Replating technique

Just as with the in situ technique, reconstruction experiments with HPRT" Lesch-Nyhan (LN) cells accompanied each replating mutagenesis experiment to determine the efficiency of recovery of AG- or TG-resistant cells in the presence of nonresistant cells. That is, for each individual determination, a known number of LN cells (CRL 1112, American Type Culture Collection) were seeded into flasks or dishes containing either no other cells or cells seeded at the same density as in the experimental dishes or bulk culture vessels. The cloning efficiency of the LN cells in the presence of the experimental cells, divided by their cloning efficiency when seeded into empty dishes, determined the efficiency of recovery of mutant colonies. Our studies with the in situ technique, in which loosely formed miniature colonies are present at the time selection with AG begins, showed that if the total number of cells present in 60-mm-diameter dishes reached 30,000, recovery decreased to approximately 65%. With the replating technique, in which individual cells are plated directly into selective medium at the end of the expression period, a density of 500-750 attached cells/cm for TG and 1000-1500/cm in AG resulted in approximately 85% recovery (data not shown). Therefore, cells were plated at or below these respective lower densities. 

Scrape and scratch loading techniques Scrape loading. By scrape loading, adherent cells are scraped off their substratum with a cell scraper in the presence of the macro-molecule(s) (up to 500 kD mol weight) to be loaded (McNeil et al 1984). Thus loaded cells are obtained in suspension, and have to be replated and allowed to spread before microscopic observation. 

Copper-containing deposits may be found in process equipment any time that the fluids come in contact with copper-base alloys. While most copper alloys are relatively resistant to corrosion, very small amounts of copper will dissolve, and then may replate on contact with an iron-base ailoy. Copper that is plated onto the steel will accelerate corrosion because of the galvanic nature of the iron/copper couple. The copper acts as the cathode for the oxidizing agent, while the anodic iron corrodes. Many different techniques have been developed for removing copper deposits. Included are sections on alkaline oxidizers, HCI/complexers, and chelating agent solvents. 

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