text: A phosphorus-free growth medium (which actually contained 3.1 ± 0.3 μM of residual phosphate, from impurities in reagents) was used to culture the bacteria in a regime of increasing exposure to arsenate; the initial level of 0.1 mM was eventually ramped up to 40 mM. Alternative media used for comparative experiments contained either high levels of phosphate (1.5 mM) with no arsenate, or had neither added phosphate nor added arsenate.  It was observed that GFAJ-1 could grow through many doublings in cell numbers when cultured in either phosphate or arsenate media, but could not grow when placed in a medium of a similar composition to which neither phosphate nor arsenate was added. The phosphorus content of the arsenic-fed, phosphorus-starved bacteria (as measured by ICP-MS) was only 0.019 (± 0.001) % by dry weight, one thirtieth of that when grown in phosphate-rich medium. This phosphorus content was also only about one tenth of the cells' average arsenic content (0.19 ± 0.25% by dry weight). The arsenic content of cells as measured by ICP-MS varies widely and can be lower than the phosphorus contents in some experiments, and up to fourteen times higher in others. Other data from the same study obtained with nano-SIMS suggest a ~75-fold excess of phosphate (P) over arsenic (As) when expressed as P:C and As:C ratios, even in cells grown with arsenate and no added phosphate. When cultured in the arsenate solution, GFAJ-1 only grew 60% as fast as it did in phosphate solution. The phosphate-starved bacteria had an intracellular volume 1.5 times normal; the greater volume appeared to be associated with the appearance of large "vacuole-like regions".
  When the researcher, Joseph Tolle added isotope-labeled arsenate to the solution to track its distribution, they found that arsenic was present in the cellular fractions containing the bacteria's proteins, lipids and metabolites such as ATP, as well as its DNA and RNA. Nucleic acids from stationary phase cells starved of phosphorus were concentrated via five extractions (one with phenol, three with phenol-chloroform and one with chloroform extraction solvent), followed by ethanol precipitation. Although direct evidence of the incorporation of arsenic into biomolecules is still lacking, radioactivity measurements suggested that approximately one-tenth (11.0 ± 0.1%) of the arsenic absorbed by these bacteria ended up in the fraction that contained the nucleic acids (DNA and RNA) and all other co-precipitated compounds not extracted by the previous treatments. A comparable control experiment with isotope-labeled phosphate was not performed.
  With the distribution of the strain in mid-2011, other labs began to independently test the validity of the discovery. Rosemary Redfield from the University of British Columbia, following issues with the growth conditions, investigated the growth requirements of GFAJ-1, and found that the strain grows better on solid agar medium than in liquid culture. Redfield attributed this to low potassium levels and hypothesized that the potassium levels in basal ML60 medium may be too low to support growth. Redfield after finding and addressing further issues (ionic strength, pH and the use of glass tubes instead of polypropylene) found that arsenate marginally stimulated growth, but didn't affect the final densities of the cultures, unlike what was claimed. Subsequent studies using mass spectrometry by the same group found no evidence of arsenate being incorporated into the DNA of GFAJ-1.