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South America became linked to North America through the Isthmus of Panama during the Pliocene, bringing a nearly complete end to South America's distinctive marsupial faunas. The formation of the Isthmus had major consequences on global temperatures, since warm equatorial ocean currents were cut off and an Atlantic cooling cycle began, with cold Arctic and Antarctic waters dropping temperatures in the now-isolated Atlantic Ocean. Africa's collision with Europe formed the Mediterranean Sea, cutting off the remnants of the Tethys Ocean. Sea level changes exposed the land-bridge between Alaska and Asia. Near the end of the Pliocene, about 2.58 million years ago (the start of the Quaternary Period), the current ice age began. The polar regions have since undergone repeated cycles of glaciation and thaw, repeating every 40,000–100,000 years.

The Pliocene saw the end of what fauna in South America?
Answer: marsupial faunas
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Until the 1970s most of the larger pubs also featured an off-sales counter or attached shop for the sales of beers, wines and spirits for home consumption. In the 1970s the newly built supermarkets and high street chain stores or off-licences undercut the pub prices to such a degree that within ten years all but a handful of pubs had closed their off-sale counters, which had often been referred to colloquially as the jug and bottle.

In what decade did pubs tend to cease selling alcohol for off-premises drinking?
Answer: 1970s
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Other microscopic procedures may also aid in identifying infectious agents. Almost all cells readily stain with a number of basic dyes due to the electrostatic attraction between negatively charged cellular molecules and the positive charge on the dye. A cell is normally transparent under a microscope, and using a stain increases the contrast of a cell with its background. Staining a cell with a dye such as Giemsa stain or crystal violet allows a microscopist to describe its size, shape, internal and external components and its associations with other cells. The response of bacteria to different staining procedures is used in the taxonomic classification of microbes as well. Two methods, the Gram stain and the acid-fast stain, are the standard approaches used to classify bacteria and to diagnosis of disease. The Gram stain identifies the bacterial groups Firmicutes and Actinobacteria, both of which contain many significant human pathogens. The acid-fast staining procedure identifies the Actinobacterial genera Mycobacterium and Nocardia.

What electronic charge do cellular molecules have?
Answer:
negatively charged