumed that fracturing to find the complementary parts of the fractured occurred along both surfaces of the membrane (11, bacteria . 12) . The aim of the present investigation on B . subtilis has been twofold : (a) to verify whether Thin Sectioning of Fractured Bacteria the concept of a unique fracture plane applies to its plasma membrane ; and (b) to attempt to locate Bacteria contained in agar blocks (16) were fixed in osmium tetroxide and posttreated with uranyl the fracture plane in or at the membrane . An acetate (12, 16) . An agar block was placed on a answer to the first question has been sought by scratched copper disc and, after freezing in liquid making complementary freeze-fracture replicas as Freon 22 (E . I . du Pont de Nemours & Co., Inc ., has been done recently for other membranes (5, Wilmington, Del.), was affixed on the object stage 17, 21, 22) . The location of the fracture plane precooled to -150 °C. A cut was made with the has been investigated by making thin sections of microtome knife at -100 °C. The fractured agar freeze-fractured cells (3, 4, 8) . block was then thawed and dehydrated in a graded series of acetone (12, 16) . A flat embedding was done

UNIQUENESS AND LOCATION OF THE FRACTURE PLANE IN THE PLASMA MEMBRANE OF BACILLUS SUBTILIS