Filterable Substance Research Articles

Production of a bacteriolysin by a hemolytic Escherichia coli strain.

Nonhemolytic Escherichia coli were outnumbered by hemolytic E. coli within 24 h after being inoculated in a mixed culture at an initial ratio of 200 nonhemolytic to 1 hemolytic organism. The hemolytic strain was found to produce a cell-free, filterable substance which causes lysis of nonhemolytic E. coli B when grown in liquid cultures but not when grown on agar plates. The bacteriolysin is inactivated by boiling, by freezing and thawing, and by incubation with trypsin. The inability to inhibit growth on an agar plate, dependence on cell concentration for its effect, lysis of the sensitive cells, and appearance of phage particles in the cell lysates suggest that this substance is not like colicins or microcins previously described. After lysis of E. coli B, bacteriophage particles were visible in transmission electron micrographs of material pelleted by ultracentrifugation. However, no bacteriophage were observed in pellets from the bacteriolysin-containing supernatants before lysis of E. coli B. Failure to find bacteriophage in these preparations, and the fact that some bacteriolysin activity remains in the supernatant solution after centrifugation at 150,000 X g for 6.5 h, indicate that the bacteriolysin is not itself a bacteriophage. Exposure of E. coli B to UV light and mitomycin C did not induce production of a temperate phage. The properties of this system, in which a cell-free substance produced by one strain of bacteria causes lysis of another strain, appear to differ from those of the various types of bacteriocins and bacteriophages described to date.

Effects of floods on nutrient and metal concentrations in a coastal plain stream

Water quality in Creeping Swamp, a coastal plain stream in North Carolina, was monitored during two floods in 1976. The concentrations of most of the filterable substances studied remained relatively constant, particularly at the upstream station, CP‐20, which drained the least disturbed portion of the watershed. Concentrations of NO3 and dissolved organic carbon increased, and concentrations of Cl decreased during the floods at CP‐20. At the downstream station, CP‐10, which received effluents from a hog farm, concentrations of the filterable forms of N, P, K, and Fe increased markedly during floods. Indications were that increases in concentration of filterable substances during floods may be a better indicator of watershed disturbance than ambient concentrations. Despite the dominance of overland flow in the hydrologic regime and the abundance of organic material in the stream channel and floodplain, concentration patterns of the filterable constituents of streamflow during floods in Creeping Swamp are similar to the patterns in streams draining upland watersheds, where contact between runoff waters and the inorganic strata of watershed soils is likely to be much greater. The concentrations of particulate forms of phosphorus, silicon, and most of the metals studied increased during the rising limb of the hydrograph, peaked prior to peak streamflow, and decreased thereafter. The contribution of floods to total annual flux of P, Fe, Si, and Al in Creeping Swamp is especially great because of large increases in concentrations of the particulate forms of these elements as streamflow increases.

Growth of blue-green algae in the Manukau (New Zealand) oxidation ponds—II. Experimental studies on algal interaction

An experimental approach was made to the interaction between the blue-green algae Anahaena and Microcystis and the normal oxidation pond dominant Chlorella. Methods employed were dialysis tubing, mixed culture with assay by in vivo absorbance, and by interaction tubes in which the algae were separated by a filter membrane. No dialysable inhibitor material in the oxidation ponds was detected with in situ experiments. In mixed culture Chlorella inhibits Anabaena only when starting concentration of Chlorella is high and that of Anabaena is low. On the other hand at all concentrations Anabaena quickly dominated Chlorella. These results were confirmed in interaction tubes where the algae were separated and the results extended to show Microcystis also inhibits Chlorella growth. Filtrate experiments indicate that both Microcystis and Anabaena produce a filterable substance capable of suppressing Chlorella growth but that Chlorella does not exert any influence on blue-green algae by way of soluble materials.

Gas contaminant inhibition of mammary gland differentiation in vitro.

A gas mixture of 95 percent 02/5 percent CO2 from several commercial sources was contaminated with a filterable substance (s) that inhibited mammary gland morphologic differentiation in vitro.

Demonstration in Tissue Culture of Lymphocyte-Mediated Immunity to Tuberculosis

We have shown that splenic lymphocytes from mice immunized with viable attenuated mycobacterial cells (H37Ra) or ribonucleic acid prepared from these cells will bring about inhibition of multiplication of virulent tubercle bacilli (H37Rv) within normal mouse peritoneal macrophages in tissue culture. Apparently these lymphocytes, when stimulated with viable virulent tubercle bacilli, elaborate a filterable substance(s) which is responsible for this intracellular growth inhibitory effect.

