Elimination of bacteriophages from tissue culture serum by affinity chromatography.

Use of immunoadsorbents to remove bacteriophages from tissue culture serum was investigated. Immune globulins from rabbit antiserum prepared against phi V-1 phage were immobilized by covalent linkage to activated porous silica glass derivatives of p-aminoarylamine and to Sepharose-4B. Chromatographic columns of each material were used to filter samples of a fetal bovine serum into which had been introduced 8100 plaque-forming units of the phage per ml. Efficiency of removal was determined by plaque assays of phi V-1 phage recovered in the effluent fluids. Activated but uncoupled matrices nonspecifically removed from 49 to 59% of the phages introduced into the experimental serum. A reduction of 35 to 37% in phage content occurred in the serum after filtration through columns coupled to nonantibody protein. With specific immune globulins attached, the Sepharose-4B matrix reduced the concentration of phage in the serum below a detectable quantity. Noapparent alterations occurred in the growth-promoting property of serum filtered through the Sepharose-4B immunoadsorbent as measured by cloning efficiency of BHK-21, WI-38, and FRhL-2 cells. These experiments serve as a model system for use of immunoadsorbents for selective removal of bacteriophages and perhaps other extraneous microbial agents from tissue culture serum.

Bacteriological activity in unfiltered calf sera collected for tissue culture use.

Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found were Bacillus species and streptococci and 63% of the sera coagulated Limulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain of Escherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.

Persistence of bacteriophages in experimentally infected cell cultures.

A bacteriophage, isolated from a live measles vaccine and designated varphiV-1(L), was intentionally introduced into cell culture systems. The bacterial virus persisted in the cell cultures after daily medium changes and trypsinization, and in cell lysates prepared by freezing and thawing. The results suggest that phages contaminating cell cultures may be carried over into the final product during the manufacture of viral vaccines.

Survival of animal tissue cells in primary culture in the absence of serum.

The ability of cells from tissues of several species of animals to survive in primary culture without serum was tested. Of the species tested, cells from the kidneys of Macaca mulatta (rhesus) and Cercopithecus aethiops (vervet) monkeys and chicken embryo cells not only survived under these conditions, but indeed developed into confluent monolayer cultures. The addition of either serum or its globulin or albumin fraction enhanced the development of cell monolayers and permitted those cells unable to survive in the absence of serum to do so. Certain specific protein trypsin-inhibitors not of serum origin were unable to provide conditions necessary for cell survival or growth when used in place of serum proteins.

Evaluation of three cell culture systems as substrates for influenza virus assay.

The propagation of various influenza virus strains in primary rhesus monkey kidney (RMK), primary hamster kidney (HK), and the MDCK-USD canine kidney (CK) cell cultures was compared. Virus-infected cultures were examined for cytopathic effect (CPE) and by performing the hemadsorption (HAd) technique. The highest HAd titers were found most often in HK, followed by RMK and CK. However, the CK cell line provided the best substrate for detecting CPE. Although influenza B strains tended to grow to higher titers, these strains produced less CPE than did the A strains.

Adaptation of a roller-tube apparatus to accommodate cultivation of cells in roller-bottles.

We have adapted the standard roller-tube apparatus to production of large scale cell culture by modifying the drum to accept bottles.

Short-term storage at 4 C of trypsinized tissues.

Cells dispersed from tissues of certain animals remained viable for several days at 4 C although the per cent of viable cells varied among the different species.