Contamination Of Culture Medium Research Articles

Chemicals eluting from disposable plastic syringes and syringe filters alter neurite growth, axogenesis and the microtubule cytoskeleton in cultured hippocampal neurons.

Cultures of dissociated hippocampal neurons are often used to study neuronal cell biology. We report that the development of these neurons is strongly affected by chemicals leaching from commonly used disposable medical-grade syringes and syringe filters. Contamination of culture medium by bioactive substance(s) from syringes and filters occurred with multiple manufacturing lots and filter types under normal use conditions and resulted in changes to neurite growth, axon formation and the neuronal microtubule cytoskeleton. The effects on neuronal morphology were concentration dependent and significant effects were detected even after substantial dilution of the contaminated medium. Gas chromatography-mass spectrometry analyses revealed many chemicals eluting from the syringes and filters. Three of these chemicals (stearic acid, palmitic acid and 1,2-ethanediol monoacetate) were tested but showed no effects on neurite growth. Similar changes in neuronal morphology were seen with high concentrations of bisphenol A and dibutyl phthalate, two hormonally active plasticisers. Although no such compounds were detected by gas chromatography–mass spectrometry, unknown plasticisers in leachates may affect neurites. This is the first study to show that leachates from laboratory consumables can alter the growth of cultured hippocampal neurons. We highlight important considerations to ensure leachate contamination does not compromise cell biology experiments.

Small doses, big troubles: Modeling growth dynamics of organisms affecting microalgal production cultures in closed photobioreactors

The destruction of mass cultures of microalgae by biological contamination of culture medium is a pervasive and expensive problem, in industry and research. A mathematical model has been formulated that attempts to explain contaminant growth dynamics in closed photobioreactors (PBRs). The model simulates an initial growth phase without PBR dilution, followed by a production phase in which culture is intermittently removed. Contaminants can be introduced at any of these stages. The model shows how exponential growth from low initial inocula can lead to “explosive” growth in the population of contaminants, appearing days to weeks after inoculation. Principal influences are contaminant growth rate, PBR dilution rate, and the size of initial contaminant inoculum. Predictions corresponded closely with observed behavior of two contaminants, Uronema sp. and Neoparamoeba sp., found in operating PBRs. A simple, cheap and effective protocol was developed for short-term prediction of contamination in PBRs, using microscopy and archived samples.

‘Spiromed’: a rapid and inexpensive Spiroplasma isolation technique

An isolation technique for Spiroplasma species (class: Mollicutes) was developed which did not require the labour and cost intensive process of sample filtration. Isolation of spiroplasmas depended upon the ability of these organisms to resist high concentrations of a variety of antibiotics during 45 min prior to subculture in spiroplasma growth medium SP4 enriched with penicillin, sodium hypochlorite and mycostatin. Some 1450 insect samples were treated without contamination of culture media by nondesirable bacteria or fungi, while 96 water samples were associated with an acceptable contamination rate of 6%. The latter figure could probably have been improved upon had the concentration of penicillin in the final culture medium been increased. Eleven Spiroplasma strains or species grew successfully using this isolation technique. Utilisation of 96-well plastic titration plates meant that large numbers of arthropods could be processed rapidly. Non significant difference was observed in the isolation rate achieved using ‘Spiromed’ and the classical approach of filtration and culture in SP4 medium.

Lipopolysaccharide-Dependent and Lipopolysaccharide-Independent Pathways of Monocyte Desensitisation to Lipopolysaccharides

The present study demonstrates that with time in culture blood monocytes (MO) lose their ability to express procoagulant activity (PCA) and secrete tumor necrosis factor-alpha (TNF alpha) in culture medium in response to lipopolysaccharide (LPS) stimulation. Thus, upon 10 micrograms/ml LPS stimulation for 4 hours 2-day-old MO produced lower levels of PCA and TNF alpha than fresh MO. The decrease in responsiveness was not caused by cell death, since in the case of TNF alpha it was fully reversible by interferon-gamma (IFN-gamma). Compared with cells pre-incubated in medium alone, the responsiveness of MO pre-incubated in LPS was further decreased. Thus, in MO LPS pre-incubation was followed by an LPS refractory state. It was expected that the decrease in responsiveness induced by cultivation in medium alone was mediated by LPS contamination of culture medium. However, as we were unable to prevent this decrease by neutralizing LPS contamination of the culture medium with polymyxin B, the loss in LPS-induced activities of cultured MO is likely to be mediated by culture conditions other than LPS contamination. Taken together the present data demonstrate that LPS-dependent as well as LPS-independent pathways of MO desensitization to LPS exist.

The influence of contamination of culture medium with hepatitis B virus on the outcome of in vitro fertilization pregnancies

Heat-inactivated human serum is added to the culture medium used for in vitro fertilization and other forms of assisted conception. Because one batch of pooled serum contained hepatitis B virus, an epidemic occurred among women participating in the treatment program. Seventy-nine women had serologic proof of hepatitis B infection. This incident gave the opportunity to study the effect of hepatitis B virus on pregnancy outcome and the newborn. The situation is unique because the preimplantation embryo was exposed to hepatitis B virus or the pregnancy was complicated by a (sub)clinical infection. Twenty-four women were or became pregnant while having an acute hepatitis B infection. Five pregnancies ended in abortion. The remaining 19 pregnancies ended in the birth of 24 children. No evidence for any harmful effect of exposure to hepatitis B virus in the embryonic or fetal period on the newborn could be found.