The Experimental Transmission of a Virus Causing Hypergammaglobulinemia in Mink: Sources and Modes of Infection

Hypergammaglobulinemia (Aleutian disease or AD) of mink has been produced by the inoculation of afifected tissue suspensions and cell-free filtrates (Henson et al, 1962b, 1963a; Karstad and Pridem, 1962; Russell et al, 1962; Trautwein et al, 1962; P. G. White, personal communication). The filterable substance is particulate, since it can nearly all be removed from aqueous suspension by centrifugation at 95,000 rcf for 1 hour (Henson et al, 1963a). The causal agent (which we will provisionally call a virus) in the form of a crudely ground tissue suspension is resistant to 0.3(^) formalin for 2 weeks, but not for 40 weeks, at 5 C (Henson et al, 1962b). The present paper confirms the demonstration of the virus in the urine (Kenyon et al, 1963) and forms part of an investigation of the natural history of the disease among mink.

Biochemical and Genetical Studies on Variants of the Mouse Strain L Cells in Cell Culture

The effects of various chemical and physical agents (mitomycin C, 8-azaguanine and UV-irradiation) on the cell multiplication and DNA, RNA and protein syntheses of original mouse fibroblastic strain L cells in vitro were determined.The variant cells resistant to these agents were isolated from the original L cells and some detailed properties of these variants were described. Mitomycin C-resistant cells (LMit cells), which were obtained by several successive treatments of mitomycin C in gradual higher levels showed smaller size in appearance than the original L cells and contained less amounts of protein, although amounts of DNA and RNA contained in LMit cells were not different from those in original L cells. 8-azaguanine-resistant cells (L8-AZ cells) showed no marked difference in the cell morphology, cell multiplication rate and contents of protein, DNA and RNA, from those in the original L cells.Properties of UV-resistant cells (LUV cells) were almost comparable with those of LMit cells in every point of view.The appearance of these resistant cells seems to depend on adaptation to or selection by these respective agents rather than on mutagenic actions of these agents.The cross-resistant phenomenon was found between LMit cells and LUV cells, whereas it was not found between LMit and L8-AZ and between LUV and L8-AZ. These facts suggest that effects of mitomycin C may have something common to those of UV-irradiation, which differ from those of 8-azaguanine.It was found that the multiplication of LMit cells and of LUV cells was accelerated by filterable substances produced from L cells or from L8-AZ cells, whereas the multiplication of L cells and of L8-AZ cells was not affected by filterable substances from cells of any variants.

FUNDAMENTAL STUDIES ON A SALTANT OF HELMINTHOSPORIUM SATIVUM

A saltant of H. sativum disappeared from six different soils in which it was cultured in the laboratory within four weeks, whereas the indigenous flora persisted for nine weeks. During this nine week period numbers of bacteria showed no obvious trend, but numbers of fungi decreased. When cultured in sterilized portions of the above soils, the saltant showed a downward trend in numbers in the nine week period, a trend that was more obvious in some cultures than in others. Twenty-two of 221 isolates of fungi from soil, grain, and air, 45 of 286 isolates of Streptomyces, and 33 of 193 isolates of other bacteria produced evidence of antagonism to the saltant in spot inoculation studies. Filtrates prepared from two of the fungal isolates suppressed the growth of the saltant, as did also autoclaved portions of the nitrates. On the contrary, filtrates from three cultures of bacteria failed to suppress growth of the saltant, even though cultures of the bacteria did. The same 22 fungal isolates and 25 of the Streptomyces, when introduced separately into sterile soil along with the saltant, reduced the disease rating of barley seedlings below that produced by the saltant alone. The direct-count method of estimating numbers of spores of the saltant in a suspension was found to be reliable. Estimates based on the direct count were significantly greater than those made by the plate method. Estimates based on numbers of the saltant developing on potato dextrose agar containing small amounts of sterile soil were smaller than those made on the same medium without the soil. Even though the soil in the medium changed the pH of the medium, the difference in pH was considered not to be the primary factor in producing the lower estimates. Soil, either natural or heat sterilized, would contain the thermostable filterable substance or substances found to be produced by two species of fungi and probably produced by other species, likewise normally present in soil.

Hemolytic Action of Mouse Mammary Carcinoma Filtrate on Mouse Erythrocytes in vitro.

