Aerobic respiratory activity of the yeastlike phase of Sporotrichum schenckii as affected by high hydrogen ion concentrations

Summary Six strains ofSporotrichum schenckii were studied in regard to the effect of hydrogen ion concentration on certain aspects of the aerobic respiratory activity of the yeastlike phase.Optimal oxygen uptake of endogenous respiration occurred at pH 2.0, although no effects were observed on the oxygen-carbon dioxide exchange ratio (respiratory quotient) at this pH value when compared to the R.Q. obtained at pH 7.0. Endogenous respiratory activity at both pH 2 and 7 was markedly sensitive to the presence of certain respiratory inhibitors.Optimal respiratory activity using glucose as substrate occurred at pH 7.0 .On the other hand, oxidation of pyruvate as substrate proceeded at significant rates only at pH values below pH 4.0. With decreasing hydrogen ion concentration, accumulation of this organic acid occurred when glucose was employed as substrate. With the exception of acetate, none of the organic acid respiratory intermediates were found to stimulate respiration.The results reported herein suggest that the respiratory activity of the yeastlike phase ofS. schenckii differs in several respects from that observed for the yeastlike phases ofHistoplasma capsulatum andBlastomyces dermatitidis.From the Research Laboratory, Veterans Administration Hospital, Kansas City, Missouri, and The Department of Microbiology, Kansas University Medical Center, Kansas City, Kansas.Supported in part by USPHS Grant AI 03485.     

Rapid Schedules for Koh Clearing and Alizarin Red S Staining of Fetal Rat Bone

Various schedules for staining fetal rat skeleton with alizarin red S were tested to determine a procedure that would produce a completely cleared and well-stained specimen in a short period of time. A 2 day procedure is presented which can produce specimens that are satisfactory but not completely transparent. A 7 day procedure produces cleared and stained specimens which can be well visualized with a dissecting microscope (30×). Fetal rats of 21 days gestation were fixed in 10% formalin for at least 1 wk. The specimens were skinned and eviscerated and then dehydrated in 2 changes of acetone for 12 hr (8 ml per gram body weight). The specimens were then placed in 1% KOM-alizarin red S (6 mg/liter) or 3 days, followed by 10% KOH-alizarin red S for 3 days. Finally, the specimens were placed in a mixture of benzyl alcohol, ethanol, and glycerol (1:2:2) (4 ml per gram body weight) for 12 hr, and then transferred to pure glycerol for storage.     

Pseudoisocyanin Staining of Insulin and Specificity of Empirical Islet Cell Stains

Two closely related pseudoisocyanins, N,N'-diethyl-6,6'-dichlorpseudoisocyanin chloride and N, N'-diethylpseudoisocyanin chloride, were tested for their metachromatic staining behavior with oxidized insulin. N,N'-diethyl-6,6-dichlorpseudoisocyanin chloride gave nonspecific metachromasia with collagen, mucus, and mast cells of adult tissues; almost all tissues of rat embryos exhibited nonspecific staining. Nonspecific reactions were rarely observed in adult or fetal tissues with the extremely labile metachromasia of N, N'-diethylpseudoiso-cyanin chloride. When oxidation time and temperatures are carefully controlled, this reagent apears to be highly specific for insulin-containing cells and can be used as a selective stain for beta cells. Paraffin sections of formalin fixed material were oxidized 45 sec at 28-29 C in freshly prepared acidified permanganic (2.5% KMnO₄, 1; 5% H₂SO₄, 1; distilled water, 7—parts by volume), decolorized 30 sec in 5% oxalic acid, and washed 5 min in running tap water. After rinsing in 2 changes of distilled water, sections were stained 20 min in a 36 mg/100 ml aqueous solution of N, N'-diethylpseudoisocyanin chloride. Sections were then washed in running tap water until the albumen adhesive was decolorized, and mounted in Karo syrup diluted with an equal amount of distilled water. The insulin-containing cells are stained light to dark purple; all other tissue components, various shades of red. N, N'-diethylpseudoisocyanin chloride was used as a reference for evaluating the specificity of 5 commonly used empirical methods for demonstrating alpha and beta cells in pancreatic islets. Cells exhibiting pseudo isocyanin metachromasia were stained selectively by aldehyde-fuchsin, Heidenhain's azan, and chrome-hematoxylin. Aldehyde-Iuchsin was the only empirical stain tested which gave results comparable to pseudoisocyanin for clarity and definition of beta cells. After oxidation in acidified permanganate, azocarmine and phosphotungstic acid-hematoxylin differentially stained alpha cells; cells demonstrated by these two methods did not exhibit pseudoisocyanin metachromasia. This histochemical procedure can precede empirical methods which require preliminary oxidation in acidified permanganate or it can follow empirical methods which do not extract the insulin nor alter its intramolecular disulfide bonds.     

