The persistence of the distemper virus in continuous human cells]

Two cultures chronically infected with distemper virus (HEP-2 and L-41) were obtained. The cultures produced a small-plaque cell-associated virus and a virus-specific antigen which was demonstrated by the fluorescence antibody technique in 40%-60% of the cells. The chronically infected cells produced interferon as judged by their resistance to superinfections with heterologous viruses. The virus-carrier state was characterized by temperature sensitivity.     

Stimulation of thymus cells by the calcium ionophore A23187 in the presence of LPS and 2-ME: on the mode of action of A23187 and 2-ME

The stimulation of murine spleen and thymus cells by the calcium ionophore A23187 was studied using a serum-free culture technique. In cultures of spleen cells, rabbit IgG was capable of replacing A23187 in bringing about the stimulation of cells. Low concentrations of A23187 were inhibitory to proliferating B lymphocytes. In cultures of thymus cells a stimulating activity of A23187 was observed only if LPS was present in the medium.     

Subchorionic fibrin cultures for bacteriologic study of the placenta

Previous bacteriologic studies of the placenta have been hampered by a high rate of contamination of vaginal flora. In the present study, cultures of the subchorionic fibrin layer of the placenta were compared to conventional swab-cultures of the surface of the fetal membranes for recovery of aerobic and anaerobic bacteria. Parallel cultures of membrane surface and subchorionic fibrin (SCF) were done in the placentas of 33 deliveries suspected clinically of being complicated by infection (CD). Placentas from 46 uncomplicated deliveries were similarly cultured to serve as controls. SCF cultures were positive in 11 of 33 CD placentas and in only 1 of 46 controls (P less than 0.001). SCF cultures showed contaminating vaginal flora in 2 of 79 while surface cultures showed vaginal contamination in 16 of 79 (P less than 0.01). Recovery of pathogens was similar by the two methods: 10 of 33 and 14 of 33 respectively. One hundred ninety-one additional CD placentas were cultured by the SCF technique. Forty-nine of the 224 SCF cultures yielded pathogens with a predominance of group B hemolytic streptococci, anaerobic gram positive cocci and anaerobic gram negative rods (81 total isolates). Escherichia coli was isolated in 5 cases. Cultures showing exclusively contaminating vaginal flora were obtained from only 3 of the 224 placentas. Subchorionic fibrin cultures combine technical simplicity, low rate of contamination and excellent recovery of pathogens. The bacterial types found by this method are the predominant species that cause endometritis, pelvic infections, and neonatal septicemia. Subchorionic fibrin culture is a useful technique for the bacteriologic diagnosis of infection in the placenta.     

Egtved virus: Demonstration of virus antigen by the fluorescent antibody technique in tissues of rainbow trout affected by viral haemorrhagic septicaemia and in cell cultures infected with Egtved virus

Summary The use of a direct fluorescent antibody technique for the demonstration of Egtved virus in tissue sections from diseased trout and in infected cell cultures is described.Specific fluorescence indicating the presence of Egtved virus could be demonstrated in kidney, spleen, liver, and heart from fish attacked by VHS.In cell cultures Egtved virus could be demonstrated eight hours after infection as a granular, mainly cytoplasmic fluorescence resembling the one described for vesicular stomatitis virus.Another trout pathogen, IPN virus, could be demonstrated in cell cultures by means of a similar technique. No cross-reaction between VHS and IPN conjugates was observed.     

Comparison of the capillary tube and explant methods for the in vitro assay of tuberculin

Purified antigens prepared from mycobacteria were tested for nonspecific toxicity and tuberculin activity by two in vitro methods. One technique utilized measurements of macrophage migration in 4-day-old cultures of spleen from normal and tuberculin-sensitive rabbits. The other method was a modification of the capillary tube technique. To obtain enough peritoneal macrophages for good quantitation, changes were made in the method of harvesting cells, and in some instances exudates from two or more Wright strain no. 13 guinea pigs were combined. The capillary tube method was as sensitive as the explant method for detecting nonspecific toxicity. Each tuberculin assay included a group of control cultures of cells from sensitive animals, test groups containing two widely spaced concentrations of a standard tuberculin, PPD-S, and one or more concentration of the tuberculin to be tested. Macrophages from tuberculin-sensitive animals were regularly inhibited by 0.25 μg of PPD-S per ml with both in vitro methods. The potency of the test tuberculins relative to that of PPD-S was somewhat greater in capillary tube assays than in explant cultures. A purified tuberculopolysaccharide was equally inhibitory for both normal and tuberculin-sensitive cells in both culture systems.     

