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The risk of adventitious contamination and subsequent overgrowth of cell lines by unrelated cells is a potential and often recurring problem where cells are grown and studied. This problem of intraspecies and interspecies cross-contamination among human cell lines has been recognized for over 25 years; incidences of cell cross-contamination between 17 and 35% have been reported. The most useful methods to detect human cell cross-contamination are DNA fingerprinting and cytogenetic analysis, each complementing the other. Using this combination, we found that in total 14.8% of the human hematopoietic cell lines received either from the original investigator (n = 117 cell lines) or from secondary sources (n = 72 cell lines) were cross-contaminated with another hematopoietic cell line and were thus false cell cultures. Another problem relates to the fact that not every cell line established from a patient with a hematopoietic malignancy is a malignant cell line; unintended immortalization of non-malignant B cells by 'passenger' Epstein-Barr virus (EBV) leads to the establishment of B-lymphoblastoid cell lines (termed EBV+ B-LCLs), an event which is much more frequent than the establishment of a 'true' leukemia-lymphoma-myeloma cell line. These EBV+B-LCLs are most often (albeit not always) unrelated to the malignant clone. The misinterpretation of such EBV+ B-LCLs as true malignant hematopoietic cell lines (particularly in research areas investigating B cell-derived neoplasms such as myeloma) and the indiscriminate use of these cell lines may render some of the results of such studies irrelevant to the pathobiology of the disease concerned. However, a combination of markers commonly allows for an accurate determination of the nature of EBV+ B-LCLs: immunoprofile, cellular morphology, EBV status, and karyotype. In summary, the continuous need for vigilant quality and identity control procedures is emphasized by the high incidences of cross-contaminated cell lines. Most laboratories using cells cultured in vitro maintain multiple cell lines. Such cell lines should be monitored regularly for their identity and specific characteristics in order to prevent invalidation of research work due to incidents of cell line cross-contamination or misinterpretation.

The scarcity of donors for allogeneic bone marrow transplantation, the limited number of haematopoietic stem cell (HSC)/progenitors in cord blood samples and the sometimes insufficient number of mobilized peripheral blood cells collected from heavily treated cancer patients may benefit from ex vivo expansion of these cells for clinical transplantation. Depending on the clinical application, expansion of different haematopoietic cell subsets is required. HSC transplantation requires expansion of all cellular subsets including precursors, progenitors and HSCs for the short and long-term engraftment of patients. Quiescent HSCs may also be required for gene therapy by retrovirus. Finally, amplification of cells such as dendritic cells (DC) and different subsets of T and natural killer (NK) cells is required for immunotherapy. The different haematopoietic lineages are produced under different experimental conditions and the starting population is a critical parameter for the proposed clinical application. So it is essential to define the aims of haematopoietic cell expansion and to adapt the experimental conditions to obtain the required cell population. Mobilized peripheral blood cells are increasingly used as a source of haematopoietic cells. We review the biological characteristics of mobilized peripheral blood and the expansion of the different components according to the aims of their clinical use in the context of the progress currently achieved.

With the aim of developing a standardized program of clonogenic cultures, a multicenter intercomparative study of human CFU-GM, BFU-E and CFU-GEMM cultures was conducted. Aliquots of fresh mononuclear cord blood cells, as well as uniform culture materials and instructions for cell culture and for colony scoring were distributed to 28 national laboratories involved in hematopoietic research and transplantation. High interlaboratory coefficients of variation (CV) in the reported number of progenitors were found in our first intercomparative study (range 67-231%). To investigate the relevance of colony scoring in variations of the reported colony numbers, participants were invited to attend a meeting where a single culture dish was scored. In this case, the CVs ranged from 31% to 81%. A subsequent intercomparative assay was then conducted, and significant reductions in the inter-laboratory CVs were obtained with respect to the first study (CVs for colonies grown with two different media: CFU-GMs, 48% and 55%; BFU-Es, 70% and 62%; CFU-GEMMs, 70% and 51%; respectively). In most instances CVs were not significantly different from those obtained in the single plate scoring study, suggesting that the scoring process was the most relevant parameter accounting for variations in the reported colony numbers.

