Problems incubation

Occasionally, the cells prepared according to the method in Section 3.4. become badly clumped by DNA that has been released from dying cells. If this is a problem, incubate the cells for 10 min at 37°C in culture medium containing 4 tg/mL of DNAse. 

Glass or disposable plastic culture dishes are used. If glass petri dishes are employed, a humid environment must be maintained during incubation. This prevents losses of media by evaporation (the dishes have loose-fitting covers). Disposable plastic dishes have tight-fitting lids which minimize the problem of dehydration. 

Historically the production of titrate has been an important development in the pioneering of fermenter technology. It was shown back in 1893 by Wehmer that a fungus, Citromyces (now reclassified as a Penicilliutn spp.) would accumulate citric add in liquid culture. Wehmer in fact tried to scale up the process to an industrial level but there were two main problems. Firstly, the duration of the process under his conditions took far too long of the order of several weeks. Secondly, a problem was caused by Wehmer s incorrect belief that citric add only accumulated around neutral pH and lengthy incubation at this pH inevitably leads to contamination. 

A major breakthrough in the fermentation process came in 1916 - 1920 when it was found that Aspergillus niger grew well at pH values below 3.5, producing citric add in days rather than weeks. The faster incubation and highly add conditions (often below pH 2.0) also served to minimise potential problems caused by contamination. 

Numerous workers have found that measurements of serum lipase activity are useful in the diagnosis of pancreatitis (83, 84, 85). Despite this, serum lipase determinations are not usually performed in clinical laboratories, probably due to inherent problems associated with the conventional methods, based on an emulsified lipid substrate. The methods are also not very suitable for manual batch analysis nor for automation due to laborious post incubation procedures. 

It is possible to also test semi-solid antibacterial preparations on the skin itself, as described for liquid disinfectants (section 3.5.1). A portion of the skin— the backs of the fingers between the joints is a useful spot— is treated with the test organism, the preparation is then applied and after a suitable interval the area is swabbed and the swab incubated in a suitable medium. Alternatively, the method employing pig skin, described in section 3.5.1, may well be adapted to the problem of testing semi-solid skin disinfectants. 

One of the long-standing criticisms of Bis is that the incubation period required in order to confirm a satisfactory sterilization process imposes an undesirable delay on the release of the product. This problem has been overcome, with respect to steam sterilization at least, by the use of a detection system in which a spore enzyme, a-glucosidase (reflective of spore viability), converts a non-fluorescent substrate into a fluorescent product in as little as Ihour. 

A 500-ml bottle of frozen serum is thawed overnight at 4° and swirled to thoroughly mix the contents prior to incubation in a 56° waterbath for 30 min. The serum is then aliquoted to 50-ml conical tubes and stored at —20° for a year without any problem. 

There is a need to measure the activities of enzymes and to correlate these measured activities with microbial diversity in soil. It is conceptually wrong to assume a simple relationship between a single enzyme activity and microbiological activity in soil (Nannipieri et al. 2003). Most of the assays used to determine microbiological activities in soil present the same problem measuring potential rather than real activities (Nannipieri et al. 1990). Indeed, assays are generally made at optimal pH and temperature and at saturating concentration of substrate. Furthermore, synthetic rather than natural substrates are often used, and soil is incubated as a slurry (Nannipieri et al. 1990). 

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