Cent Calf Serum Research Articles

Different progress of MDCK cell death after infection by two different influenza virus isolates.

The effect of influenza strains A (H3N2) and B, isolated during the seasons of 1994 and 1995 in the Czech Republic, on MDCK cells was studied. Various concentrations of virus and conditions of nutrition were used during the cell culture. The virus replication and consequently fragmentation of genomic DNA together with cytotoxicity were investigated in the absence and presence of 10 per cent calf serum. Virus replication, regardless of type A or B, caused earlier DNA fragmentation in comparison to non-infected cells in tissue culture. The results showed that the influenza B strain had a greater cytotoxic effect on MDCK cells than influenza A. A higher infection dose of influenza A virus accelerated the onset of apoptosis; conversely, a higher infection dose of influenza B virus delayed the onset of apoptosis. The absence of serum enhanced the progress of influenza-induced apoptosis in conditions in vitro.

Effect of selenium on developing mouse teeth in culture in vitro

First molars from 2-day-old mice were cultured for 7 days in medium supplemented with 0.04, 0.4, 4.0 parts/10 6 selenium (Se) or 10 per cent calf serum. Collagen synthesis, measured after labelling with [ 3H]-proline for the last 24 h of culture, was most active at 0.4 parts/10 6 Se. Supplementation with 300 parts/10 6 Se for the first 24 h of culture caused ultrastructural changes in newly-deposited enamel and dentine matrices.

Effects of pregnant mouse serum and pregnancy hormones on the replication in vitro of murine cytomegalovirus.

When mouse embryo fibroblasts cultivated in medium containing 2 per cent pregnant mouse serum were infected with mouse cytomegalovirus they gave 3-4 fold greater plaque counts and virus yield than when the medium contained 2 per cent normal mouse serum. Plaque size was increased up to two fold. There were similar differences between 2 per cent foetal calf serum and 2 per cent calf serum. When medium containing 2 per cent dialysed fetal calf serum was supplemented with physiological concentrations of oestrogen, progesterone and corticosteroid there was a 2-3 fold increase in plaque count and virus yield. The increase was only seen when the hormones were present both during virus adsorption and throughout virus replication. Any one of the hormones added by itself gave a smaller and more variable increase in yield and/or plaque count. Growth hormone by itself gave a 2 fold increase in virus yield; when added to the three other hormones it gave a 7-13 fold increase in virus yield but little change in plaque count. The three hormones also increased the rate of infection of resident peritoneal macrophages as scored by fluorescent antibody staining after a single cycle of virus growth in vitro. These experiments suggest that mouse cytomegalovirus infecting a pregnant mouse, or reactivating in a pregnant mouse would replicate more extensively.

Enhanced cell killing by thio-TEPA and urokinase (author's transl)

The effect of urokinase on thio-TEPA toxicity has been studied in monolayer cultures of KK-47 cell line, which was established from a human bladder carinoma in our department in 1977.The results obtained are as follows:1. The cell growth curve consisted of a 2-day lag phase and a 5-day logarithmic phase followed by a stationary phase. The average doubling time for the cells on Ham's F12 medium supplemented with 20 per cent calf serum was approximately 36 hours.2. Thio-TEPA in Ham's F12 medium supplemented with 20 per cent calf serum was stable for 72 hours on incubation at 37°C. Urokinase and thio-TEPA showed no deteriorating effect on each other under some mixing conditions.3. Remarkable toxicities of thio-TEPA were observed with each exposure of 2 and 24 hours at concentrations of the agent between 10-1 and 102μg/ml. Value of IC 50 at the 2-hour exposure was approximately 10μg/ml. A potent inhibitory effect of urokinase against the propagation of the cells in vitro was observed at a concentration of 100CTAu./ml, although there was no significant change in cell growth at concentrations ranging from 10-4 to 10 CTA u./ml.4. Using methods, of assessing cell growth and 3H-thymidine uptake, a combined use of urokinase (10 CTA u./ml) and thio-TEPA (10μg/ml) resulted in a significant increase of the thio-TEPA toxicity with both the exposure times.5. A peak in 32P-thio-TEPA uptake in the cells was observed 2 hours after administration of the agent (0.1μCi/10μg/ml), and about 2-time increase in the peak was obtained by adding urokinase (10 CTA u./ml).From the results, it is quite possible to say that the increased cytotoxicity may be related with a gross change in cell permeability to thio-TEPA through plasminogen activation by urokinase.Because of the substantial effect of urokinase obtained in vitro, a possible usefulness of urokinase as an adjunct to the topical use of thio-TEPA in the treatment of bladder carcinoma is discussed.

