Shake flask preparation

Dissolve I g. of finely powdered acetanilide in 5 ml. of cold glacial acetic acid contained in a 25 ml. conical flask. Then in another small flask prepare a solution of 0 42 ml. (1 34 g.) of bromine (care ) in 6 ml. of glacial acetic acid, and add this solution slowly to the acetanilide solution, shaking the latter throughout the addition to ensure thorough mixing. Allow the final mixture to stand at room temperature for 15 minutes. Then... 

In a 1 or 1-5 htre round-bottomed flask prepare a solution of 53-5 g. of o-toluidine in 170 ml. of 48 per cent, hydrobromic acid, cool to 5° by immersion in a bath of ice and salt. Diazotise by the gradual addition of a solution of 36 -5 g. of sodium nitrite in 50 ml. of water stopper the flask after each addition and shake until all red fumes are absorbed. Keep the temperature between 5° and 10°. When the diazotisation is complete, add 2 g. of copper powder or copper bronze, attach a reflux condenser to the flask, and heat very cautiously on a water bath. Immediately evolution of gas occurs, cool the flask in crushed ice unless the... 

A substitute may be prepared thus 0 05 gram palladous chloride is placed in a special shaking flask with 50 c.c, of 50 per cent, alcohol and 1 or 2 c c. of 1 per cent, aqueous solution of gum-arabic, the weight of gum being about one-fourth the weight of the palladous chloride. On shaking this mixture in an atmosphere of hydrogen the chloride is reduced with formation of a black solution of colloidal platinum, which is rendered stable by the small quantity of gum present. 

Applications Shake-flask extraction nowadays finds only limited application in polymer/additive analysis. Carlson et al. [108] used this technique to extract antioxidants from rubber vulcanisates for identification purposes (NMR, IR, MS). Wrist-action shaking at room temperature was also used as the sample preparation step for the UV and IR determination of Ionol CP, Santonox R and oleamide extracted from pelletised polyethylene using different solvents [78]. BHT could be extracted in 98 % yield from powdered PP by shaking at room temperature for 30 min with carbon disulfide. 

Prepare 100 mL of a stock standard solution of toluene in an alkane solvent by pipetting 0.1 mL of toluene into the flask and diluting to the mark with the alkane. Use the 100-mL flask prepared in step 1. Shake well. This solution has a concentration of 0.870 g/L. 

Filter the water samples if necessary. Then prepare them for measurement by adding 1 mL of 1 N HC1 to 50 mL of each. This can be done by filling (rinse first) 50-mL volumetric flasks with the samples to the 50-mL mark and then pipetting 1.0 mL of the HCl into these flasks. Shake well. Prepare the control sample and a blank (distilled water) in the same way as the samples. 

In dry 25-mL volumetric flasks, prepare four calibration standards of isopropyl alcohol in toluene solvent that are 20, 30, 40, and 50% in alcohol concentration. Obtain an unknown from your instructor and dilute to the mark with toluene. Shake well. 

The synthesis of 6-hydroxy fluvastatin with M. rammaniana DSM 62752 gave high conversion (>95 %) in shake flask culture on 400 mL scale with 0.1 g L of fluvastatin as well as on 22 L scale in a Wave bioreactor-fed batch process at a final substrate concentration of 0.4 g L Instead of the partial purification by a second solid-phase extraction described above, 6-hydroxy fluvastatin can be obtained in high purity ( 95 %) by, for example, preparative medium-pressure liquid chromatography (MPLC) on RP18 silica gel. ... 

Hydroxy fluvastatin could be prepared analogously via biotransformation in shake flask culture with Streptomyces violascens ATCC 31560. Different media and minor variations of the process schedule had to be applied. Before supplementation of... 

Broth extracts were prepared by ethyl-acetate (EtOAc) extraction of 7-day-old shake-flask cultures using three extractions of EtOAc with volumes equivalent to the original culture volume. The samples were dried using reduced pressure and the aliquots (400 mg) of the dried materials were reconstituted in 10 ml of the HSCCC solvent system (5 ml of the lower phase and 5 ml of upper phase). 

