Seitz Filtration Research Articles

English

The commercial production of purified tetanus toxoid mainly depends on the effective separation of the bacterial toxin and toxoid from large volumes of fermentation broth of Clostridium tetani (Harvard 49205) vaccine strain. Tangential flow or cross-flow filtration system was used as rapid drive in the processing of immunobiological assays of tetanus toxin. Tetanus toxoid was prepared by detoxifying the culture filtrates of C. tetani and further purified by ultrafiltration, salt fractionation and adsorption onto aluminium phosphate. Present study deals with the separation of tetanus toxins using a microporous membrane (0.22 µm) and concentration of tetanus toxoids using an ultrafiltration membrane (30 kDa, NMWL pore size) with operational variables like average trans-membrane pressure (ATP), cross flow rate, flux. Under the best conditions, >96% recovery was achieved. Additionally, potency control of 10 batches of tetanus toxoid, prepared from the filtered toxins/toxoid lots by microporous tangential flow filtration system, was evaluated by in vitro passive haemagglutination (PHA) assay and the results obtained in the in vitro PHA were compared with in vivo toxin neutralization (TN) test. An excellent correlation between in vitro test and in vivo TN test was observed by Spearman’s correlation coefficient. It reveals that the process development in which employing available equipment and the in vitro PHA is a promising alternative to the toxic TN test in the potency assay of tetanus vaccine. Key words: Tetanus toxin, Seitz filtration, tangential flow filtration, microfiltration module, flux, tetanus toxoid, immunogenicity test.

Developments in the production and quality control of poliovirus vaccines – Historical perspectives

Using virus grown in monkey kidney cells, Salk and his colleagues developed an inactivated poliovirus vaccine (IPV) in 1952. A large-scale field trial showed the vaccine to be safe and highly immunogenic in children, but soon after the vaccine became generally available in 1955, cases of paralytic disease were reported in recipients. Investigations showed that almost all the cases occurred in children who had received vaccine from one particular manufacturer. Extensive studies attributed the disaster to problems with inactivation. Addition of a Seitz filtration step midway during formalin inactivation and extension of the inactivation period resulted in a safe vaccine. No further paralytic cases were observed following the use of several hundred million doses of this improved vaccine. Thus, IPV was safe and caused a dramatic decline in the incidence of poliomyelitis in countries where it was used. A second generation IPV is produced in fermentors using well-characterized cell strains or continuous cell lines. The major breakthrough in the development of live poliovirus vaccine was the application of tissue culture methods for virus attenuation. By 1959 several candidate live oral poliovirus vaccines (OPV) had been developed. These were clinically tested in millions of individuals and found to be safe and effective. Since the attenuated virus strains developed by Koprowski and Cox were more neurotropic in monkeys than the Sabin strains, only the latter was licensed in the USA in 1961 and endorsed shortly after by the World Health Organization (WHO). The widespread use of Sabin's OPV in many countries hastened the development of International Requirements by WHO for OPV in 1962 to define the criteria that ensured the uniformity of batches produced by different manufacturers. These have been updated continuously in light of new information and quality control procedures. Extensive field trials have shown the risk of OPV associated polio to be less than 0.3 per million doses administered.

In vitro direct haemagglutination by Corynebacterium ovis exotoxin

Haemagglutinating activity of toxigenic Corynebacterium ovis culture supernatants was estimated by titration in phosphate buffered saline diluent against sheep erythrocytes. The haemagglutination titre was closely related to the haemolysis and rabbit dermonecrosis titres of culture supernatants and to the toxigenicity of different C ovis strains. The substance responsible for haemagglutination was diffusible and its concentration was unrelated to cell yield in cultures. Corresponding inactivation of all three activities by antitoxin, toxoid formation with formalin, heat and Seitz filtration suggested that haemagglutination is another activity of C ovis exotoxin.