Stimulierung von Kulturen embryonaler Rattenzellen durch Kälberserum / Stimulation of Embryonic Rat Cells in Culture by Calf Serum

Embryonic rat cells cultured in a synthetic medium are stopped in G1 phase if serum is omitted from the culture medium. On addition of calf serum these cells resume DNA synthesis after a lag phase of 15—20 hours. In the present paper the events concerning the metabolism of adenosine phosphates after stimulation by serum have been examined. The results are the following: 1. The concentration of cAMP in cells which have been incubated for 24 hours in serum-free medium is 32 times higher than that in cells growing in medium containing 10 per cent calf serum; the accumulated cAMP is metabolized within 10 min after the addition of 10 per cent calf serum. This has been confirmed by a double labelling technique after preincubation with radioactive adenosine as well as by estimation of cAMP using a protein-binding assay. 2. A cAMP-specific phosphodiesterase is activated shortly after stimulation; the enzyme activity in dialysed cell homogenates is increased within 30 min by a factor of 2. 3. Cells incubated in serum-free and those in serum-containing medium have equal amounts of ATP. On addition of serum the ATP concentration in cells preincubated in serum-free medium decreases by 27%; the original level is restored 20—30 min later.

Standardization of human diploid cell cultivation.

Human embryonic diploid lung fibroblasts grown in Eagle's medium were exposed continually to a variety of environmental conditions over a large number of passages to observe how these conditions affected the growth and longevity of these cells in vitro. The cells grew well at temperatures between 34 and 37 C and some cells could be adapted to grow at 40 C. Very limited growth occurred at 30 to 31 C; however, confluent monolayers of cells could be maintained for months at 30 C and still give rise to actively growing cultures. Increasing the amino acid concentration in Eagle's medium or the calf serum concentration above 10% had no effect on the growth rate or longevity. One per cent calf serum could not support prolonged active growth. Trypsin concentrations between 1 and 0.1% and crystalline trypsin at 50 mug/ml showed no influence on cell growth. Ethylenediaminetetraacetic acid treatment and scraping, however, destroyed many of the cells, and the survivors grew poorly. The clonal morphology varied with age. Young cells frequently gave rise to densely packed clones, whereas older cells gave rise to clones with widely scattered cells. The cloning efficiency was high when the cells were young but decreased rapidly with successive passage. It was relatively constant from the 7th to 20th passage at about 15%.

Standardization of Human Diploid Cell Cultivation

Human embryonic diploid lung fibroblasts grown in Eagle's medium were exposed continually to a variety of environmental conditions over a large number of passages to observe how these conditions affected the growth and longevity of these cells in vitro. The cells grew well at temperatures between 34 and 37 C and some cells could be adapted to grow at 40 C. Very limited growth occurred at 30 to 31 C; however, confluent monolayers of cells could be maintained for months at 30 C and still give rise to actively growing cultures. Increasing the amino acid concentration in Eagle's medium or the calf serum concentration above 10% had no effect on the growth rate or longevity. One per cent calf serum could not support prolonged active growth. Trypsin concentrations between 1 and 0.1% and crystalline trypsin at 50 mug/ml showed no influence on cell growth. Ethylenediaminetetraacetic acid treatment and scraping, however, destroyed many of the cells, and the survivors grew poorly. The clonal morphology varied with age. Young cells frequently gave rise to densely packed clones, whereas older cells gave rise to clones with widely scattered cells. The cloning efficiency was high when the cells were young but decreased rapidly with successive passage. It was relatively constant from the 7th to 20th passage at about 15%.

Stimulation of DNA synthesis in resting stage human fibroblasts after infection with Rous sarcoma virus.