The development of media free of serum and animal or human protein is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. The main drawback associated with the use of serum or animal-derived substances for animal cell technology is the potential introduction of contaminants (adventitious agents) into the process and thus potentially into the final product. This fact led to an increased effort to replace serum-containing with serum-free media. In most cases, these media are supplemented with purified proteins, peptones, or hydrolysates, mainly of animal or human origin. Although such serum-free media are more defined than serum-containing media, the risk of the introduction of viruses by using animal-derived substances is still present, signifying that only a complete replacement of animal-derived substances by non-animal-derived products leads to a relatively safe serum-free medium. The potential replacement of these animal/human-derived substances by those of non-animal origin (e.g. plant origin) will be discussed. In several examples, the potential of serum-free media free of any animal-derived component in supporting cell growth and production of biologicals will be presented. In this context, the risk of using non-animal-derived substances in serum-free media for animal cell technology will be discussed with respect to classically used cell culture media.

Biological materials are variable by their very nature. Serum in particular is a complex mix of substances and may contain adventitious agents. Serum for pharmaceutical use is controlled on a batch to batch basis given that each batch may have differing properties. Control points for slaughterhouse obtained bovine and foetal bovine serum exist with the disease status and disease surveillance of the country of origin, veterinary inspection for clinical disease, collection methods and post collection quality control. With these control points many batches of commercially available serum are contaminated with viruses such as BVD. The use of donor serum provides a mechanism to extend Good Manufacturing Practice principles back to the farm environment giving the manufacturer the ability to increase control on production parameters in the donor animal such as disease and specific antibody status, genetic type and history, age, diet, treatments and origin of feed. As well as providing a secure supply of serum for manufacture, donor herds provide added traceability and give the opportunity for in vivo manipulation of the serum and specialised selection of the animals for specific applications with more consistent reproducibility of performance. The BSE crisis of the last 10 years has highlighted the added control that donor serum producers can exert: Many producers of donor serum applied ruminant feed bans on their animals ahead of nation-wide bans in their own countries. SPF donor herds can be set up in several ways and the purchaser should make themselves familiar with the methods used. SPF herds with accompanying seronegative status allow quality control testing of product free from the interference of specific antibodies. Such specific antibody freedom may also allow better growth of viruses for vaccine manufacture.

Growth of continuous cell lines for preparing biopharmaceuticals in the absence of animal serum has been attempted by many organizations to improve process and product quality, prevent exposure to adventitious agents, and reduce costs. Literature surveys suggest that substantial academic studies on serum-free medium have been pursued for many decades, with varying levels of success for different cell types and cell lines in terms of achieving cell growth while retaining cell function. Industrial research proceeded for at least three decades. Recent work with CHO cells and with some hybridomas has been successful in providing the basis for serially propagating cells on a large scale in suspension in the total absence of serum, while preserving the ability to prepare biopharmaceuticals. In some cases, this can be achieved not only without serum, but also without the use of other animal-derived proteins.

Serum-free formulations may be << re-engineered >> to eliminate traditional protein constituents and replace their biological function with non-protein substitutes. Non-protein additives may also be obtained from animal sources. Nutrient formulations totally free of exogenous protein and containing no materials of animal origin may be designed for high density cell culture and biological production. Cell-culture medium production requires (i) strict vendor qualification and raw material specifications; (ii) scrupulous maintenance of media kitchen facility and equipment; (iii) monitoring of process water; (iv) air-handling systems and technical personnel; (v) clearly-defined manufacturing protocols to ensure correct formulation and dispensing and (vi) validated sanitization processes to guard against cross-contamination within a multi-use facility.

Eukaryotic cells, in general, require serum for growth in vitro. Serum is a complex mixture of a large number of constituents, so the addition of serum introduces an ambiguous factor into cell cultivation. However, many commercially available sera are of a high uniform quality. Of these, foetal bovine serum is the most frequently used and is capable of supporting the growth of a wide variety of eukaryotic cells. However, with the identification of essential growth factors and nutrients required by different cells, several very effective serum-free media have been formulated. The use of these serum-free media is limited to a very narrow range of cells. Regulatory constraints generally make it impractical and uneconomic to alter existing biopharmaceutical production processes in which serum is used as a raw material.

