Georges Knaysi
1957 Journal of Bacteriology  
In spite of the tremendous volume of literature on the bacterial nucleus, there is still no agreement among bacterial cytologists on what is a sure criterion of its identity. Witness the present status of the granules of Mycobacterium, variously considered as mitochondria (Mudd et al., 1951) , nuclei (Knaysi et al., 1950; Knaysi, 1952 ), neither (Glauert and Brieger, 1955), or artifacts (Bisset, 1955. The present investigation attempts to find the reasons for these differences and demonstrates
more » ... s and demonstrates that these granules, regardless of their size or position, are real and are the cell nuclei. MATERIALS AND METHODS Most of the observations reported in this paper were made on Mycobacterium thamnopheos, although other organisms, particularly Staphylococcus flavo-cyaneus, were employed in certain phases of the investigation. The internal structure of both organisms is clearly revealed by simple staining with an acidified solution of a basic dye, such as methylene blue or thionine, regardless of the medium on which they are grown (Knaysi, 1951) . Consequently, observations on the structure and behavior of intracellular granules in both organisms were made primarily on cells stained by methods A or C previously described (Knaysi, 1955a). In method A the cells are stained with an acidified solution of methylene blue (pH 3.1-3.2), and in method C they are stained first with a slightly acidified solution of thionine, then with a solution of methylene blue buffered with a mixture of K acid phthalate and HCl using the same pH as that in method A. In all reported cases (with the exception of figure 21), the organisms were grown at various temperatures on collodion films supported by various agar media. The media most commonly used were MITG/2: MI = meat infusion, T = 1 1 The numbers and letters in parentheses after the figure number identify the negative in the author's library. Any figure without a scale has the same magnification as figure 1. per cent tryptone, and G = 1 per cent glucose; and GA/5: G = 1 per cent glucose and A = 1 per cent Na acetate; the denominators indicate the dilution. Comparison of the same cells and structures after various consecutive treatments was made according to the procedure previously described (Knaysi, 1955a (Knaysi, , 1957 : for the most part between cells and structures stained by method A or C and by the HCl-Giemsa technique; or between cells and structures in microcultures grown on a collodion film supported by an agar medium containing 0.005 per cent neotetrazolium and the same cells and structures after staining by methods A, C, or HCl-Giemsa. Sometimes the acidfast or Gram reaction was included. Emphasis was placed on comparison of these methods to that of the HCl-Giemsa method not only to satisfy those who believe the significance of this technique, but also to test further the cytological value of this procedure. Fixation with formol vapors was carried out by inverting the air-dry microculture, for a definite time, over the well of a culture slide containing a drop of commercial formalin. To fix the cells in the wet state with OS04 vapor, the floated microculture was picked up with a cover glass, thoroughly drained by placing it in a petri dish at an angle and absorbing the free water which collects at the border of the collodion film with blotting paper. The cover glass is then quickly inverted over the well of a culture slide containing a 0.25 per cent solution of OSO4. After exposure for a definite time, the cover glass is removed and allowed to dry before further treatment, or sometimes immediately inverted over a drop of the staining solution, sealed, and examined. For making a gram stain we used a 0.1 per cent solution of crystal violet for 1 min, rinsed the crystal violet with a solution containing 0.1 g of I2 and 0.2 g of KI per 100 ml, and mordanted with a fresh portion of this solution for 1 min. Differentiation was made with 95 per cent ethanol for 20 sec but was not followed by a contrast stain. The reaction of acid fastness was carried out as 12 on May 8, 2020 by guest
doi:10.1128/jb.74.1.12-21.1957 fatcat:3c5ud7hmavdmvkhn6juzrezlay