Neutralization of Human Serum β-Lysin by Sodium Polyanetholsulfonate and Sodium Amylosulfate

Normal fresh and heat-inactivated (56 degrees C, 30 min) human sera (80 vol%, i.e., 80% [vol/vol] of a 2-ml assay volume) killed Bacillus subtilis ATCC 6633 cell inocula of 1.5 x 10(4) colony-forming units per ml within 1 to 2 h after exposure. The B. subtilis assay strain proved slightly and reversibly susceptible to 5 mug of egg white lysozyme per ml. Seitz filtration of fresh human serum completely removed beta-lysin activity; significant amounts of serum lysozyme were removed as well, as determined with the bioassay strain Micrococcus lysodeikticus ATCC 4698. However, bactericidal activity of human serum via classical or alternative complement pathway activation remained intact. Addition of 0.01 M dithiothreitol to fresh human serum abolished beta-lysin activity, but not that of serum lysozyme. Chelation of fresh and heat-inactivated human serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid, but not with 0.01 M ethylenediaminetetraacetic acid, markedly retarded beta-lysin activity; however, lysozyme activity remained unaffected. Chelation of serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid + 0.01 M CaCl(2) completely abrogated beta-lysin activity, but not that of lysozyme. Absorption of human serum with 10 mg of bentonite per ml (10 min, 37 degrees C) completely removed beta-lysin and lysozyme activity, but failed to affect serum bactericidal activity against Escherichia coli control strain C. Reconstitution of 50 vol% of bentonite-absorbed serum with 40 vol% of heat-inactivated human serum restored both beta-lysin and lysozyme activity. Addition of either 63 to 500 mug of sodium polyanetholsulfonate per ml or 63 to 500 mug of sodium amylosulfate per ml to 80 vol% of fresh human serum completely neutralized beta-lysin activity for the entire observation period of 22 h.

The influence of Seitz filtration on the activity of hydatid fluid antigen.

Filtration of hydatid fluid through Seitz EK filter pads results in an initial retention of a part of sensitizing proteins on the filter disc. Filtration through membrane filters allows the passage of most of the proteins of the fluid and may be considered as a more satisfactory procedure for the preparation of antigen for Casoni skin tests. Membrane filtrates are likewise reactive in indirect haemagglutination tests.

Entamoeba histolytica: Encystation and cultivation of several isolates

Culture media consisting of Ringer extracts of colon contents or faecal material from man, including meconium from newborn babies, pig, horse, cow, dog, and fowl have, with addition of rice starch, supported isolation, maintenance, encystation, and excystation of Entamoeba histolytica. These media improve by one autoclave sterilization, but reautoclaving and Seitz filtration ruin their potency. Acriflavine eradicates some bacteria necessary for starch ingestion in these media. Individual cultures have been kept alive up to 50 days. Practically all strains isolated with bacteria from distal parts of the colon encyst spontaneously, commencing in the earliest cultural generations. Strains isolated with bacteria from proximal parts of the colon do not encyst spontaneously. Almost every well established strain can be induced to encyst by suitable methods. Two new methods have been successfully tried. One consists of adding acriflavine at inoculation to cultures in diphasic serum media with starch. In practically every such culture and in several subsequent cultures cysts appear. The second method consists of cooling cysts for several days, after which on reincubation fresh cysts appear in one or several subsequent cultures. This method was successful only with freshly isolated not old and well established strains. Reduced encystation capacity in two established strains was temporarily restored by addition of unmodified caecal bacteria. Viability of early, spontaneous cysts is short, but induced cysts are hatchable after several weeks in the refrigerator.

Sterilisation of colicine preparations.

Summary Preparations of colicines A, B, D, E2, E3 and K were exposed in separate experiments to chloroform, heat, filtration, and ultraviolet light and the effects were examined quantitatively. Chloroform at a concentration of 5 per cent. (v/v) sterilised preparations of each colicine within 5 min. and there was an acceptably low degree of inactivation. Exposure for longer periods was associated with progressive inactivation of B colicines and colicine K. Millipore filtration and ultraviolet irradiation are slightly more effective but technically more exacting methods applicable to large and small samples respectively. Seitz filtration removed all detectable colicine activity. Heating is not recommended as its effects on colicine activity are difficult to predict. The criteria of thermostability are discussed.

