Freeze-thaw effect on selected fecal indicator bacteria : Escherichia coli and Enterococcus faecalis / by Nicole A. Hawdon.

The survival of two selected fecal indicator organisms, two strains of Escherichia coli, a Gram-negative bacterium, and two strains of Enterococcus faecalis, a Gram-positive bacterium, after freezing and thawing successively for five cycles was determined using a drop plating method. It was found that all bacterial strains, when an initial concentration of 1.0 x E +08 was used, showed significant decreases in their ability to be cultured (p 0.05). The differences in cell inactivation between the two strains of each species of bacteria tested at all temperatures was not significantly different after five freeze-thaw cycles; while the difference between species was shown to be significant, depending on the temperature and condition tested (p < 0.05). When comparing small sample volume sizes (100l) to larger sample volume sizes (100ml) the observed differences were that Escherichia coli strains showed a decrease in cell culturability at both -7C and -15C when cycled in the larger volume; whereas, Enterococcus faecalis strains showed a decrease in cell culturability at -7C and an increase in cell culturability at -15C when cycled at the larger volume. Additional studies investigating culturability, cell wall integrity, and membrane damage of the bacterial strains were conducted using 100ml samples, cycled at -7, -15, and -30°C, and evaluated by three microbiological methods: drop plating, epi-fluorescent microscopy, and flow cytometry, respectively. In all instances, the plate counting method indicated that there was a decrease in cells that were culturable. Results from flow cytometry indicated a smaller decrease in cell culturability, followed, lastly, by results using the epi-fluorescent microscope. Thus, these studies would suggest that the most damage that occurs to frozen and thawed cells, when cycled five times at -7,-15 or -30°C, would be due to damages occurring at the cellular level, rather than damages occurring on the cell envelope, since less cells were able to uptake nutrients from the culture plates

Histological representation of the progression of injured muscle through repair and regeneration.

<p>(A) Uninjured tibialis anterior (TA) muscle. Injured TA muscle (B) 1 days, (C) 5 days, (D) 10 days, and (E) 28 days after barium chloride (BaCl₂) injection. Bar in E = 100 μm. Images taken at 10X magnification.</p

The effect of IL-1β treatment on IL-6 mRNA and protein levels.

<p>(A) Primary MPCs were treated with IL-1β (0.25 ng/ml) and IL-6 mRNA was determined over 24 hours. *denotes significance (p≤0.05) compared to control. # denotes significance (p≤0.05) compared to the 2 hour time point (n = 4 per time point). (B) IL-1β treatment (1 ng/ml) also increased IL-6 protein released into the media in C2C12 myoblasts. *denotes significance (p≤0.05) compared to control (n = 4–6).</p

IL-1β and IL-6 mRNA expression post-injury to the tibialis anterior (TA).

<p>TA muscle was injured using a barium chloride injection method and muscles were collected 1, 5, 10, and 28 days post-injury and analyzed for (A) IL-1β and (B) IL-6 mRNA expression levels. Data are expressed as fold-increase +/− expected high and low expression relative to the average value <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092363#pone.0092363-Livak1" target="_blank">[51]</a>. *denotes significance (p≤0.05) compared to uninjured control (n = 3–4 per time point).</p

Determining the role of NF-κB activation in IL-1β and TNF-α induced proliferation.

<p>(A) NF-κB activity was determined using the NF-κB cis-reporter construct and data are reported as the ratio of firefly to <i>Renilla</i> luminescence. C2C12 myoblasts were transfected with the NF-κB cis-reporter construct, pretreated with 50 μM PDTC for 1.5 hours, and treated with either 1 ng/ml IL-1β or 20 ng/ml TNF-α for four hours. *denotes significance (p≤0.05) compared to control (n = 4). (B) Proliferation of myoblasts was measured through BrdU incorporation in C2C12 myoblasts following a 50 μM PDTC pretreatment for 1.5 hours, and a 20 hour treatment with either 1 ng/ml IL-1β or 20 ng/ml TNF-α. *denotes significance (p≤0.05) compared to control (n = 4).</p

TNF-α promotes proliferation of myoblasts.

<p>BrdU incorporation was measured after a 24 hour TNF-α treatment (20 ng/ml). Data are expressed relative to control ± SEM. *denotes significance (p≤0.05) compared to control (n = 7).</p

The mitogenic effects of IL-1β.

<p>(A) An IL-1β dose response was performed on C2C12 myoblasts. Concentrations of 0.5 ng/ml to 1 ng/ml significantly increased proliferation in myoblasts. (B) An intermediate dose of IL-1β (0.25 ng/ml) was used to test the mitogenic effects of IL-1β on primary muscle precursor cells. Data are expressed relative to control ± SEM. *denotes significance (p≤0.05) compared to control (n = 3–5 per dose).</p