The history of cell substrates for the manufacture of biological products is directly related to a series of technical advances and challenges to the status quo, some of which were accepted quickly while others took more than a decade to resolve. The development of cell culture techniques in the 1950s opened the door to the manufacture of a wide range of biological products. The first major challenge occurred in the late 1960s when human diploid cells (HDCs) were developed and proposed as an alternative to primary cell cultures for the production of live viral vaccines such as polio, which up to that point had been produced in primary cells of various species. In the 1970s, attention was focussed on the use of continuous cell lines (CCLs) for the production of non-replicating biological products such as interferon (IFN). The next significant technical advance and challenge was the development of recombinant DNA and monoclonal antibody technologies in the 1980s, both of which required the use of CCLs. Although most of the issues relating to CCLs in the manufacture of biological products have been resolved, issues related to their use as substrates for live viral vaccines remain to be fully addressed. Those experiences in the past teach us clearly that a system in which regulatory authorities, industry, and the general biomedical community cooperate in finding solutions to problems and in reaching consensus on issues raised by technical advances is ultimately in everyone's best interest. The World Health Organization has played a major role in that regard, and it should continue to provide leadership in this area.

Animal cell lines are increasingly used in the manufacture of viral vaccines. They may play a key role in the preparation of seed stock virus and vaccine production. However, the same animal cell substrates may also be used for diagnosis of viral infection and surveillance of prevalent virus strains. Quality control of cell lines intended for use in this range of procedures is vital to ensure the absence of contaminating organisms and correct identity of the substrate cells used. Furthermore, the qualification and validation of the procedures, facilities and staff involved in the cell culture and testing are also important issues addressed in regulatory guidelines and regulations. The standards to which these activities are performed are dependent on whether the cells are intended for diagnostic and surveillance work or for seed stock virus isolation and production. This paper indicates when and how some of the relevant quality standards and quality control issues apply to cell lines intended for the different procedures involved in virus isolation and vaccine production.

The United States Code of Federal Regulations requires that all influenza virus vaccines produced for use in the United States adhere to specific regulatory standards including the demonstration of safety and efficacy. For vaccines produced in cell lines, rigorous characterization for manufacturing is particularly important. Influenza vaccines produced by the passage of viruses in mammalian cell lines will require careful evaluation to ensure the removal or inactivation of potential adventitious agents.

Infection with bovine viral diarrhoea virus (BVDV) and other viruses is frequent in the bovine population. In utero infection leads to virus and antibody contamination of foetal and other serum used in cell culture production. The use of contaminated cells for vaccine production may result in contaminated vaccines, which may lead to seroconversion or disease in the vaccinated animal. Contaminated serum or cell cultures may also interfere with the diagnosis of viral infections. Methods for the detection of BVDV and other viruses in serum, cell cultures, seed viruses and vaccines at the CVB-L, and the frequency of detection are described. Reasons for continued use of serum in cell culture production, and the risks of using serum, are discussed.

Serum-free culture is used routinely for many cell types, especially if transformed, e.g. CHO, hybridoma and recombinant myeloma cell lines. Serum-free medium reduces operating costs and process variability, and removes a potential source of infectious agents. The removal of the components of serum and the elimination of other animal-derived raw materials are proving more challenging. Nevertheless, careful risk/benefit analyses to target R&D toward critical materials is presenting several solutions in this area. A risk/benefit analysis is required for each component replacing serum or serum derivatives. Specific serum proteins used in serum-free media can be eliminated. At present large scale protein-free processes are not routine.

The American Association of Blood Banks requires routine culture of hematopoietic progenitor cells prior to bone marrow transplantation. We sought to evaluate the cost of that requirement and the incidence and clinical significance of positive cultures. We performed a retrospective analysis of transplant recipients at our institution. Of the 605 patients for whom 1,934 consecutive cultures of harvests were done between December 1992 and February 1996, 11 had positive cultures. Six patients received a culture-positive harvest with no adverse effects. The total cost of cultures was $35,660 (U.S. $). In North America and worldwide in 1995, routine culture of harvests would have prevented 7.9 and 18.9 cases of bacteremia, respectively, at a cost of $95,000 per bacteremia prevented. We conclude that routine culture of hematopoietic progenitor cells yields low rates of positivity and that infusion of contaminated harvests rarely results in clinically adverse outcomes.

Veterinary diagnostic test kits are used in clinical practice and production medicine as a basis for prescribing vaccination and/or treatment regimens. These kits are also used by state and federal animal health officials to control or regulate the movement of animals. The regulation of such kits, which generally takes the form of establishing requirements for licensure, ensures that the kits conform to minimum standards for sensitivity, specificity and reproducibility. To qualify for licensing, diagnostic test kits should have consistent performance characteristics.