Frenching of tobacco in Australian soils and in soil leachates

In Australia frenching occurred consistently in tobacco plants grown in sandy soils that tend to remain wet for long periods. At Katherine, N.T. and Manjimup, W.A., plants grown in soils taken from a depth of 0–6 in. frenched readily, but in soils from a depth of 6–12 in. frenching symptoms in plants were less severe, appeared later, or were absent. Cultivated soils were shown to cause frenching in tobacco plants more readily than uncultivated soils. Under glasshouse conditions frenching occurred in "active soils" at a wide range of temperatures (18–45°C); high soil temperatures increased the severity and decreased the time to appearance of symptoms. Various minerals added to the soil did not influence frenching, but excess nitrogen delayed the onset of symptoms up to 3 weeks. Prolonged storage of active soils did not affect their ability to induce frenching symptoms in plants. Frenching could not be transmitted from plant to plant by grafting, mechanical means, or dodder, but could be readily transmitted from active to inactive media by inoculation. Leachates of soil from Katherine and sand from Molonglo, A.C.T. induced frenching in plants. Severity of frenching was decreased by centrifugation, flocculation, or Seitz filtration of the leachates. Heat and chemical sterilization of active media (leachates, soils, sand, and vermiculite) eliminated their ability to induce frenching in plants. A biological origin of frenching disorder is suggested.

Factors Affecting Isolation and Survival of Bacteriophages for Lactic Streptococci

Summary Comparisons were made of the abilities of Streptococcus lactis, Streptococcus cremoris and Streptococcus diacetilactis bacteriophages to endure various laboratory isolation and storage treatments. Neutralization of Cottage cheese whey containing phages offered no survival advantage over unneutralized samples; refrigeration of neutral and acid whey samples increased the survival of only the S. diacetilactis phage. About 50% of phages could be recovered from milk coagulated by addition of lactic acid to provide casein-free, clear, phage-containing whey. Sterilization of phage lysates by membrane filtration allowed collection of 92 to 99% phages; Seitz filtration allow collection of a maximum of 24%. Whey samples containing phages could be concentrated by removing water, using polyethylene glycol. Lyophilization decreased phage titers at least 50%.

Separation and Purification of β-Lysin from Normal Serum

Summary By use of Seitz filtration, elution, back filtration, and differential ultracentrifugation techniques, it has been possible to purify β-lysin. The degree of purification when measured as specific bactericidal activity/unit nitrogen was from 1000 to 5000 times that of normal serum. Separation of β-lysin from serum or purified preparations by Millipore filtration was primarily a mechanical process. A comparison of Millipore and Seitz filtration procedures revealed that the latter technique was more efficient for the purification of β-lysin. β-lysin was separated from both agglutinating antibody and complement and was not dependent on either of these serum components for bactericidal activity.

SEPARATION OF SV40 FROM POLIOVIRUS BY EXTRACTION WITH 1-BUTANOL.

SummaryPoliovirus and SV40 in tissue culture fluids are neither removed nor inactivated by extraction with diethyl ether, chloroform or fluorocarbon. However, similar treatment with 1-butanol removes large amounts of SV40 from aqueous suspensions. The infectious titer of poliovirus is practically unaffected by the same procedure. That the action of 1-butanol on SV40 is one of extraction into the solvent phase rather than virus inactivation has been demonstrated in two ways. First, an addition of 1-butanol sufficient to saturate the aqueous suspension did not reduce the infectious titer significantly. Second, the virus was recovered quantitatively from an emulsion of equal volumes of 1-butanol and virus fluid. However, when the aqueous and solvent phases were separated, the titer in the aqueous layer was reduced by at least 4 logs (base 10). Extraction of samples of rejected Sabin polio vaccine/high in adventitious SV4Q, with an equal volume of 1-butanol and subsequent Seitz filtration reduced the concentr...

ANALYSES OF WOUND EXUDATES FOR CLOSTRIDIAL TOXINS.

