Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory.

Cell culture is a delicate skill necessary for growing human, animal, and insect cells, or other tissues, in a controlled environment. The goal of the protocol is to emphasize the correct techniques used in a research laboratory to prevent contamination from fungi and bacteria. Special emphasis is placed on avoiding mycoplasma contamination, a major concern in the cell culture room due to its small size and resistance to most antibiotics used for cell culture. These same techniques ensure continuous growth and maintain healthy cells. For new and experienced cell culture users alike, it's important to consistently adhere to these best practices to mitigate the risk of contamination. Once a year, laboratories should review cell culture best practices and follow-up with a discussion or additional training if needed. Taking early action to prevent contamination in the first place will save time and money, as compared to cleaning up after contamination occurs. Universal best practices keep cell cultures healthy, thereby reducing the need to constantly thaw new cells, purchase expensive cell culture media, and reducing the amount of incubator decontamination and downtime.

Streptococcus pyogenes Sortase Mutants Are Highly Susceptible to Killing by Host Factors Due to Aberrant Envelope Physiology.

Cell wall anchored virulence factors are critical for infection and colonization of the host by Gram-positive bacteria. Such proteins have an N-terminal leader sequence and a C-terminal sorting signal, composed of an LPXTG motif, a hydrophobic stretch, and a few positively charged amino acids. The sorting signal halts translocation across the membrane, allowing sortase to cleave the LPXTG motif, leading to surface anchoring. Deletion of sortase prevents the anchoring of virulence factors to the wall; the effects on bacterial physiology however, have not been thoroughly characterized. Here we show that deletion of Streptococcus pyogenes sortase A leads to accumulation of sorting intermediates, particularly at the septum, altering cellular morphology and physiology, and compromising membrane integrity. Such cells are highly sensitive to cathelicidin, and are rapidly killed in blood and plasma. These phenomena are not a loss-of-function effect caused by the absence of anchored surface proteins, but specifically result from the accumulation of sorting intermediates. Reduction in the level of sorting intermediates leads to a return of the sortase mutant to normal morphology, while expression of M protein with an altered LPXTG motif in wild type cells leads to toxicity in the host environment, similar to that observed in the sortase mutant. These unanticipated effects suggest that inhibition of sortase by small-molecule inhibitors could similarly lead to the rapid elimination of pathogens from an infected host, making such inhibitors much better anti-bacterial agents than previously believed.