Edward D. DeLamater

A Staining and Dehydrating Procedure for the handling of Microörganisms

A new staining and dehydrating procedure for microorganisms is presented. It is based on the observation of Atwood and Orinstein (1949) that azure A or thionin, in the presence of SO2, stains chromosomes intensely and apparently selectively. The staining process is as follows: Cover-slip smears are hydrolyzed in 1 N HCl at 60°C. for the previously ascertained optimal hydrolysis time. They are then briefly rinsed in distilled water and transferred to either of the two following stains, (1) 0.25% thionin (10 ml.) to which thionyl chloride (1 drop), as a source of SO2, is added; (2) 0.25% azure A (10 ml.) to which thionyl chloride (2 drops), as a source of SO2, is added. Neither of these stains decolorizes, i.e., forms a leuco base, in the presence of SO2. Staining is allowed to proceed for a minimum of two hours.Following staining, the preparations are rinsed in distilled water, drained on filter paper, and immediately immersed in absolute alcohol which has been previously chilled to about -50°C. by packing...

A Non-Vacuum Freezing-Dehydrating Technic for Histology, Autoradiography and Microbial Cytology

A method for the freezing-dehydration of tissue and microbial cells is described which circumvents the need for high vacuum for the purpose of removing water from the cells by direct vaporization of ice. The present method utilizes the dissolution of ice from frozen tissue and cells by immersion in chemical dehydrating agents which remain liquid at low temperatures. Choice of the particular agent depends upon the desired result. Glycols are recommended if fixation is to be avoided, or water-soluble isotopes retained in the cells; alcohols or acetone are used if fixation is not undesirable. Specific applications of the method are given for use with tissues and with microbial cells. It has been demonstrated that radioactive phosphorus (P32) is not leached out of the tissues during the procedure.

The Squirrel as a New Host to a Ringworm Fungus

SUMMARYThe squirrel is described for the first time as host to an already well known ringworm fungus, T. mentagrophytes (gypseum) (Robin-Sabouraud).

Eidamella Spinosa (Matruchot and Dassonville) Refound

Matruchot and Dassonville (11) published their account of Eidamella spinosa in 1901 for the first and only time that this organism has been reported. In the spring of 1936 Dr. N. F. Conant of the Department of Bacteriology, Duke Medical College, gave to the writer a culture of an obviously gymnoascaceous fungus, which he had cultured from a fingernail while working at the Beth Israel Hospital in Boston in 1935. The fungus was at that time tentatively identified as Eidam1ella spinosa (Mat. & Dass.). Since then the organism has been extensively studied in this laboratory and compared with the very lucid description of the two original authors and with other members of the family Gymnoascaceae. Cultures of the original strain have not been available, but there can be no doubt of its identity with the fungus described as causing a skin condition in a dog in France in 1901, although certain minute differences are noticeable, as will be shown below. These are, however, taken to be within the limits of variation of the form, or as due to differences in observation. Matruchot and Dassonville considered Eidamella spinosa to be a true dermatophyte, although they were unable to reproduce typical lesions by inoculation. They did, however, isolate it more than once from the original lesion. The writer has also been unable to produce typical ringworm lesions with the present cultures, even though he has attempted to inoculate both himself and guinea pigs. The fact, however, remains that this fungus, the two times that it has been found, has occurred in definitely pathological conditions. This fact along with the knowledge that to experimentally induce infection with ringworm fungi is very difficult, can be taken as highly suggestive, though by no means final. The

A cytological and chemical analysis of the bacterial nucleus. I. A method for the isolation of nuclei from Bacillus megaterium and the cytology of the isolated structures.

Abstract The clearing action of sodium glycocholate on cells of B. megaterium is described. This bile salt also renders the organism more fragile and amenable to rupture by sonication or mechanical (Mickle) disruption, permitting the liberation and subsequent isolation of the nuclei and chromosomes. These have been studied by means of routine cytologic and cytochemical procedures and by phase contrast microscopy. The results indicate that the bacterial nucleus is a complex structure containing chromosomes, granules suggesting centrioles, and a spindle-like element. The stages observed suggest a mitotic cycle, and conform to those previously described by other means in intact cells. The significance of the findings is discussed.

A cytological and chemical analysis of the bacterial nucleus. II. A study of the cytochemical changes involved during the isolation of bacterial nuclear material by means of bile salts and high-frequency sound waves.

Abstract A method for the isolation of bacterial nuclei has been described. The use of sodium glycocholate to clear the cells, makes them more susceptible to breakup by sonic waves. By using filtration and differential centrifugation, it is possible to isolate an intact nuclear structure. The chemical aspects of the isolation procedure are discussed, and it is believed that the methods used do not cause a depolymerization of the nuclear material, and that by carefully controlling the process, there are minimal losses of vital components from the cell. The isolated structures take up recognized nuclear stains, and are identical to those seen in whole cells.

A CHEMICALLY SPECIFIC DOUBLE-STAINING TECHNIQUE FOR DESOXYRIBOSENUCLEIC ACID AND OTHER ALDEHYDE-YIELDING OR ALDEHYDE-CONTAINING SUBSTANCES

A chemically specific double staining procedure has been devised for Chlamydomonas, which depends upon the staining of the phosphoric acid moiety of the chromosomal DNA in hydrochloric acid hydrolyzed cells, with the subsequent transfer of the basic dye (azure A) to the aldehyde radicals of the DNA in the presence of sulfurous acid. Subsequent treatment with Schiff reagent stains the primary zygote membrane magenta by combining with its free aldehyde groups. The method as outlined is simple and useful with potentials for application to other types of cells.

Studies on the cytochemistry of alkaline phosphatase in various bacteria.

Abstract The Gomori staining reaction for alkaline phosphatase was carried out in several bacteria. Fixation with acetone did not interfere with the staining reaction, whereas pretreatment with acid citrate buffer proved inhibitory to the enzyme. The optimum pH zone of the enzyme in B. subtilis (pH 8.8–9.2) was found to be close to that at which the staining reaction was carried out (pH 9.4). The intensity of staining was proportional to the concentration of calcium in the incubating mixture. No concomitant change in localization was found. The addition of manganese to the culture medium significantly increased the staining intensity in ten species. The localization of the stain in seven species was in cytoplasmic granules which appeared to correspond to those described as mitochondria by previous authors. Staining of cell walls and of the entire cytoplasm was also found.

[Non-occurrence of spirochetes in cerebrospinal fluid of patients with multiple sclerosis].

Summary Spinal fluids from 32 patients not having multiple sclerosis, 22 patients having multiple sclerosis, and 3 patients having probable multiple sclerosis, were cultured according to the method of Ichelson except that the distilled water used was made in our laboratories. All cultures, carried a minimum of 18 months, were negative for spirochetes.