Article

Studies on the physiological conditions prevailing in tissue culture

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Abstract

An analysis of some of the physiological factors active in Maitland tissue cultures has been presented in the hope that it may be of some value in clarifying the principles underlying tissue cultures in general. It has been found that the empirically determined necessity of using relatively small amounts of tissue in such cultures is dependent upon the fact that excessive tissue leads to a rapid change of reaction toward the acid side. Whereas tissue may remain viable in an environment as alkaline as pH 9 and over, viability is rapidly destroyed when the reaction approaches pH 6. Evidence is presented to indicate that the changes in electrode potentials which take place in Maitland cultures are not, as has been suggested, the determining factors upon which virus multiplication depends, although they may, of course, be incidentally important. It has been shown that there are fundamental differences between those conditions in Maitland cultures which favor the multiplication of a typical virus and those upon which the growth of the Rickettsiae of typhus fever depends. The virus which we have studied (equine encephalitis virus, western type) multiplies during the period of active tissue metabolism. The maximum virus titrations are obtained at about the time at which metabolism has come to a standstill. Thereafter the virus not only ceases to increase but rapidly deteriorates. The period of viability of the tissue cells themselves is shortened by several days in the presence of virus multiplication. There is some evidence that a temporary acceleration of oxygen uptake takes place during the time of active virus multiplication. Technical difficulties in controlling such experiments prevent certainty in regard to this point. In contrast with the conditions determining the growth of a virus agent in the Maitland cultures the multiplication of Rickettsiae does not begin to any determinable extent until after active cell metabolism has either become stabilized or has ceased. The Rickettsiae continue to grow at a time when the cells are no longer viable. It appears likely that these organisms find the most favorable conditions for growth in cells which are no longer metabolically active but in which some delicately heat-susceptible elements have not yet been disturbed. As a consequence of these observations, frozen and preserved embryonic tissues have been successfully used for Rickettsia cultivation. A report on these experiments will be made in a separate communication.

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... Conditions which favor the multiplication of the particular host cell also favor the growth of rickettsiae. These findings are in contrast to some of the earlier studies of Zinsser and Schoenbach (21), in which nonviable cells were thought to represent a better growth medium for the rickettsiae. The studies of Hoppes et al. also suggest differences in the requirements of rickettsiae and animal viruses, in that there are a number of examples in which tissue culture cells support the propagation of viruses in simple media (22,23). ...
... Early studies, qualitative in nature, indicated that rickettsial growth was best in slowly metabolizing cell (33). However, the quantitative estimation of growth of R. orientalis in more simplified system established the role of adequately nourished cells to support multiplication of the rickettsia (6). ...
Article
A technique is described for measuring the oxygen uptake of embryonate eggs. Statistical analysis has shown that the method is reliable and accurate. Determinations were made on groups of 15 to 20 eggs, in order to average out individual biological variations. Reduction of the CO(2) tension and relative humidity to approximately zero previous to analysis has been found to be desirable. The oxygen consumption of normal and typhus-infected eggs, untreated and treated with agents previously found to inhibit or enhance rickettsial growth has been studied. Rickettsial infection caused a slight but significant increase in the rate of oxygen consumption on the 4th day after inoculation, followed by a rapid drop in the rate as the infection developed. The evidence suggests that low concentrations of rickettsial toxins may stimulate respiration, while higher concentrations depress respiration and lead eventually to embryonic death. PABA, which is rickettsiostatic, markedly increased the oxygen uptake of normal eggs, the effect appearing 4 days after injection and lasting for about 4 days. Thereafter, the rate fell below that of the untreated eggs. In typhus-infected eggs, PABA had similar effects, but the oxygen consumption reached much higher levels. A possible explanation of this fact is suggested. MABA and OABA, which are not rickettsiostatic, did not increase oxygen uptake; in fact they depressed cellular respiration moderately, OABA being more active in this way than MABA. These two compounds may compete with PABA for a position in some respiratory enzyme system. Potassium cyanide, which enhances rickettsial growth, caused, in concentrations not lethal to the embryos, a moderate drop in the oxygen consumption of normal eggs, the effect starting almost immediately after injection and lasting usually for 9 days. In infected eggs, its effect was more striking. It is probable that rickettsial toxins and KCN act synergistically to depress cellular respiration. When PABA and KCN were injected simultaneously, the stimulating effect of PABA on respiration predominated. The resulting level of oxygen consumption, though lower than that resulting from PABA alone, was still high enough to inhibit rickettsial growth. As far as our results go, they support the hypothesis that, within certain limits, rickettsial growth is inversely proportional to the respiratory rate of the host cells, regardless of the factors which determine that rate. It is not yet clear that PABA owes its rickettsiostatic action to its ability to increase cellular respiration, but this assumption seems reasonable as a working hypothesis. The respiratory mechanism in which PABA participates is not as yet known. Although PABA forms part of the folic acid molecule, folic acid itself, in concentrations corresponding to effective doses of PABA, did not increase cellular respiration or show rickettsiostatic action.