In a 1- or 1.5-litre round-bottomed flask prepare a solution of 53.5 g (0.5 mol) of o-toluidine in 170 ml of 40 per cent w/w hydrobromic acid cool to 5 °C by immersion in a bath of ice and salt. Diazotise by the gradual addition of a solution of 36.5 g (0.53 mol) of sodium nitrite in 50 ml of water stopper the flask after each addition and shake until all red fumes are absorbed. Keep the temperature between 5 and 10 °C. When the diazotisation is complete, add 2g of copper powder or copper bronze (Section 4.2.79, p. 426), attach a reflux condenser to the flask and heat very cautiously on a water bath. Immediately evolution of gas occurs, cool the flask in crushed ice unless the flask is rapidly removed from the water bath, the reaction may become so violent that the contents may be shot out of the flask. When the vigorous evolution of nitrogen moderates, heat the flask on a water bath for 30 minutes. Then dilute with 400 ml of water, and steam distil the mixture until about 750 ml of distillate are collected. Render the distillate alkaline with 10 per cent sodium hydroxide solution (about 50 ml) and separate the lower red layer of crude o-bromotoluene. Wash it with two 20 ml portions of concentrated sulphuric acid (which removes most of the colour) and then twice with water. Dry with magnesium sulphate or anhydrous calcium chloride, and distil from a flask fitted with a lagged fractionating column. Collect the o-bromotoluene at 178— 181 °C. The yield is 40 g (47%). The spectral features are noted in Expts 6.72 and 6.81. 

Yandenburg et al. [92] compared extraction of additive Irganox 1010 from freeze-ground polypropylene polymer by pressurized fluid extraction (PFE) and MAE with reflux, ultrasonic, shake-flask, and Soxhlet extraction. PFE and MAE were faster than any conventional method with comparable extraction efficiency. The times to reach 90% recovery by PFE using propan-2-ol at 150°C and acetone at 140°C were 5 and 6 minutes, respectively. Reflux with chloroform was found to be the fastest method performed under atmospheric pressure with 90% recovery in 24 minutes. Reflux with cyclohexane propan-2-ol (1 1) required 38 minutes. Ultrasonic, shake-flask, and Soxhlet extraction required about 80 minutes (90%) extraction). The total sample preparation time for PFE was 15 minutes, MAE 28 minutes, and reflux with chloroform was 45 minutes. 

Seed preparation The primary seed fermentation is performed using shaking-flask culture techniques. Once grown, the suspension is then transferred to further seed stages. The purpose of the seed preparation is to generate enough inoculums for the production of a fermenter. 

The shake-flask method is based on the phase solubility technique that was developed 40 years ago and is still the most reliable and widely used method for solubility measurement today (Higuchi and Connors, 1965). The method can be divided into hve steps sample preparation, equilibration, separation of phases, analysis of the saturated solution and residual solid, and data analysis and interpretation (Yalkowsky and Banerjee, 1992, Winnike, 2005). 

Prepare 1 L Superbroth as described in Subheading 2.4. into at least a 2-L Erlenmeyer or bacterial shake flask. Add 1 mL 100 mg/mL ampicillin (see Note 26 and 27). 

An alternative to transformation each time a protein is to be produced is to prepare a master cell bank. To produce a master cell bank, a single colony obtained from the transformation in step 5, Subheading 3.2., is cultured in a shake-flask with 200 mL of superbroth containing 100 pg/mL ampicillin (or 15 pg/mL chloramphenicol). When the culture reaches an OD650 of 3, the cells are placed on ice, mixed with a 50% volume of 86% glycerol in water, and frozen at-80°C in 1.5 mL aliquots. Future fermentations can be started from a single aliquot for more reproducible results. 

Fermenter sterility testing requires a room with a laminar flow hood to prepare plates, tubes and shake flasks. Space needs to be provided for incubators and microscopes. Since it is very important to identify when infection occurs in large scale production, microscopic examination of shake flasks is usually preferred because a large sample can be used, and it gives the fastest response. Similarly, stereo microscopes are used for reading spiral streaks on agar plates before the naked eye can see colonies. 

The focus in Chapters 7 and 8 is on the specific sample preparation approaches available for the extraction of organic compounds from environmental matrices, principally soil and water. Chapter 7 is concerned with the role of Soxhlet, ultrasonic and shake-flask extraction on the removal of organic compounds from solid (soil) matrices. These techniques are contrasted with newer developments in sample preparation for organic compound extraction, namely supercritical fluid extraction, microwave-assisted extraction and pressurized fluid extraction. Chapter 8 is arranged in a similar manner. Initially, details are provided on the use of solvent extraction for organic compounds removal from aqueous samples. This is followed by descriptions of the newer approaches, namely solid-phase extraction and solid-phase microextraction. 

The P. pastoris otMnPl-1 inoculum was prepared with 100 ml YPD medium in a 500 ml shake flask at 250 ipm and 30 °C. After 24 h, the inoculum was separated through centrifugation and washed with sterile normal saline solution (0.9% NaCl). The washed inoculum was then added to each flask to achieve an initial cell density of approximately 0.15 g/1 dry cell weight. 

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