Limited usefulness of quantitative culture of blood drawn through the device for diagnosis of intravascular-device-related bacteremia.

The use of a differential quantitative blood culture technique (Isolator) to diagnose intravascular-device-related bacteremia (IDRB) was studied prospectively. During septic episodes in 44 patients, blood was obtained simultaneously through the suspected infected device and from a peripheral venipuncture. The blood samples were processed by the Isolator technique, which enables easy quantification of microorganisms. The cannula was removed, and its tip was cultured semiquantitatively. Of the 52 cannulas studied, 15 were the cause of IDRB, but only 7 of these showed a significantly higher bacterial count in blood obtained through the device compared with peripheral blood. The bacterial count was higher in blood drawn through the device than in peripheral blood in four of six cases that did not fulfill the definition of IDRB. Some blood cultures obtained through the device were positive despite negative cultures of peripheral blood and cannula tips (six cannulas). Quantitative blood cultures were not useful in diagnosing IDRB in this study.     

Rapid diagnosis of typhoid fever through identification of Salmonella typhi within 18 hours of specimen acquisition by culture of the mononuclear cell-platelet fraction of blood.

Detection of Salmonella typhi in blood by culture of the mononuclear cell-platelet layer was compared with other methods currently used for the diagnosis of typhoid fever. Colonies of S. typhi were present in all mononuclear cell-platelet layer-positive cultures within 18 h of plating and were identified within an additional 10 min by a coagglutination technique. In contrast, identification of all positive cultures by conventional blood culture required 3 days.     

Synchronization culture of amniotic fluid cells using excess thymidine block followed by deoxycytidine release and its application to high-resolution banding analysis of chromosomes

We describe a simple synchronization culture technique of amniotic fluid (AF) cells to yield many earlier mitotic divisions with extended chromosomes. AF cell samples obtained by amniocentesis were cultured in the usual manner. Thirty hours after the first subculture, they were exposed to excess thymidine (0.5 mM). This cell cycle block was released by adding deoxycytidine (10 microM) 18 hr after synchronization. At exactly 7.5 hr after the release, the cultures were treated with Colcemid (0.02 microgram/ml) for 20 min then harvested. The mitotic index and the ratio of cells in the earlier mitotic stages were much higher in the synchronized cultures than in the control cultures. The same favorable effects were obtained also in AF cell cultures by combining this technique with ethidium bromide or actinomycin D treatment. The technique was less toxic to the cells, and was simple and reproducible. It was successfully applied to prenatal cytogenetic diagnosis of 2 families with a subtle inherited chromosome abnormality, so it is recommended for high-resolution banding analysis of AF cells and possibly chorionic villus samples.     

Use of phorbol-12,13-dibutyrate as a mitogen in the cytogenetic analysis of tumors with low mitotic indexes

Solid tumors, particularly those involving the colon, breast, and lung, are the most common tumors in humans. However, many technical difficulties exist in obtaining analyzable chromosomes from these tumors, including the inability to stimulate cell division. Phorbol-12,13-dibutyrate (PDBu) is a tumor promoter that activates a variety of cellular responses, including proliferation. Using flow cytometry, we have demonstrated that PDBu acts as a mitogen in primary cultures of colon tumor cells. Based on these results, we developed a short-term culture technique that greatly improves the yield of analyzable metaphases from colon tumors. Stimulated cultures consistently contained at least ten times more metaphases than unstimulated cultures, and chromosome morphology was improved. By modifying this technique with the addition of the calcium ionophore A23187, we have successfully obtained analyzable chromosomes from the peripheral blood of normal individuals, chronic lymphocytic leukemia patients, and a nodular small cell lymphoma patient. These results demonstrate that mitogenic stimulation by PDBu is a valuable technique in the cytogenetic analysis of colon tumors. By using PDBu alone or in combination with other agents, this technique may also be applicable to many other tumors that are difficult to karyotype because of an inability to obtain mitoses.     