ROUS sarcoma virus (RSV) suspensions can induce the synthesis of deoxyribonucleic acid (DNA) and mitosis in resting stage human fibroblasts1. The virus can also induce DNA synthesis in undividing myotubes2 and DNA synthesis and mitosis in resting stage chick embryo fibroblasts3,4. The stimulatory activity may not be identifiable with the virus particle, for when the virus suspension was treated with RSV anti-serum the focus forming capacity was inhibited but the stimulatory action was not eliminated1. We have now extended these findings. The WI-38 human embryonic line5 was used maintained in Eagle's minimal essential medium6 supplemented with 10 per cent calf serum and aureomycin (50 µg/ml.). RSV suspensions were obtained from the supernatants of infected chick embryo fibroblastic cultures, and had a titre of 105 FFU/ml. DNA synthesis was estimated by adding 0.1 µCi/ml. of labelled thymidine (3H-TdR) (specific activity, 14 Ci/mmole) to the cultures. The cells were fixed 24 h later, DNA was extracted7 and the radioactivity incorporated was measured in a liquid scintillation counter.

Radiation-induced cytogenetic damage in the HeLa cell.

The contributions to mammalian cytogenetic technic by Hsu (1) and Moorhead et al. (2) have stimulated efforts to quantitate cellular radiosensitivity by analysis of radiation-induced chromosome aberrations. Experience with human tissue irradiated in vivo during occupational exposure (3, 4), internal deposition of radionuclides (5–7), accidental exposure (8–10), and radiation therapy (11, 12); and experiments with human tissue (13–15) and cultured mammalian cells (16–18) irradiated in vitro have shown that cytogenetic analysis can provide a sensitive measure of radiobiological damage. The results have supported the generally accepted view that the nucleus of a cell is the principal target-site responsible for radiation-induced cell death. Quantitative measurements of radiation-induced single-hit and multi-hit chromosome aberrations have been made following the in vitro irradiation of human leukocytes (14, 15), human and monkey kidney cells (13, 19), human skin (13), and Chinese hamster cells (16, 17). The data from these studies were analyzed by employing a quadratic dose-response relationship, Y = a + bD + cD2, while assuming that the coefficients b and c were zero for multi-hit and single-hit aberrations, respectively. The reported single-hit aberration frequencies ranged from 0.0032 to 0.24 aberrations per cell-rad, and the reported multi-hit aberration frequencies ranged from 0.9 × 10−6 to 5.2 × 106−6 aberrations per cell-rad squared. Spontaneous aberration rates were reported to range from zero to 0.35 aberrations per cell. The HeLa cell has been used extensively in many areas of radiobiological research; there have been no reports, however, describing structural chromosome aberrations in the HeLa cell following irradiation. This deficiency is probably due to the great difficulty of analyzing chromosome damage in HeLa cells because of the large and variable number of chromosomes per cell. The present investigation was undertaken to quantitate radiation-induced chromosome aberrations in the HeLa cell in an effort to further characterize the radiosensitivity of this mammalian cell line. Materials and Methods HeLa cells of Puck's S3 clonal line (20) were cultivated in the laboratory on a seven-day growth schedule in sealed 30-ml tissue-culture flasks. Growth was maintained with Eagle's basal medium, supplemented with 8 per cent calf serum; this was changed every other day. A dense monolayer of cells was formed in seven days, yielding approximately 106 viable cells. Prior to irradiation the cells were removed from the growth surface by gentle shaking in a 0.02 per cent Versene (ethylene-diaminetetraacetic acid) solution. The cells were centrifuged, the supernatant was drained off, and they were resuspended in Eagle's basal medium without serum in 12 × 75 mm tubes. In order to ensure aerobic conditions, cell suspensions were aerated with oxygen for fifteen minutes prior to and during irradiation.

Radiosensitivity of a series of cultured primary fibrosarcomata.