Noyes, Howard E. (Walter Reed Army Institute of Research, Washington, D.C.), William L. Pritchard, Floyd B. Brinkley, and Janice A. Mendelson. Analyses of wound exudates for clostridial toxins. J. Bacteriol. 87:623-629. 1964.-Earlier studies indicated that death of goats with traumatic wounds of the hindquarter could be related to the number of clostridia in the wounds, and that toxicity of wound exudates for mice and guinea pigs could be partially neutralized by commercial trivalent gas gangrene antitoxin. This report describes in vitro and in vivo analyses of wound exudates for known clostridial toxins. Wounds were produced by detonation of high-explosive pellets. Wound exudates were obtained by cold saline extraction of both necrotic tissues and gauze sponges used to cover the wounds. Exudates were sterilized by Seitz filtration in the cold. In vitro tests were used to measure alpha-, theta-, and mu-toxins of Clostridium perfringens and the epsilon-toxin of C. novyi. Mouse protection tests, employing commercial typing antisera, were used to analyze exudates for other clostridial toxins. Lethality of wound exudates for mice could be related to (i) the numbers of clostridia present in the wound, (ii) survival time of the goats, and (iii) positive lecithovitellin (LV) tests of the exudates. However, the LV tests could not be neutralized by antitoxin specific for C. perfringens alpha-toxin. Mice were not protected by typing antisera specific for types A, C, or D C. perfringens or C. septicum but were protected by antisera specific for type B C. perfringens and types A and B C. novyi.

BACTERIOLOGY OF SPOILAGE OF FISH MUSCLE. I. STERILE PRESS JUICE AS A SUITABLE EXPERIMENTAL MEDIUM.

A sterile raw fish muscle press juice, diluted 1:4 with saline, has been prepared. This dilution greatly facilitated Seitz filtration and affected the spoilage properties of the medium only negligibly. At 5.5 C, the spoilage pattern of naturally contaminated diluted juice was almost identical to that of naturally contaminated fillets. This was shown by comparing the quantitative and qualitative aspects of the bacterial flora on the two substrates and by measuring the production of volatile reducing substances (VRS) and of trimethylamine (TMA). With the sterile raw muscle press juice, some preliminary data showed that individual members of the genera Achromobacter and Pseudomonas differ markedly in their spoilage capabilities: some grew but did not produce spoilage detectable either organoleptically or chemically; others gave rise to strong off odors and to high levels of VRS and TMA.

Toxic Effect of Staphylococcal Lysins for Goldfish1

Goldfish died within 24 hr after intraperitoneal injections of 0.2 ml of Seitz filtrates of hemolytic Staphylococcus aureus cultures grown on Dolman and Wilson medium under increased CO(2) pressure for 72 to 96 hr. Two lethal toxins differing in heat sensitivity, antigenicity, and degree of toxicity were demonstrated. Studies of the relationship between the lethal factors and the hemolysins in the filtrates suggested that alpha- and beta-lysin were responsible for the lethal effects. Filtrates of nonhemolytic staphylococcal cultures were innocuous. Goldfish were suitable animals for detecting toxicity in staphylococcal culture filtrates and for quantitative studies of the toxins. The results were highly reproducible.

Toxic effect of staphylococcal lysins for goldfish.

Goldfish died within 24 hr after intraperitoneal injections of 0.2 ml of Seitz filtrates of hemolytic Staphylococcus aureus cultures grown on Dolman and Wilson medium under increased CO(2) pressure for 72 to 96 hr. Two lethal toxins differing in heat sensitivity, antigenicity, and degree of toxicity were demonstrated. Studies of the relationship between the lethal factors and the hemolysins in the filtrates suggested that alpha- and beta-lysin were responsible for the lethal effects. Filtrates of nonhemolytic staphylococcal cultures were innocuous. Goldfish were suitable animals for detecting toxicity in staphylococcal culture filtrates and for quantitative studies of the toxins. The results were highly reproducible.

An Evaluation of β-Propiolactone for the Sterilization of Fermentation Media

Twenty-five bacterial species were cultured in basal broth plus 1 of 19 different carbohydrates which were sterilized by Seitz filtration, autoclaving (112 C, 10 min), or exposure to 0.2% β-propiolactone (BPL). No significant differences were found either in the visual observations for acid and gas, pH, or titrable acidity determinations after 3 days of incubation with any of the three preparations tested. An effort was made to further determine the effect of BPL and heat on carbohydrates by assaying for glucose before and after treatment. Results indicated that glucose was not degraded by 0.2% BPL, however, it was shown that autoclave temperatures caused extensive degradation. Statistical treatment of the results from Warburg studies indicated that BPL-treated glucose showed no appreciable toxic effects, although the actual oxygen uptake was not as great as with Seitz- or autoclave-treated glucose. The application of the BPL sterilization process was discussed.

