However, these studies came under fire with regards to whether or not these cells were both alive and not from a contamination source.  Schippers et al. 2005 were the most recent to address this question, using new  techniques on cores from leg 201 of the Ocean drilling Program to show that the cells at least 400m plus depth are in fact alive and in situ.  Four counting methods were used in the study: (1) microscopic cell counts using unspecific fluorescent DNA (RNA) stains such as acridine orange; (2) sequences of high-molecular-weight prokaryotic DNA; (3) cultivation of diverse bacteria from subsurface sediments; and (4) bacterial activities measured with radiotracers.  Only a small fraction of counted cells have been culturable, leading to questions as to whether or not these cells were alive.  Also, acridine orange can still potentially stain DNA from dead or inactive bacteria.  RNA, however, is degraded much quicker in cells that are inactive due to starvation.  Therefore, living cells were determined using a 1. a technique using ribosomal RNA as a target for the technique known as catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) and 2. real-time polymerase chain reaction (Q-PCR) quantification of 16S ribosomal DNA genes.  The Q-PCR data from Schippers et al. showed that total numbers of prokaryotes in the direct cell counts were similar to the bacteria counted and showed about one to one-third fewer Archea.  The CARD-FISH data did not show a decrease in cell numbers with depth, as is expected and demonstrated in other counts.  It also detected about one-tenth fewer cells in the ocean margin and one third less cells in open ocean sediments.  This difference is attributed to better DNA preservation in the open-ocean sediments, or to the greater abundance of other electron acceptors in the open-ocean in the higher sediments (Schippers et al 2005).

Symbols to the left are as follows:  1. Squares- CARDFISH, 2. Crosses - Q-PCR, 3. Circles - traditional stain cell counts.  Different counting methods yield different results at both ocean margin sites (top pair) and open-ocean sites (Bottom pair), with CARDFISH data being up to two orders of magnitude lower.  Also, CARDFISH data do not show the logarithmic decrease as exhibited in the other counts.  (Schippers et al. 2005).