Almost all the very large volume of work that followed the introduction by Puck and Marcus (1) in 1956 of the clonal-survival technic for determining the radiosensitivity of cells in tissue culture has been concerned with established cell lines. In the hands of an investigator in one laboratory this technic yielded strikingly reproducible results for a particular cell line. Very little work has been done with primary cultures of cells. The possibility of using the technic to assay the radiosensitivity of individual clinical tumors before deciding on a particular course of radiotherapy is very attractive, however, and with this in mind the present experiment was designed to determine the radiosensitivities of primary cultures of cells from a series of morphologically similar tumors in rats. Methods For this experiment Chester Beatty Research Institute colony-bred albino rats six weeks old were employed. In each animal a 10-mg pellet of 3:4 benzpyrene was implanted subcutaneously in the right flank. Tumors became palpable after five months, and no animals were used in which the tumor took longer than one year to appear. When the tumors were between 1.5 and 2.0 cm in diameter, they were biopsied aseptically under ether anesthesia by making an incision under the skin and removing approximately 0.5 g of tissue. The specimen was first cut with scissors into fragments of about 1 to 2 mm3 and then suspended in 15 ml of a 0.25 per cent w∕v solution of trypsin3 (1:250) in medium 199 at 37°C; this was agitated with a finger pump by the method of Boyse (2) for thirty minutes. The cell suspension was left to stand for one minute at room temperature to allow residual lumps to settle, and then the supernatant cell suspension was withdrawn and the remaining fragments put through the trypsinizing procedure a second time. The cells from the combined supernates were precipitated by gentle centrifugation and resuspended in culture medium which consisted of a 1:1 mixture of medium 199 and a medium due to Fischer (3) supplemented with 15 per cent calf serum (Difco). The cells were then introduced into medical flat bottles; this step allowed the viable cells to adhere to glass, enabling the dead cells, fragments, and erythrocytes to be subsequently removed, and it also provided assurance that up to this stage the culture was free from infection. The cultures were kept in a 37°C incubator flushed with a water-saturated atmosphere of 5 per cent CO2, 95 per cent air. Radiation survival curves were obtained as quickly as possible after growth was established at a steady rate (this usually took about ten days). For irradiation the cells were removed from the bottle by incubating at 37°C with the 0.25 per cent trypsin solution for ten to twenty minutes (depending on the time they took to detach from the glass).

Factors affecting the expression of blood group antigen a in cultured cells

It has been shown that the maximum proportion of A antigen positive cells in heterogeneous culture of RK 13 rabbit kidney cells is about 40 per cent. This is produced in medium containing 10 or 20 per cent calf serum. Increasing or reducing the amount of calf serum leads to a reduction in the growth rate and proportion of antigen-positive cells. Addition of the four constituent sugars of the A blood group determinant did not increase the proportion of A-positive cells, but omission of d-Glucose and increase in the amount of d-Galactose lead to a decrease in the growth rate and some decrease in the proportion of positive cells. The cells were tested with eight different anti-A sera and with cattle anti-J, sheep anti-R and pig anti-A antisera.

Effects of N-methyl-N-nitrosourethane on cells in tissue culture.

A COMMUNICATION on the morphological conversion of cells in vitro by N-nitrosomethylurea by Sanders and Burford1 prompts me to report similar experiments performed about 5 years ago in which the related and very effective carcinogen, N-methyl-N-nitrosourethane (MNU)2 was used. Its action in vitro was tested on various mammalian cells grown in tissue culture. These included the Syrian hamster fibroblast BHK 21/13 C cell line3 made available by Professor M. Stoker, and also fresh cultures of trypsinized baby (or foetal) rat heart fibroblasts. The cultures were maintained in Roux bottles, in Eagle's medium (containing double concentration of amino-acids and vitamins) and Hanks tryptose phosphate broth with 10 per cent calf serum. The cultures were trypsinized and sub-cultured once a week or more often. Explants, 24 h or 48 h old, were used for treatment with MNU of concentrations 1 × 10−2 – 1 × 10−5 molar. For this purpose, the medium was removed from the cultures and replaced by MNU dissolved in ethanol (1 : 10 v/v) and diluted to the desired concentration with phosphate buffer. After 30 or 60 min at 37° C, the MNU solution was decanted, the cultures were washed once with phosphate buffer, and left in fresh medium at 37° C.

Isolation of noninfectious particles containing Rous sarcoma virus RNA from the medium of Rous sarcoma virus-transformed nonproducer cells.