Visualization of horse sickness virus by the fluorescent antibody technique

Horse sickness virus was grown in tissue cultures of monkey kidney cells, and virus was detected by a direct fluorescent antibody technique. Virus was detected at 8 hours in or around the nucleus of cells, and at 24 and 48 hours after infection it was also seen in the cytoplasm.     

Direct electron-microscopy of organ cultures for the detection and characterization of viruses

Summary Organ cultures were inoculated with viruses belonging to five different morphological groups. Cells and debris were abraded from the cultures four days later and examined by a negative contrast technique. All but the smallest particles were readily recognised. This method has been of value in visualising some recently isolated viruses.     

Agarose Embedding: A New Method for the Ultrastructural Examination of the In-Situ Morphology of Cell Cultures

A new technique for the ultrastructural examination of the in-situ morphology of cell cultures is described. By embedding the cell cultures in situ in agarose before embedding them in epoxy resin, numerous agarose blocks with the cells on top of the blocks can be obtained and used for different types of investigation, including electron microscopy. Use of this technique enables many of the problems encountered with earlier methods to be avoided.     

Evaluation ofprimary blood monocyte and bone marrow cell culture for the isolation of Rickettsia rickettsii.

Rickettsia rickettsii was isolated from experimentally infected guinea pigs by culture of blood monocytes and bone marrow cells, and from experimentally infected rhesus monkeys by blood monocyte culture. Rickettsiae were identified in monocyte-macrophage monolayers stained by Giménez or flourescent antibody techniques. A total of 78 culture attempts were made from 20 guinea pigs and 16 monkeys. The success of isolation of R. rickettsii in culture was positively correlated with the numbers of rickettsiae present in the blood and bone marrow. in cultures derived from infected guinea pigs, rickettsiae were usually observed after 5 to 7 days of culture, and in monkeys monocyte cultures they were usually observed within 3 to 5 days. Positive cultures were derived from guinea pigs and monkeys as early as the first day of fever and 1 to 3 days before the appearance of other clinical signs. Monocyte cultures became negative with the resolution of rickettsemia and concomitantly with the appearance of serum antibody. Monocyte culture isolation of R. rickettsii may be as sensitive for the detection of rickettsiae in blood and marrow as the intraperitoneal inoculation of guinea pigs or the plaque assay technique. Because of the simplicity of the method and because rickettsiae were often identified within 3 to 5 days after initiation, the monocyte culture technique may be useful in the early diagnois of human rickettsial disease.     

Elastase effect on the extracellular matrix of rat aortic smooth muscle cells in culture

The effect of elastase on the extracellular matrix of neonatal rat aortic smooth muscle cell cultures was monitored both chemically and ultrastructurally. Porcine pancreatic elastase was shown to decrease the elastin content in these cultures. Although chemically no distinction could be made between the elastin remaining in the culture matrix after elastase when compared to that in the nontreated cultures, the elastin was dramatically altered morphologically. The elastin assumed a "mottled" appearance after elastase treatment similar to that seen in vivo in emphysema models. A highly sensitive immunogold staining technique was used to detect elastin at the earliest stages of accumulation. Pulse experiments demonstrated an increase in protein synthesis by the cells 20 hr after elastase exposure. The culture system described here provides a model for probing in vivo elastase effects on elastin-containing tissues.     

Nature of the Giant Cell in Hodgkin's Disease

A new technique for the ultrastructural examination of the in-situ morphology of cell cultures is described. By embedding the cell cultures in situ in agarose before embedding them in epoxy resin, numerous agarose blocks with the cells on top of the blocks can be obtained and used for different types of investigation, including electron microscopy. Use of this technique enables many of the problems encountered with earlier methods to be avoided.     

