Extended incubation

Bacteria in thawed ice samples from Ellesmere Island (79°38 N, 74°23 W) produced CO2 and CH4 during extended incubation at 4°C in a diluted complex medium (yeast extract, casamino acids, starch, and glucose) (Skidmore et al. 2000). It was suggested that the results demonstrated that the subglacial environment beneath a polythermal glacier provided an acceptable habitat for microbial life. 

Mesocosms placed in shallow Finnish lakes were used to evaluate changes brought about by extended incubation of biologically treated bleachery effluent from mills that used chloride dioxide. The mesocosms had a volume of ca. 2 m and were constructed of translucent polyethere or black polyethene to simulate dark reactions. The experiments were carried out at ambient temperatures throughout the year, and sum parameters were used to trace the fate of the organically bound chlorine. In view of previous studies on the molecular mass distribution of effluents (Jokela and Salkinoja-Salonen 1992), this was measured as an additional marker. Important featmes were that (a) sedimentation occurred exclusively within the water mass within the mesocosm, (b) the atmospheric input could be estimated... 

The time lag between exposure to a biological agent and the appearance of symptoms may serve as both a plus and a minus for terrorists. An incubation period allows terrorists time to attack and escape before an alarm is sounded. Conversely, an extended incubation period may diffuse the impact that the terrorists hope to achieve to bring attention to their causes (Tierno, 2002).2 The routes of exposure to biological agents vary, but may include ... 

Except for B. subtilis, streak slants (one or more) of appropriate agar (PDA for C. albicans and A. niger, SCDA for all others) with specific microorganisms from stock culture. Incubate at 30 1°C for 48 to 72 h, except A. niger, which may need extended incubation for good spomlation. 

The arabinans are highly branched polymers of a-L-arabinofuranosyl residues having a-(l - 3) and a-( 1 - 5) linkages. Beet arabinan was hydrolyzed to the extent of only 3% by endo-a-L-arabinofurananase,142 in agreement with the highly branched structure. On treatment with a-L-arabinofuranosidase,72 a polymer of a-( 1 - 5)-linked L-arabinose could be precipitated from solution. This was hydrolyzed by endo-a-L-arabinofurananase to the extent of 23%, with release of a series of L-arabino-oligosaccharides initially and, on extended incubation, of L-arabinose and (l- 5)-a-L-arabinobiose,142 providing further evidence for the structure of the arabinan substrate. Partially debranched arabinan was hydrolyzed by endo-a-L-arabinofurananase at 16 times the rate for native arabinan.143... 

Pretreatment of polycarbonate filters with ethanolic NaOH enhances responsiveness towards IL-8, possibly by expression of anionic carboxylate ions. Because NaOH dissolves polycarbonates, and therefore lowers filter thickness and enhances pore size, do not extend incubation time and concentration of NaOH ... 

Several comprehensive studies of N assimilation in the North Pacific trades biome have been conducted over the past several decades. Gundersen and his colleagues (1974, 1976) were the first to estabhsh N2 fixation as a source of new N to the open ocean ecosystem, and concluded that it was a more important source of fixed N than wet deposition from the atmosphere (see Case Studies section). They also made measurements of the rates of nitrification, denitrification and assimilatory nitrate-reduction. These latter experiments involved the addition of fairly high concentrations of exogenous N substrates (NH4 , N02, NOa ) and extended incubations (days to months), so the rates reported must be viewed as potential rates at best. 

Glucose oxidase is highly specific for -D-glucose. As noted earlier, 36% and 64% of glucose in solution are in the a- and P forms, respectively. Complete reaction of glucose therefore requires mutarotation of the a- to p-form. Some commercial preparations of glucose oxidase contain an enzyme— mutarotase—that accelerates this reaction. Otherwise, extended incubation time allows spontaneous conversion. 

Figure kk. Aeeumulation of methanethiol from methionase activity on methionine (lOOmM) in an aerobic environment during extended incubation at 30°C FDE=freeze-dried enzyme preparation. 

Figure 5. Accumulation of A) methanethiol and B) total methanethiol (as the sum of methanethiol plus dimethyl disulfide) from methioninase activity (constant concentration, 2.5 mg freeze-dried extract/ml) on varying concentrations of methionine in an aerobic environment during extended incubation at 30°C.

If solubility becomes a problem for a particular compound, DMSO can be added (up to 1%) to media. BSA can also be added to media up to 0.2% (or higher, as determined) but should not be used for extended incubations due to potential bacterial growth. Further additions to media for compound screening have also been described (4). For long-term treatments with compounds, one should consider omitting PTU treatment, as this may interact with some compounds and cause complications. 

The sensitivity of the assay using this fluorogenic substrate was evaluated by conducting a series of enzyme dilution experiments (10). A detection level as low as 30 ng/mL after incubation of only 3-5 minutes was measured. With extended incubation time (2-3 h), it was estimated that the assay could detect renin at 0.5 ng/mL. 

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