Stocks of the Brvan high-titer strain of Rous sarcoma virus (RSV) have been shown to contain avian leukosis viruses as well as RSV.I Chick embryo fibroblasts infected with the leukosis viruses associated with the Bryan stocks, Rous associated virus-I (RAV-1) or Rous assoc iated virus-2 (RAV-2), do not transform but do produce virus, whereas cells infected with RSV transform but do not produce infectious virus.2 When RSV-infected transformed cells are superinfected with RAV-1 or RAV-2, the transformed cells not only produce the superinfecting virus but also produce infectious RSV which has the growth characteristics,3 antigenic specificity,4 and host range of the superinfecting virus.5 The RSV-transformed cells which do not produce virus have been named nonproducer (NP) cells, and the avian leukosis viruses associated with the Bryan stock, helper viruses. Viral coat antigen cannot be detected in NP cells,2 4 and it has been hypothesized that the absence of coat arntigen in RSV-infected cells prevents synthesis of infectious RSV and that the role of the helper virus in the production of infectious RSV is to provide coat antigen.2 The finding that NP cells do not produce infectious virus raised the possibility that the carcinogenic action of RSV nmight be related to the apparent absence of late functions in virus replication in infected cells.2 Recently, Dougherty and DiStefano6 have observed particles with the appearance of avian leukosis viruses in electron micrographs of RSV-transformed NP cells and have suggested that NP cells may be producing non infectious virus. The experiments presented here approach the question of virus production by the NP cell with the aid of radioactive tracers7 and demonstrate that the NP cell is producing noninfectious particles with the physical characteristics of the avian leukosis viruses. The nucleic acid of the particles is characterized and found indistinguisbable in sedimentation velocity and base composition from the RNA of RSV + RAV. Materials and Methods.--Isolation and care of NP cells: Chick embryo cells from 10-day-old embryos of strain 13 or 813 white leghorn chickens which were susceptible to infections by both group 1 and group 2 avian leukosis viruses were used throughout the course of this work. The methods used for culturing chick enmbryo cells9 and assaying for focus-forming units of RSV9 and interfering units of RAVy have been described. Clones of NP cells were isolated according to the method of Trager and Rubin8 from secondary chick embryo fibroblasts infected with the Bryan high-titer straini of RiSV. The isolated clones were grown on X-irradiated chick embryo fibroblast feeder layers (4 X 105 cells/60-mrn plate) in a rich medium: 82 per cent 199A, 10 per cent tryptose phosphate broth, 4 per cent calf serum, 1 per cent chicken serum, 2 per cent 2.8 per cent NaHCO3, 1 per cent beef embryo extract, which had been incubated at 57?C for 45 minutes to inactivate any contaminating leukosis viruses. Culture medium was changed daily and the cultures