In this paper we determined the prevalence of mycoplasma contamination in 17 cell lines. Eighty per cent of the laboratories that currently use cell culture techniques participated in this study. Hoechst 33258 dye was used to detect mycoplasma contamination. The relationship between culture maintenance conditions and the presence of mycoplasma were analyzed, considering the use of antibiotics in the culture media, fetal calf serum (FCS) quality, culture media processing, use of disponsable labware, type of laminar flow cabinet, quantity of operators, and cell culture system. Thirty-five per cent of the analyzed cell lines showed mycoplasma contamination. Those lines belonged to 2 of the 8 surveyed laboratories. When confronting the working conditions versus mycoplasma contamination, 66% of the laboratories that employ non-certified FCS or reuse their labware, show mycoplasma contamination. Mycoplasma presence was found in 50% of the laboratories that use closed culture system, or more than one operator. Laboratories that process their culture media or that include antibiotic in the growing media, show a 40% contamination. The results obtained help to establish working conditions necessary to avoid introducing or spreading the microorganism.

Based on the formation kinetics of monoclonal antibody, the bench-scale culture of hybridoma cells in a bioreactor was optimized by the application of perfusion culture mode, the supplement of potassium acetate, and the fortification of nutrients. When the bioreactor was operated with the daily perfusion of medium of 1/2 working volume, viable cell density in bioreactor was 11 x 10(5) cells/ml and antibody concentration 28 mg/L. After supplement with 1 g/L potassium acetate, the cell density kept constant and antibody production was improved to 38 mg/L. The fortification of nutrients such as amino acids and vitamins raised the cell density up to 42 x 10(5) cells/ml and, responsively, antibody concentration to 94 mg/L.

The subject of the chapter is cell culture contamination. Contamination may enter the cell culture system as a physical, chemical, and/or biological component of the environment. The potential sources and consequences of cell culture contamination are unique to the cell culture system and the contaminant. A basic understanding of cell culture contamination is necessary to appreciate the need to develop and practice standardized cell culture procedures. General sources, consequences, and preventative measures are discussed for physical and chemical contamination based on current technology. Mycoplasmal contamination is the focus of the discussion on biological contamination and its impact on cell cultures. The introduction of other biological contaminants should be controlled by the institution of cell culture management procedures needed to minimize the incidence of mycoplasmal contamination. The need to eliminate the routine use of antibiotics in cell culture systems and institute routine testing to detect contamination is emphasized. More rapid detection of contamination should reduce the incidence of cross-contamination and minimize the consequences of any contamination event.

The properties of baby hamster kidney (BHK 21) cells are modified by passage in suspension culture. The suspended cells differ from monolayer cells in the surface expression of some integrin chains involved in attachment of foot-and-mouth disease virus (FMDV), in particular the progressive down-regulation of both alpha5 and alphaV integrin chains. This down-regulation is correlated with the loss of actin stress fibres. FMDV particles from these cells are unstable towards the aziridine used in inactivating the virus for vaccine production. Moreover, growth of virus in suspended cells can lead to the selection of antigenic variants which differ from those produced in monolayer cells.

The analysis of the gel electrophoresis banding patterns and relative migration distances for the individual isoforms of intracellular enzymes, such as lactate dehydrogenase, purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase, is used routinely in the biopharmaceutical industry for confirmation of cell line species of origin. In the present study, the sensitivity of the technique (AuthentiKit, Innovative Chemistry, Marshfield, MA) for determining interspecies cell line cross-contamination was examined. Extracts were prepared from a CHO-K1 line (AA8, Chinese hamster), MRC-5 (human) cells, and L929 (mouse) cells and from several proportional mixtures of the various binary combinations of cells. The isoenzymes were analyzed according to standard procedures for the technique. Contamination of MRC-5 cells with CHO-K1 or with L929 cells was clearly detectable with each enzyme analyzed. Similarly, the contamination of L929 or CHO-K1 cells with MRC-5 cells was readily apparent with each enzyme. On the other hand, contamination of CHO-K1 cells with L929 cells was only detected with lactate dehydrogenase analysis, and contamination of L929 cells with CHO-K1 cells was not detected with any of the four enzymes examined. For the latter case, the analysis of an additional enzyme (peptidase B) was required. The results indicate that interspecies cross-contamination should be detectable with isoenzyme analysis if the contaminating cells represent at least 10% of the total cell population.