In Vitro Studies on the Antiviral Activity of 1,3-Bis(2-Chloroethyl)-1-Nitrosourea

Chemotherapy experiments carried out in vitro demonstrated that 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was active against lymphocytic choriomeningitis virus and had an equivocal antiviral effect on Semliki Forest, herpes simplex, and vaccinia viruses. No antiviral effect was observed with BCNU against western equine encephalomyelitis, polio, and parainfluenza (HA-1) viruses. Activity of the drug was determined by inhibition of viral-induced cytopathogenic effect in KB or chick embryo cells and by reduction of virus titer in cell culture supernatant fluid. Maximal activity against the viruses was observed when drug and virus were incubated together for 30 min prior to addition to cells; essentially no activity could be demonstrated if BCNU and virus were added to cells with no prior incubation.     

Production of a Pyrrole Antibiotic by a Marine Bacterium

Evidence is presented for the isolation and identification of bacteria able to synthesize an unusual antibiotic containing five bromine atoms per molecule. The identification and taxonomic position of these bacteria was made by use of a computer in conjunction with traditional methods. These microorganisms and closely related strains have been isolated on various occasions from tropical water in the vicinity of Puerto Rico. One bacterium, a pseudomonad, has been given the name Pseudomonas bromoutilis because of its distinctive capability. The antibiotic has been extracted, purified, and obtained in crystal form, and its structure has been determined. Although clinical tests of its properties were not encouraging, it may be of significant value and interest from an ecological standpoint.     

Preservation of Serratia marcescens by High-Vacuum Lyophilization

Water-washed Serratia marcescens (ATCC strain 14041) cells were lyophilized in an all-glass system capable of evacuation to pressures of less than 5 x 10(-6) torr. Lyophilization at the lowest pressures resulted in 50 to 65% survival for unstabilized washed organisms compared with 10 to 20% when the cells were lyophilized at pressures of about 2.5 x 10(-2) torr. At the latter pressures, 45 to 65% survival was obtained when NaCl or Naylor-Smith stabilizer was added to the cell suspensions before lyophilization. However, the stabilizers failed to increase significantly the levels of survival compared with water suspension when cells were lyophilized at pressures less than 10(-5) torr. The high survival rates obtained by the high-vacuum technique may be attributed to the reduction of traces of molecular oxygen which has been reported to be destructive of the dried bacteria. Survival of unstabilized dried S. marcescens after 1-day storage increased markedly with decreasing sealing pressure. Under the highest vacuum attained, survival of the dried bacteria was not impaired by storage for up to 1 month at Dry Ice temperatures; at higher temperatures, viability losses occurred. Exposure of the dried unstabilized bacteria to dry air resulted in rapid viability loss. The inactivation could be stopped almost immediately by evacuation to pressures of less than 10(-5) torr, but the evacuation failed to reverse the viability losses that occurred during exposure.     

Psychological Implications of Cystic Fibrosis

A psychological and psychiatric study of 11 children with cystic fibrosis revealed major psychological problems in all of them. Among the parents of the majority of these children, marked psychopathology and gross marital discord were noted. Popular literature concerning cystic fibrosis had a negative effect on the child''s attitude toward the disease. Virtually all of these patients showed a preoccupation with death. In this study, the necessity of psychiatric consultation as an integral part of current intensive treatment programs in cystic fibrosis clinics was demonstrated.