Article
Changes in the metabolism of the chick embryo host cell produced by p-aminobenzoic acid or sodium cyanide did not influence the multiplication of psittacosis or Newcastle virus. A suspension of psittacosis virus was unable to catalyse oxygen uptake in presence of glutamic acid, casein hydrolysate, succinic or pyruvic acids. The results suggest that, in its relation to certain metabolic activities of the host cell, psittacosis virus more nearly resembles the true viruses than the Rickettsiae. The systematic position of the psittacosis-lymphogranuloma group of infective agents is still a matter of dispute. Some authorities, impressed by the re- semblances of these agents to the Rickettsiae, have suggested that they should be grouped with them. Lillie (1 930), who was one of the first to make a morpho- logical study of psittacosis virus, stated that he considered it to be a rickettsia, naming it Rickettsia psittaci; others retain these agents with the viruses. A third view is that they form a group by themselves, occupying a position intermediate between the rickettsiae and what are termed the ' true viruses '. Admittedly the agents of the psittacosis-lymphogranuloma group resemble the rickettsiae in some respects. They have a similar affinity for basic dyes which appears to be related to their high content of deoxyribonucleic acid (Lkpine & Sautter, 1946). Although, in general, they are smaller than the rickettsiae they are not dissimilar in this respect from R. burneti. They produce intra- cellular colonies which resemble those of the rickettsiae rather than the inclusions of the true viruses, and their susceptibility to sulphonamides and some natural antibiotics, though differing in some respects from that of the rickettsiae, suggests a closer resemblance to the latter than to the viruses. There are, however, points of difference. The agents of the psittacosis-lympho- granuloma group do not appear in bacillary form as do the rickettsiae, and when multiplying they pass through a cycle of development not seen in the rickettsiae. A more important dissimilarity is the difference in host range: the rickettsiae are essentially parasites of arthropods, though some species have also acquired mammalian hosts, whereas none of the viruses of the psittacosis-lymphogranuloma group has an insect host, The recent work of Greiff & Pinkerton (1948) may well have a bearing on the question of the relationship of the psittacosis-lymphogranuloma group to the viruses on the one hand, and the rickettsiae on the other. These workers found that injection of p-aminobenzoic acid (PAB) into the developing hen's egg increased the metabolism of the cells of the embryo as measured by respiratory exchange, making them in consequence unsuited to the multiplication of typhus
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By injection into typhus-infected yolk sacs, a number of agents were tested for possible inhibition or acceleration of rickettsial growth. The previously reported rickettsiostatic activity of penicillin was further confirmed. Para-aminobenzoic acid, in single injections of 6.6 mg. and 3.3 mg. giving initial concentrations of approximately 1:6000 and 1:12,000 was found to have rickettsiostatic activity approximately equal to that of penicillin. No conclusion could be drawn regarding the possibility of a synergistic action of para-aminobenzoic acid and penicillin. Para-aminobenzoic acid neutralized with sodium hydroxide was found to be as effective as the acid itself, when given in single injections of 6.6 mg. Sodium benzoate, as Well as the ortho and meta forms of aminobenzoic acid were found to be ineffective when given in similar amounts. Para-aminobenzoic acid, when added to the food in a concentration of 3 per cent, was shown to have a remarkably effective chemotherapeutic action on murine typhus infection in mice. Sodium fluoride was found at times to accelerate the growth of rickettsiae in the yolk sac, and to cause heavy infection under conditions such that the controls showed practically no multiplication of the organism. When rickettsiostatic substances (penicillin and para-aminobenzoic acid) were combined with sodium fluoride, their rickettsiostatic activity was not demonstrably changed. Other agents studied and found not to affect rickettsial multiplication are listed. The possible mechanisms involved in the observed inhibition and stimulation of rickettsial growth under these conditions are discussed.
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Groups of embryonated eggs infected with the PR8 strain of influenza virus A were incubated at 34 degrees , 37.5 degrees , and 40 degrees C. At frequent intervals, for periods ranging up to 96 hours, pooled allantoic fluids were tested simultaneously for infectivity and hemagglutination. After about 12 hours of virus growth, fluids often showed infectivity titres greater than 10(-5), but were incapable of causing hemagglutination. At later time intervals, marked disagreement between the two tests for viral activity was noted at all temperatures, but most strikingly at 40 degrees C. Hemagglutination titres were highest and best sustained in eggs incubated at 34 degrees C., while incubation at 37.5 degrees C. resulted in the highest and best sustained infectivity titres. Hemagglutination titre determinations do not reflect accurately the rate of influenza virus multiplication. Possible reasons for the lack of correspondence between hemagglutination and infectivity are discussed.