Approximately 20 years ago the first child conceived with in vitro fertilization (IVF) and embryo transfer was born in England. Although overall pregnancy rates and delivery rates after IVF have improved over the years, success between IVF clinics can vary as much as tenfold. The factors that influence the success rate include type of patient, ovarian stimulation protocol, quality of oocytes, culture conditions, handling of gametes and embryos, quality of embryos, embryo transfer technique, and endometrial receptivity. Culture conditions and handling of gametes and embryos will be reviewed with the hope of improving success of human IVF. The ongoing clinical pregnancy rates in our program increased from about 30% in 1995 and 1996 to about 50% in 1997. Improved embryo culture conditions and embryo cryopreservation technology should not only increase fresh but also frozen-thawed embryo transfer pregnancy. This will make IVF efficient and cost-effective while achieving high overall pregnancy rates with lower multiple pregnancy rates.

The presence of residual host cell proteins in protein pharmaceuticals obtained from continuous mammalian cell lines has been a point of concern. Clinical experience with different biopharmaceuticals shows that residual protein in these highly purified products does not represent a danger to the health of the patients.

Cell cross-contamination in cell cultures is a common problem during cell culturing and use. Contamination invalidates research results, compromises the comparison of results between laboratories, reduces reproducibility required in industrial production of cell lines, and may lead to unusable therapeutic products. The problem can be solved by increasing the awareness of its seriousness and by introducing regular quality control of cell cross-contamination in every laboratory where cells are grown and used.

For many years the debate surrounding the potential danger of contaminating cellular DNA in biological products has focussed on the possibility that this DNA could give rise to tumours in the recipient host. Current knowledge of the nature and the fate of this DNA after in vivo administration allows us to conclude that this material represents a minimal risk to the patient.

Media, chemicals, sera and protocols used for in vitro production of bovine embryos are different from one laboratory to another. This paper describes some of the critical steps required to produce embryos in vitro, suggests quality control measures to lower variations in blastocyst yield and describes different tests which may be used when implementing new procedures in a routine production system.

As infertility treatments evolve and techniques are developed to improve the fertilization and pregnancy rates, it is clear that the in vitro environment in which the gametes/embryos are cultured is less than perfect when compared to the in vivo counterpart. This becomes a serious problem for couples who seek to have children but are limited by unsuccessful attempts to become pregnant and a technology that is behind in mimicking the in vivo environment that would enhance gamete interaction and embryo development. In an attempt to begin to correlate the in vivo and in vitro microenvironment of the gametes/embryos and to recognize the potential of the fallopian tube as more then a highway for oocytes to transverse in their pathway to the uterus, coculturing of tubal cells along with gametes and embryos was introduced to the in vitro fertilization community. This report is an attempt to compile the research and results of those attempting to improve pregnancy rates by improving in vitro culture conditions via co-culture of cells with gametes and/or embryos.

Large-scale cell culture operations for biotechnology products use millions of litres of complex media and gases as well as huge quantities of organic and inorganic raw materials. These raw materials must always be assumed to contain contamination by adventitious agents such as Minute Virus of Mice (MVM). Genentech has had experience in dealing with two such contaminations. Although the source of these contaminations was not positively identified, there was strong evidence to suggest that the virus entered through a raw material used in cell culture. Analytical methods that can be used to detect the presence of viruses can be used as an early warning system and as part of a strategy to devise barriers against such contamination. Methods such as a polymerase chain reaction (PCR) assay and a cell culture-based infectivity assay have been found to be efficacious in providing early detection of MVM infection. In any contamination, timely interactions with regulatory authorities are vital in minimizing delays in product manufacture.

The informations obtained using in vitro folliculogenesis in rodents have shown that ovulation of preantral follicles or granulosa-cumulus complexes can be obtained after a few days of in vitro culture and that live young can be obtained following fertilization and development of these oocytes. In addition, initiation of follicular growth from primordial follicles could be achieved in organ culture in vitro. Hence, when the two techniques were combined, all folliculogenesis (from the primordial stage to ovulation) could be achieved in vitro. The situation is more complex in primates/human owing to the lower number of follicles initiating growth per day and to the longer duration of folliculogenesis. A few reports suggest that preantral follicles can survive and grow in culture for a few days and one study demonstrates that in 2 months, follicles can grow from 150 microns to 1.6 mm in diameter. The short term interest of such studies is to improve our understanding of the mechanisms involved in basal folliculogenesis. In the longer term, such techniques may prove useful to maintain some reproductive potential to women submitted to total body irradiation, to improve the management of reproduction of women suffering from early menopause or to generate oocyte banks to donate oocytes.

