Cell harvesting/washing

The technique is easy and fast to use, involving only cell harvesting, washing and drying of the biomass on an IR-transparent carrier as sample preparation, before the spectra are recorded and evaluated. An instrument dedicated for organism identification is available and has been used for the cited process monitoring experiments with complete spectrometer hardware and software, model IFS 28/B from Bruker (Milton, ON, Canada or Bruker Optik, Karlsruhe, Germany). The whole procedure takes less than an hour. 

Cells harvest, wash with PBS, store cell pellet at -20°C. 

Cell Harvesting/Washing. In recent years, the use or microorganism-based fermentations for the production of chemical products has greatly expanded. Interferon, insulin, novel antibiotics, cytotoxic antitumor agents, numerous enzymes, fine chemicals, and even fuels have been produced in carefully controlled fermentations. A key step in the fermentation cycle is the separation of the cells or cellular debris from the liquid phase of the fermentation broth-commonly called "cell harvesting". 

Subsequently 36 strains of aerobic endospore-forming bacteria, consisting of six Bacillus species and one Brevibacillus species could be discriminated using cluster analysis of ESMS spectra acquired in the positive ion mode (m/z 200-2000).57 The analysis was carried out on harvested, washed bacterial cells suspended in aqueous acidic acetonitrile. The cell suspensions were infused directly into the ionization chamber of the mass spectrometer (LCT, Micromass) using a syringe pump. Replicates of the experiment were performed over a period of six months to randomize variations in the measurements due to possible confounding factors such as instrumental drift. Principal components analysis (PCA) was used to reduce the dimensionality of the data, fol-... 

Oligonucleotide probes were labeled at their 3 end using terminal transferase and digoxigenin-11 -UTP, according to the manufacturer s recommendations (Roche Pharmaceuticals). Ten ml of cells are pre-fixed in 3.7% formaldehyde for 15 min at room temperature. Five ml of cells are harvested and resuspended in 6 ml of 4% paraformaldehyde, 0.1 MKPO4 (pH 6.5), and 5 mM MgCb. After 3 h, the cells are washed twice in solution B (1.2 M sorbitol, 0.1 M KPO4 (pH 6.5)). The cells are then resuspended in 2.8 ml... 

After an additional 48 h, the cells were washed, harvested and assayed for specific 125I-toxin binding as described elsewhere (5). Values are the mean of triplicate determinations and there was less than 10% variation amongst the three determinations. 

Mix the washed NS-0 cells harvested from the 2-T flasks with one quarter of the splenocytes from one spleen. 

C, 5% C02 for 60 h and the CaPC>4 precipitate is allowed to remain on the cells during this incubation period. At the end of the incubation the culture media is discarded, cells are washed with PBS, and harvested using PBS containing 5 mM EDTA. The collected cells are centrifuged at 1000 x g for 10 min, and stored at -20 °C until purified. 

To prepare the inoculum, 1000-mL Erlenmeyer flasks containing 200 mL of medium were inoculated from a fresh agar slant and incubated at 30 2°C in a rotary shaker at 200 rpm. After 65 h, yeast cells were harvested by centrifuging at 2000 rpm for 15 min. The cells were washed twice and resuspended in saline solution (0.85% NaCl). The obtained suspensions were used for preparation of the gel beads. 

Harvest an exponentially growing suspension of CHO cells and wash them in isoleucine free Eagle s medium. 

Cells are harvested, washed and stored on ice in HEPES buffered HBSS. Cells are stored at a concentration such that 20 pL contains the number appro-... 

Protein Production, Isolation, and Purification. The expression and purification of chicken lysozyme mutant proteins in yeast are performed as described by Malcolm et al. with the following modifications. The 50-ml minimal medium second seed yeast culture is used to inoculate a 2.8-liter Fembach flask containing 500 ml of 1% yeast extract/2% Bacto-peptone/ 8% glucose (w/v) medium and is then incubated for 7 - 9 days at 30°. Cells are harvested, washed twice with 60 ml of 0.5 M NaCl, and collected by centrifugation. The supernatants are pooled, diluted 5-fold with deionized water, and loaded onto a 20-ml column of CM Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 0.1 M potassium phosphate, pH 6.24. The column is washed with the same buffer, and lysozyme is eluted with 0.5 M NaCl/0.1 M potassium phosphate, pH 6.24. Fractions are assayed by activity (decrease in A450 of Micrococcus lysodeikticus cell wall suspensions per minute). Fractions containing lysozyme are concentrated in Centricon-10 (Amicon, Danvers, MA) filter units, washed with 0.1 M potassium phosphate buffer, pH 6.24, and stored at 4°. The protein concentration is determined from e 1 = 26.4.15... 

Cell harvesters were developed to capture multiple samples of cells on membrane filters, wash away unincorporated isotopes, and prepare samples for liquid scintillation counting on special equipment developed to process and count multiple samples. Despite miniaturization and improvements in efficiency of this technique, the disadvantages of multiple liquid handling steps and increasing costs for disposal of radioactive waste materials severely limit its usefulness. Although specific applications require measuring DNA synthesis as a marker for cell proliferation, much better choices are available for detecting viable cell number for HTS. 

For the preparation of carbonyl-free solvents see Section 5.3.2.2.) The cells are washed three times in serum-free medium and harvested by scraping. The harvested cells are washed again with PBS, pH 7.4. Cell disruption is effected by light sonication and after centrifugation (600 x g, 15 min) the insoluble material is removed. The supernatant fraction is retained and the protein concentration assessed by the Lowry assay. 

Harvest the cells and wash three times with cold (4°C) PBS. 

Incubated cells are washed three times with lx PBS and harvested. 

Bacteria that remained viable after 48 freeze-thaw cycles were used to initiate new cultures and these were subjected to additional freeze-thaw cycles as described. Individual isolates, which had been previously identified, as well as the control cultures, were also subjected to freeze-thaw cycles. Occasionally, single isolates in 10% TSB would supercool rather than freeze at temperatures close to 0°C, and to ensure that all samples froze at the same temperature, a few sterilized Agl crystals were added to the vials at the start of the experiments. For some experiments, cells were harvested by centrifugation (6,000 xg for 10 min) and the cell pellet washed with 10% TSB and kept at 0°C until analysis. Spent media was obtained by centrifuging, as above, and filtering (0.45 pm). 

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