Observations on Keratinization of Human Skin in vitro

In spite of extensive investigations, epidermal keratinization remains relatively poorly understood. A number of recent studies have helped define some of the components involved in this basic process (1). The chemical composition of keratin, the role of keratohyalin granules, the influence of dermal components, the nature of both the intra- and extracellular cementing substance, and the role of cholesterol and mucopolysaccharides have received considerable attention.The purpose of this study was to investigate the possible influence of mucopolysaccharides, their components, or both, on the process of keratinization of adult human skin in vitro. Experiments with human skin in organ culture are well suited to the study of this basic process and allow more control and isolation of the factors involved (2, 3).MATERIALS AND METHODSThe modified Trowell technic was used (4). Normal skin specimens from the arm, leg or trunk were obtained either by use of local xylocaine anesthesia or at the time of surgery for other conditions performed under general anesthesia. The specimens were cut into approximately 2 square millimeter pieces after removal of the subcutaneous fat. Several specimens were grown with the full thickness dermis attached.The specimens were placed in small Petri dishes on open mesh paper (tea bag) resting on stainless steel platforms, 2 by 2 centimeters and 3 millimeters high. Usually 3 or 4 specimens of skin were grown in a single Petri dish.The medium was Eagle's with an Earle base containing glutamine and 10 per cent calf serum. Ten cubic centimeters of media were used, changed every second or third day. Initially and throughout the experimental period the pH was adjusted to 7.2 with 5 per cent carbon dioxide in air when necessary. Several Petri dishes were incubated at37°C within large staining dishes sealed with Saran wrap.The additives and their concentrations were as follows :heparin sodium 0.25 mg, 0.5 mg or 1.0 mg/ccmepesulfate (Treburon) 0.25 mg, 0.5 mg or 1.0 mg/ccglucosamine hydrochloride 0.25 mg, 0.5 mg or 1.0 mg/ccgalactosamine hydrochloride 0.25 mg, 0.5 mg or 1.0 mg/ccN acetyl-glucosamine 055 mg, 0.5 mg or 1.0 mg/ccN acetylgalactosamine 0.25 mg, 0.5 mg or 1.0 mg/ccchondroitin sulfate 0.25 mg, 0.5 mg or 1.0 mg/cchyaluronic acid 055 mg, or 1.0 mg/ccFor every experiment expiants were grown in plain media under the same conditions and parallel observations were made. The sequential histological findings were compared with the original epidermis and with those of the expiants grown in media containing additives.Additional controls included :glucose 1 mg or 5 mg/ccheparin sodium and protamine sulfate 1 mg. each/cc.protamine sulfate 0.5 mg or 1 mg/cc.sodium sulfate 1 mg/cccysteine 0.5 mg or 1 mg/cc Specimens were routinely removed at 3, 6, 9, 12, 15, 18 and 21 days intervals. Some were maintained through 35 days.The specimens were fixed in formalin, dehydrated and serially sectioned in the routine fashion. Special stains included Barnett-Seligman, toluidine blue at pH 2.5 and 5.0, colloidal iron-periodic acid-Schiff, Alcian blue at pH 0.45 and 2.5 and aldehyde fuchsin.In order to determine the uptake of radioactive sulfur S85< in expiants grown in the presence of glucosamine hydrochloride (1 mg/cc) media, 2 microcuries of sodium sulfate containing S85< (NasS O*) were added to media with and without glucosamine hydrochloride. For these studies 1 cc of growth media in disposable dishes§ was used. Some expiants were continuously exposed to the radioactive sulfate, while others were pulsed at 2 day intervals inand Cox, A. J.: Organ culture of human skin. J. Invest. Derm., 44' 151, 1965.

Freeze-drying of Certain Viruses

IT is important that a virus reference laboratory be able to hold viruses in the infectious state for long periods and to transmit them long distances. We have experienced difficulty with some newly isolated viruses of the respiratory tract. Some preparations of para-influenza viruses and rhinoviruses have become non-infectious after being frozen at below −;60° C in the presence of 2 per cent bovine plasma albumen or 2 per cent calf serum respectively for a year or more; the former viruses rapidly become non-infectious at room temperature1 and so have been transported in the frozen state or as infected tissue cultures. In earlier experiments virus was preserved by freeze-drying, but much infectivity was lost. In recent experiments viruses seem to have been better stabilized, and the results are reported here.

Lens cells of the calf in continuous culture.

HUMAN and animal cells of various types in continuous culture are useful for studies of cellular biology and virus-cell interactions1. The need for strains of primary epithelial cells suggested the calf lens as a suitable tissue for investigating the possibility of establishing such cells in continuous culture. On December 28, 1956, both eyes were removed from a 3-months-old, male calf immediately after the death of the animal. Within 5 min. after enucleation, the lenses were removed from the eyes and were washed repeatedly with Hanks's balanced salt solution. The anterior capsule was dissected by circular incision posterior to the equator and the stroma was removed from the capsule; the epithelium remained attached to the capsule. The capsule was supplied with 2 ml. of growth medium. The latter consisted of Hanks's solution, 30 per cent calf serum inactivated at 56° C. for 30 min., 0.5 per cent lactalbumin hydrolysate and 5,000 units of penicillin and 5 mgm. of dihydrostreptomycin/100 ml. The lens capsule was gently pipetted up and down in a coarse pipette so that small fragments, about 1 mm. in diameter, were obtained. The fragments of both lens capsules were planted without plasma in two screw-cap bottles of 200-ml. size. They were allowed to be attached to the glass for 3 min. Afterwards, the flasks were provided with 4 ml. of growth medium and they were incubated stationary at 36° C. After 7 days, 4 ml. of fresh growth medium was added per flask without removal of the old medium. Thereafter, 75 per cent of the culture fluid was renewed every seventh day for two weeks and then every third or fourth day.