During the last 20 years, numerous studies have been performed to improve culture conditions in order to allow in vitro growth and meiotic maturation of oocytes isolated from primordial to antral follicles. The main objective of these studies is to define optimal culture conditions which will allow to increase the recovery rate of oocytes that can be successfully fertilized. This paper reviews the more recent advances obtained when studying mouse oocyte development in vitro.

Continuous cell lines offer a level of reproducibility, and thus standardization, which cannot normally be achieved using primary cells. However, even with continuous cell lines adoption of correct cell banking and appropriate quality control procedures are critical to the provision of reliable, reproducible and safe cell stocks. These procedures enable establishment of cryopreserved stocks of pure cultures of correct identity and phenotype which are free from adventitious agents. In addition to quality control techniques, culture conditions and growth medium used often require standardization. In particular different sources of serum, growth factors and cell attachment substrates may lead to significant variation in the 'performance' of cell lines. To ensure a high degree of reliability it is essential to obtain cells from authenticated and quality controlled sources. In culture collections, the principles of correct cell banking should be applied with appropriate quality control for which the minimum standard should be confirmation of viability and mycoplasma testing. Such approaches will afford increased confidence in research data and avoid the waste of time and resources which result from the use of cross-contaminated or infected cells.

The combination of co-cultivation of hepatocytes and epithelial cell lines with a newly developed perifusion system was used for in vitro studies on drug metabolism and cytotoxicity. This approach improved the viability and enhanced the induction of the biotransforming capacity of the hepatocytes. As demonstrated for the induction of 7-ethoxyresorufin O-deethylase activity by 3-methylcholanthrene or benzanthracene, co-cultured hepatocytes in the perifusion system responded more sensitively to these inducers than without perifusion, most likely owing to stable (steady-state) concentrations of the inducers under the former conditions and rapidly declining concentrations under the latter conditions. The perifusion approach rendered it possible to determine the kinetics of drug metabolism during single or sequential incubations. After induction with 3-methylcholanthrene and phenobarbital, phase I metabolism of lonazolac to the monohydroxylated product in perifused co-cultures closely (87%) approached the values reported for the in vivo production, whereas in stationary co-cultures only 52% could be reached. Likewise, cytotoxic effects could be detected more precisely in the perifused co-cultures. If cells were pretreated with 0.2 mmol/L galactosamine for 3 h, perifusion with increasing concentrations of menadione differentially killed epithelial RL-ET-14 cells and hepatocytes at low and high concentrations, respectively, while in stationary co-cultures no differential effect was observed and only the higher concentrations were cytotoxic for both cells. Prevention by incubation with S-adenosylmethionine of menadione cytotoxicity up to a menadione concentration of 250 micromol/L was seen only in the perifused co-cultures, whereas in stationary cultures only a slight shift of the cytotoxic concentration exerting 50% cell damage to higher values was noted. These results demonstrate the versatile application of perifused co-cultures for studies on drug metabolism including induction of cytochrome P450-dependent enzymes and steady-state kinetics of biotransformation, as well as cytotoxic and protective effects of different drugs.

The possibility of air-droplet cell transmission during cell transfer was experimentally confirmed, this appearing to be the most probable route of cross contamination of cell cultures. The consequences of such contamination are assessed and measures reducing the risk of intercellular contamination proposed.

In an effort to determine how long a wide spectrum of organisms will survive in either an isotonic or a hypertonic blood culture system, all clinical blood culture flasks were subcultured on a daily basis for the first seven days of incubation and again on the fourteenth day. This subculture included all those flasks found to harbor organisms on previous subculture. Organisms such as members of the Enterobacteriaeceae and Pseudomonadaceae, as well as Staphylococcus aureus, Bacteroides spp., Eubacterium and Candida spp., survive for at least 14 days in both isotonic and hypertonic blood culture systems. However, organisms such as the various streptococci, H. influenzae, and N. meningitidis start to die off in hypertonic media within four days and in isotonic media within five days. Of 47 isolates of S. pneumoniae, 9 of N. meningitidis and 14 of H. influenzae obtained from the hypertonic system, only 21, 3 and 2 isolates, respectively, were still recoverable on the seventh day of incubation. In this study, parallel culture methods were used to compare the two systems, with the result that the hypertonic system allowed for recovery in 356 instances while the isotonic system allowed recovery in only 319 instances. It would appear from the results of this study that many organisms tend to die off more rapidly in a hypertonic system than in an isotonic system; however, fewer recoveries occur with the isotonic system.