An Evaluation of β-Propiolactone for the Sterilization of Fermentation Media1

Twenty-five bacterial species were cultured in basal broth plus 1 of 19 different carbohydrates which were sterilized by Seitz filtration, autoclaving (112 C, 10 min), or exposure to 0.2% beta-propiolactone (BPL). No significant differences were found either in the visual observations for acid and gas, pH, or titrable acidity determinations after 3 days of incubation with any of the three preparations tested. An effort was made to further determine the effect of BPL and heat on carbohydrates by assaying for glucose before and after treatment. Results indicated that glucose was not degraded by 0.2% BPL, however, it was shown that autoclave temperatures caused extensive degradation. Statistical treatment of the results from Warburg studies indicated that BPL-treated glucose showed no appreciable toxic effects, although the actual oxygen uptake was not as great as with Seitz- or autoclave-treated glucose. The application of the BPL sterilization process was discussed.

Allergenic food extracts. I. Methods of preparation: Results of a questionnaire

The majority of allergists making their own food extracts used fresh uncooked food as the source of their raw material. The food was then ground or cut into smaller particles. The majority did not defat fruits and vegetables, but did defat meats, fish, nuts, grains, and spices. The majority used a weight-by-volume method of preparing their extracts and made dilutions of same for intracutaneous testing. Either alkaline extracting fluid (Coca's solution), glycerin saline, or buffered saline was used by the allergists in general for extracting purposes. Almost one third (forty-eight) of the allergists did not concentrate food extracts. Among these were twenty-three who used 50 per cent glycerin as an extracting fluid, which rendered it difficult to concentrate. Nearly all physicians sterilized their extracts by filtration through a Seitz type of pad. Several allergists did no sterility tests subsequent to Seitz filtration. Twenty-nine physicians incubated for intervals up to three days. Three-fourths (seventy-one) of the physicians reporting did not check the pH of the extract before or after Seitz infiltration. Most of these physicians did not dialyze the extract for pH adjustment. A weight-by-volume method of standardization for food extracts was used by a majority (sixty-nine) of the allergists. There were variations in procedure in that some used a dry weight and others a wet weight as the basis for the weight-by-volume method of standardization. A few concentrated their extracts, but most did not. The strength of the testing solution showed wide variations, even among persons using the same method of extraction and standardization. The numerous variables that have been presented are not being subjected to critical analysis. Further investigation is necessary to determine the effect of each of these variables upon the allergenicity of the final extract.

Role of lipid in hemagglutination by Candida albicans and Saccharomyces cerevisiae.

SummaryThe erythrocyte sensitizing antigen in saline extracts from yeast and yeastlike organisms was inactivated by a number of procedures including exhaustive deproteinization wtih chloroform-n-butyl alcohol mixtures, Seitz filtration, albumin blocking and treatment with lipase. The erythrocyte sensitizing antigen from S. cerevisiae was found to be more heat-labile and more readily inactivated by lipase than the C. albicans antigen.

A Study of the Separation of Fibrinogen and Antihemophilic Factor (AHF) in Canine, Porcine, and Human Plasmas

Summary1. Studies are reported on attempts to separate antihemophilic factor (AHF) from fibrinogen in plasma by selective adsorption with fuller’s earth, aluminum hydroxide gel, and Seitz filtration.2. Fuller’s earth adsorption of canine plasma resulted in almost complete removal of fibrinogen without loss of AHF. By the use of this procedure as part of the fractionation procedure, potent AHF fractions free of fibrinogen have been obtained.3. Al(OH)3 adsorption of fibrinogen from bovine, canine, and human plasmas is greater than the adsorption of AHF. Al(OH)3 adsorption offers promise for the preparation of fibrinogen-poor AHF fractions of human or bovine plasma.4. Seitz filtration of plasma causes loss of AHF with little or no loss of fibrinogen.