The evolution of tissue/cell culture was very much a work in progress at this time. Explants had earlier been embedded in plasma clots and maintained with various fluid media while cells migrated outward in a single layer that could be viewed with a microscope. Primary cell cultures were prepared by digesting animal tissue to component cells, using trypsin or other enzymes, often enhanced with versene (ethylene-diamine-tetracetate) as a chelator. The cells thus freed were washed and planted in sealed glass vessels in medium typically based on either Hanks' or Earle's balanced salt solution-these had a physiologically balanced content of cations (sodium, potassium, calcium, and magnesium) with chloride ions and a phosphate buffer system, plus glucose. Adjustment of pH was done with varying levels of sodium bicarbonate: Earle's solution was formulated for higher levels of bicarbonate, which was useful as cell populations built to a level where their Food Environ Virol (2010) 2:1-23 3 metabolic acid needed more neutralization. Cell culture vessels (e.g., flasks, bottles, tubes) were sealed because the bicarbonate buffer equilibrated with CO 2 in the vessel's airspace: if the CO 2 escaped, then the sodium bicarbonate became sodium hydroxide, and the pH climbed and killed the cells. When incubators were invented that maintained a 5% CO 2 atmosphere in their interiors, cells could be grown in unsealed containers (e.g., Petri plates), but there were (are) always risks that the controls would fail, resulting in the death of the all of the cultures. Various media with CO 2 -free buffer systems have been devised, but CO 2 has not yet been entirely replaced. One formulation substituted galactose for glucose so as to inhibit acid production by the cultured cells. Nitrogen sources could be as simple as enzymatic hydrolyzate of bovine lactalbumin, which worked well with various primary cultures and was inexpensive and autoclavable. Medium 199 was a pioneer synthetic medium that contained virtually all the known chemical constituents of mammalian tissue, including nucleic acid bases; most of its ingredients are not known to be required by cells in vitro, and it was a huge project to compound from individual chemicals, but it is still used to some extent now that it can be bought from catalogs. A turning point was the research by Harry Eagle to determine the specific nutritional needs (amino acids and vitamins) of a selected cell line. He devised Basal Medium Eagle in a version for his line of HeLa cells, and another for his line of L-cells. The next step was the development of Minimum [sic] Essential Medium, which would meet the needs of a variety of cell lines. An additional solution of ''Nonessential Amino Acids'' was devised for planting new cultures and for lines that had special needs. Some researchers use these nonessential additives routinely. Another persistent component of cell culture media is animal blood serum-often, but not always, derived from fetal calves at present. Serum-free media have been developed, but the majority of medium formulations still include serum. The degree to which bovine serum enhances growth of cells of other animal species is remarkable.