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Zusammenfassung Glukose ist unbedingte Voraussetzung zur Synthese von Ornithosis-Virus sowohl beim Embryo wie beim Dottersackin vitro. Nach erfolgter Infektion vermehrt sich das Virus auch ohne Glukose. Die beiden Glukosestoffwechsel unterscheiden sich deutlich.
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This chapter is devoted to a discussion of three topics: The discovery of the major etiologic agents of rickettsial disease, their vectors, and mechanisms of transmission; The major technological developments that have facilitated the study, control, and treatment of rickettsiae and rickettsial diseases; and the evolution of the concept of rickettsia from a microbial entity which is neither a typical bacterium nor a typical virus to a well-characterized set of bacteria. Contents of Volume I include: Etiology; Rocky Mountain Spotted Fever (RMSF); Epidemic and Endemic Typhus Fevers; Trench Fever; Scrub Typhus; Q Fevers; Rickettsialpox; Staining Rickettsiae; Preantibiotic Chemotherapy; Early Vaccines; The Yolk Sac Revolution; Rickettsiae as Organisms and Rickettsial Physiology. Reprints.
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Súkrn Popísala sa kultiváciaCoxiella burneti vo vyrastených tkanivových kultúrach zo zltkových vakov. V našej úprave metodiky podl’a Weissa a Pietrycka (1956) je mozno sledovat’ rozmnozenieC. burneti vo vyrastených tkanivových kultúrach bezprostredne po infekcii tkaniva. Infekčný titerC. burneti na tkanivových kultúrach bol 10−8,15, resp. 10−8,39. Informatívne pokusy ukazujú, ze adsorpcia koxiel na povrch vnimavych buniek sa uskutočnila uz málo minút (5 minút) po kontakteC. burneti s tkanivom a ze je najlepšia pri teplote 37 °C. Mikroskopicky sa sledovalo rozmnozenie koxiel pričom sa poukázalo na prítomnost’ drobných, slabšie farbitel’ných bodkovitých čiastočiek na 2.–3. deň infekcie. Summary The author describes the culture ofCoxiella burneti in well developed yolk sac tissue cultures. Using a modification of the method of Weiss et al. (1956) it was possible to observe proliferation ofC. burneti in the fully grown tissue cultures immediately after infection of the tissue. The infection titre ofG. burneti on the tissue cultures was 10−8,15 and 10−8,39. Preliminary experiments showed that adsorption of the coxiellae on to the surface of sensitive cells takes only a few minutes (five) after contact of the organism with the tissue and that the most satisfactory results are obtained with a temperature of 37 °C. The author made a microscopic study of proliferation of the coxiellae and draw attention to the presence of minute, weakly stanining pin-point particles on the second and third day of infection.
Chapter
Durch die Untersuchungen von Carrel (34) mit dem Rousschen Hühnersarkom und die von Maitland und Maitland (184) mit dem Vaccinevirus wurde die Gewebekulturtechnik in die Virologie eingeführt. Schwierigkeiten der Technik wie auch die Fragestellungen, mit denen diese und auch spätere Untersuchungen durchgeführt wurden, verhinderten jedoch zunächst eine allgemeine Anwendung. Durch die überwiegende Verwendung der modifizierten Maitland-Technik war es nicht möglich, die Methode der Gewebekultur als solche selbsttragend zu gestalten. Der Nachweis der Virusvermehrung in der Gewebekultur mußte fast immer mittels des Tierversuches geführt werden. Die Fragestellung beschränkte sich, abgesehen von morphologischen Untersuchungen, die zum Studium der Viruszellbeziehungen durchgeführt wurden, weitgehend darauf, festzustellen, welche Gewebearten einer bestimmten Tierspecies einer gegebenen Virusart Vermehrungsmöglichkeiten bieten, die äußeren Kulturbedingungen festzulegen und die Veränderungen, die eine Virusart durch serienweise Gewebekulturpassagen erfährt, zu erfassen.
Article
1. Rickettsia prowazeki can be cultivated for many generations in vitro, without diminution in numbers or virulence, in media similar to those described by Maitland, Rivers, and others for the cultivation of certain viruses. In all probability, such cultures can be maintained indefinitely. 2. It has been impossible, thus far, to cultivate the typhus rickettsia without employing living tissue.
Article
Cells survive for at least 5 days and at times are capable of multiplying in a mixture of serum and Tyrode's solution used by Maitland for the cultivation in vitro of vaccine virus.
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