Ethanol equipment sterilization

The tissue of interest should be removed from the animals using equipment that has been treated to ensure that it is RNase-free. The surface hair of the animals can be soaked in 70% ethanol to minimize the inclusion of hair or dander in the isolated tissues. A sterile disposable Petri plate, placed on top of a bed of crushed ice, is a suitable RNase-free surface for microdissection. Immediately after isolation from the animal, individual samples of tissue can be flash frozen by immersion in liquid nitrogen and stored in cryovials in liquid nitrogen or at -70°C until all samples from an experimental set have been obtained. RNA extraction and preparation of single-stranded cDNA can then be performed simultaneously on all samples. This will minimize differences between RNA populations that result from differences in sample preparation. 

An 8-d old agar slant of Helminthosporium kusanoi Nisik. obtained from CBS (see Table 1) and maintained on oatmeal/ agar, is used to inoculate two 2-L conical flasks each containing 500 mL of nutrient medium. Incubation of the flasks is carried out for 2 d on a rotary shaker (250 rpm, 2.5 cm stroke). The culture obtained is used to inoculate 15 L of nutrient medium in a stainless steel fermentor equipped with stirrer and air inlet (200 rpm. 10 L air/min. 28 °C). 24 h after inoculation, 7.5 g (0.027 mol) of 19-nortestosterone (3) are added as a suspension in 100 mL of sterile water. 48 h laler, conversion of the substrate is complete (monitored by TLC CHC13 acetone 3 1). The mycelial mass is removed by filtration and the filtrate (pH 6.80) is extracted with four 4-L portions of 4-methyl-2-pentanone. After concentration to 100 mL under reduced pressure, the solution is treated with 0.5 g of charcoal. Concentration to 20 mL, cooling and filtration gives crude 4 yield 4.05 g (54%). Recrystallization is from 1.2-dichloroethane/aq ethanol yield 2.30 g (30.6%) mp 213 JC. 

Equipment may be sterilized or disinfected by heat, chemical disinfection or a combination of both. Many tanks and reaction vessels are sterilized by steam under pressure, and small pieces of equipment and fittings may be autoclaved, but it is important that the steam has access to all surfaces. Equipment used to manufacture and pack dry powder is often sterilized by dry heat. Chemical disinfectants commonly include sodium hypochlorite and organochlorines at 50-100 ppm free residual chlorine, QACs (0.1-0.2%), 70% (v/v) ethanol in water and 1% (v/v) formaldehyde solution. The... 

Processes for adenovirus purification typically end with concentration, formulation, and sterile filtration operations [40, 80,106]. Concentration and formulation are usually carried out in ultra-filtration units equipped with 100-300-kDA membranes [40,106]. The exact composition of the formulation buffer will depend on the intended application, mode of administration (injectable, aerosol), and required short-term and shelf stability [104,123]. A typical liquid formulation may include an aqueous buffer supplemented with cryoprotectants (e.g., sucrose) and stabilizers such as the nonionic-surfactant polysorbate-80, the chelating agent EDTA, and the oxidation inhibitors ethanol and histidine [123]. Filtration under sterile conditions is typically performed with 0.22-pm membranes [103,106]. 

When preparing a dilution series, it is important to maintain aseptic conditions (Section 13.8). For example, dilution blanks must be flamed when opened to transfer sample (Fig. 13.2). Sterile 1.0 mL pipettes (0.1 mL graduations) or variable volume pipettors equipped with sterile plastic tips must be used to aseptically transfer liquid between dilution blanks. Sterile pipettes or pipette tips should be used only once, and then a new pipette or tip is used. Pipettors can be sterilized by wiping down their surfaces with 70% v/v ethanol and should be calibrated yearly. 

The number of pasteurization units required to obtain a certain level of destruction can be calculated from yeast thermoresistance criteria in a given medium. Laboratory studies anticipated that 0.05 pasteurization units would be sufficient for the destruction of yeasts in a dry wine at 12% vol.ethanol. In practice, 0.5 pasteurization units are required to sterilize such a wine. The uneven heat supplied by industrial heating equipment and the existence of particularly resistant yeast strains at the final stages of fermentation can explain this difference. Table 9.5 shows the necessary conditions for the destruction of germs according to the constitution of the wine. 

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