Although the association of cancer with anemia has long been observed, no satisfactory explanation of the mechanism of this relationship has yet been furnished.1 Experiments reported in this study appear to suggest that, at least in the case of mammary carcinoma of mice, a direct destructive action of a filterable substance contained in the tumor cells, on the erythrocytes of the host, should be taken into consideration. Thus, filtrates prepared from mouse mammary carcinomas were found to destroy mouse erythrocytes in vitro. The following technic was employed to demonstrate this hemolytic property of the tumor extracts:Materials and Methods. Tumor extracts. Ten different mammary carcinomas that developed spontaneously in old C3H females, and 2 transplanted tumors that originated as mammary carcinomas in C3H females and have been carried through successive transplantations in these animals,2 were used as the source of the tumor extracts. A freshly prepared extract from a single tumor was used for each test...

Studies on Cultures and Broth Filtrates of Staphylococci

Abstract I. In previous reports (1), a toxin producing strain of staphylococcus has been described. This strain, “Ha,” produces in 10 day broth cultures a filterable substance that kills rabbits in twenty-four hours or less. The m.l.d. is about 0.1 cc. per kilogram of body weight. Whole cultures kill in a similar fashion. Burnet (2) has studied the lethal properties of similar strains and in particular, one strain, “Wood.” Filtrates of broth cultures of this strain kill rabbits in about five minutes after the intravenous injection of small amounts. A sublethal amount of filtrate, injected intracutaneously, produces an antiserum that neutralizes some, if not all, of the toxic properties of the filtrate. It is the belief of Burnet (2) that intracutaneous immunization produces the most potent antiserum. Further to confirm or disprove the findings of Burnet and to determine the precipitating and protective properties of filtrate Ha antiserums, the following studies were undertaken.

AN EXPERIMENTAL STUDY OF PAROTITIS (MUMPS)

Cats injected into the parotid gland and testicle with a bacterial sterile filtrate of the salivary secretion of children in the active stage of parotitis or mumps can be made to develop a pathological condition having several points of resemblance to the condition present in mumps in human beings. After an incubation stage of from five to eight days definite changes have been noted in the temperature, blood leukocytes, and inoculated organs. The temperature rise begins within twenty-four hours of the inoculations and reaches a maximum in from seven to fourteen days. The febrile rise fluctuates between 1 degrees and 2.5 degrees C. The white blood cells begin to increase on the second day following the inoculation. The first change is a polymorphonuclear leukocytosis which precedes the glandular swellings. This initial rise is followed by a decline, after which the lymphocytes increase. The increase is confined to the small lymphocytes, which increase to from 7 to 10 per cent of their initial number. The inoculated glands become swollen and tender. The swelling and tenderness become apparent from the fifth to the ninth days and persist for a variable period. The parotid changes are less constant or less obvious than are the testicular. The latter are constant and endure from eight to twelve days. The rise of temperature and the leukocytosis precede the glandular swelling, but all the changes reach the maximum at about the same time, after which they decline gradually. What may be regarded as normal conditions are reestablished in four weeks or less. The intraparotid and intratesticular injections of extracts of normal parotid gland and testicles may cause a mild rise of temperature and leukocytosis of brief duration, but swelling and tenderness are absent. The white cells increased are the polymorphonuclears and not the lymphocytes. The intraparotid and intratesticular injections of filtrates of normal saliva may cause a mild rise of temperature of very brief duration, but leukocytosis, swelling, and tenderness do not appear. The histological changes in the parotid gland when present consist chiefly of edema of the interlobular connective tissue with mononuclear interstitial infiltration about the ducts and elsewhere. In cases of long duration the ducts may be dilated. But in some instances the swollen gland while showing congestion and edema in gross showed inconspicuous changes under the microscope. The histological changes in the testicle are of two kinds: inconstant changes of cellular invasion between the tubules and swelling or even multiplication of the interstitial cells, constant ones consisting of degeneration of the epithelium and interference with spermatogenesis, a condition to which we have applied the term "spermatorrhexis." The pathological conditions set up by the filtrate derived from the salivary secretion of cases of acute parotitis are intensified by successive transfers through a small series of cats of the extract and emulsion of the parotid gland and testicle previously inoculated. The pathological changes are also prevented or reduced when the extract or emulsion is previously incubated with a quantity of blood serum obtained from a cat which has survived inoculation. Normal serum, on the other hand, has no such inhibiting effect. The deduction from these experiments is to the effect that the salivary secretion in parotitis or mumps contains a filterable substance capable of setting up a series of definite pathological conditions when inoculated into the testicle and parotid glands of cats. Whether this active material is a microorganism and if so whether it is the specific microbic cause of parotitis or mumps remains to be ascertained.