Demonstration of age-dependent capsular material on Pasteurella haemolytica serotype 1

Extracellular capsular material was demonstrated on early log-phase cells of Pasteurella haemolytica serotype 1 by the fluorescent-antibody and several capsular staining techniques. The presence of this material was shown to be age dependent. Wide capsules were demonstrable on cells from 2- to 12-h cultures, whereas cells from 16- to 22-h cultures had very little cell-associated capsular material. The Maneval technique most clearly demonstrated the presence of capsules on cells from young (6-h) cultures when compared with other capsule staining techniques.     

Study of the One-Step Growth Curve of Equine Infectious Anemia Virus by Immunofluorescence

Primary horse leukocyte cultures were inoculated with 2 or 10 50% tissue culture infective doses (TCID(50)) of equine infectious anemia (EIA) virus per cell, and the titer of cell-associated and fluid-phase virus was determined from 1 to 72 hr postinoculation (PI). Cover slips were collected from 4 to 72 hr PI and stained for EIA viral antigen by the indirect immunofluorescent (FA) technique. Viral replication was detected after a latent period of approximately 18 to 24 hr and reached peak titers of approximately 10(4.5) to 10(6) TCID(50)/0.5 ml from 48 to 72 hr PI. The fluid phase contained 10(1) to 10(2) TCID(50)/0.5 ml more virus than the cells. Viral antigen was first detected by FA from 18 to 24 hr PI. Approximately 75% of the cells contained antigen in their cytoplasm 72 hr PI. The FA technique is a sensitive method for detecting EIA virus in horse leukocyte cultures.     

Experimental mixed infection with two tick-borne viruses and interferon-mediated interference.

Tick-borne encephalitis virus (TEV), a Flavivirus, and Lipovník virus (LV), a member of the Kemerovo group and complex and possible member of the Rioviridae, were used to infect chick embryo cell (CEC) cultures. LV reproduction was inhibited by preinfection with TEV, mainly in aged cultures. In young CEC cultures, TEV production was stimulated slightly and interferon was sometimes depressed by this superinfection; on the contrary, in aged cultures the superinfecting LV stimulated interferon also when infective LV was not produced because of interference. Double staining by the fluorescent antibody (FA) technique localized both viral antigens often in the same cells. Interference between TEV and LV (and vice versa) was observed in cerebrally infected adult mice. The pathogenicity of LV, when injected first, was increased by TEV superinfection. Examination by the FA technique confirmed interference between TEV and LV also in suckling mouse brains and demonstrated both viral antigens in the same brains when TEV and LV were injected simultaneously.     

Comparison of the quantitative direct plating method and the BACTEC procedure for rapid diagnosis of Haemophilus influenzae bacteremia in children.

The efficacy and reproducibility of the quantitative direct plating (QDP) method and the semi-automated BACTEC radiometric system (BBL Microbiology Systems, Cockeysville, Md.) were analyzed for the rapid diagnosis of Haemophilus influenzae bacteremia on the basis of 41 positive cultures from 35 patients. The QDP method detected 61% and BACTEC only 19% of the positive cultures within the first 12 h. Similarly, the QDP procedure yielded growth of H. influenzae in 56% of the cultures before a positive growth index reading was obtained with the BACTEC method. The observation that the quantitative procedure recovered H. influenzae in 88% of all positive cultures, using only 1 ml or less of blood, is attributed to the fact that 68% of the cultures had counts in excess of 100 colony-forming units per ml. The reproducibility of the QDP method was documented by the fact that duplicate blood cultures taken within a few hours of each other yielded comparable results on the number of bacteria at low (1 to 100/ml), moderate (100 to 1,000/ml), and high (greater than 1,000/ml) levels of bacteremia. We concluded that the QDP method is a valuable, simple, and inexpensive supplementary technique to the semi-automated BACTEC procedure for the rapid diagnosis of H.influenzae bacteremia in children.     

Recovery of Mycobacterium avium-M. intracellulare from blood specimens by using the routine BACTEC 6B blood culture system

We describe a simple technique for recovery of Mycobacterium avium-M. intracellulare from blood culture specimens by using the BACTEC 460, a system used for routine blood cultures in many hospital laboratories. A total of 26 of 215 blood specimens (12%) from 11 of 48 patients (23%) with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex yielded isolates of M. avium-M. intracellulare. Acid-fast staining of prepared specimens was positive for 16 of 26 cultures that ultimately yielded the organism.