Establishment of two human cell strains from kidney and reticulosarcoma in lung.

Two strains of human epithelial-like cells derived from normal kidney and from reticulosarcoma of lung are described; the cells are called T-1 and T-4 cells, respectively. The T-1 cell strain has been propagated in continuous culture for more than 21/2 years, the T-4 cell strain for more than one year. Both cell strains have been grown in media containing 40 per cent human serum, 5 per cent horse serum and 5 per cent calf serum, respectively. The cell strains have been found to propagate a number of viruses.

Utilization of serum proteins by normal and poliovirus infected monkey kidney cells

The quantitative utilization of serum protein by epithelial-like cells in monkey kidney cell cultures in vitro has been studied. It was found that all electrophoretically separable serum components were utilized by the cells during the most active growth phase, namely the first 3 days. During the second three days of growth very little protein was utilized. After infection of the cells serum protein utilization was restricted to the first day, namely the period of virus multiplication. A continuous monolayer of epithelial like cells was obtained in 6 days without the 3 day routine fluid change. Since approximately half of the 10 per cent added calf serum was utilized it appears that the addition of 5 per cent calf serum would eliminate the necessity of a fluid change.

Day-by-day response of vaccinated chimpanzees to poliomyelitic infection.

or more 1 ml doses of commercial trivalent formol inactivated poliomyelitis vaccine supplied through the courtesy of the National Foundation for Infantile Paralysis. The experiments were begun in 1954 with the low potency preparations then available, but subsequently the animals received a booster dose on February 22, 1955, of a somewhat better preparation kindly supplied by Eli Lilly and Company. Since this time much better vaccines have become available, but these were the best obtainable at the time. On March 21, 1955, the chimpanzees received an oral challenge of Brunhilde cord virus (ca 25,000 PD50). Throat swabs and sera were collected daily for the first 10 days following exposure and somewhat less frequently for the following two weeks. Two swabs were obtained and stored individually from the tonsillar areas of each animal, while stools of approximately alternate days were pooled as weekly specimens (collections on Monday, Wednesday, and Friday). All the materials for virus assay were suspended in a menstruum composed essentially of balanced salt solution, to which had been added 0.5 per cent lactalbumen hydrolysate and 0.12 per cent crystalline bovalbumen.1 In some cases 2 per cent calf serum was also included. Stools were diluted at 1:3 before clarification in the Spinco centrifuge at 34,000 x gravity for one hour; swabs were placed in 2 ml of balanced salt solution at collection to be eluted later and similarly clarified without further treatment. All specimens were tumbled overnight at 40 C with 20 per cent anaesthetic ether. After settling, the top

Multiplication in vitro of Koch Bodies of Theileria annulata

THE following simple method modified from Jacoby (1944) was found suitable for the growth in vitro of Theileria annulata. A small fragment of infected calf's spleen or lymphatic gland is placed on a sterile glass coverslip and a drop each of calf plasma and chickens' embryonic extract are added. After coagulation the coverslip is sealed to the bottom of a large Carrel flask by a drop of plasma and embryonic extract; 3–5 c.c. of a mixture of 30–40 per cent calf serum in Tyrode is then added and the flask closed and incubated. On this medium Koch bodies survive for at least twelve days, but do not multiply. If fragments of normal spleen are placed in juxtaposition to the infected fragment at intervals of 3–5 days, Koch bodies survive for 15–18 days, but there is no obvious multiplication. The addition of glutamine (3 γ per c.c), pyridoxin (0·6 γ per c.c.), inositol (4 γ per c.c.) and riboflavine (0·4 γ per c.c.) to the mixture of serum and Tyrode induces multiplication of Koch bodies, which was observed in ten successive fragments of normal calf spleen during a period of two months. The addition of these factors to the transplant, in which Koch bodies have survived without multiplication for eighteen days, immediately induced multiplication of the surviving parasites.