Pipetting process

For MPN determination, sterile pipettes calibrated in 0.1-ml increments are used. Other equipment includes sterile screw-top dilution bottles containing 99 ml of water and a rack containing six sets of five lactose broth fermentation tubes. A sterile pipette is used to transfer 1.0-ml portions of the sample into each of five fermentation tubes. This is followed by dispensing 0.1 ml into a second set of five. For the next higher dilution (the third), only 0.01 ml of sample water is required. This small quantity is very difficult to pipette accurately, so 1.0 ml of sample is placed in a dilution bottle containing 99 ml of sterile water and mixed. The 1.0-ml portions containing 0.01 ml of the surface water sample are then pipetted into the third set of five tubes. The fourth set receives 0.1 ml from this same dilution bottle. The process is then carried one more step by transferring 1.0 ml from the first dilution bottle into 99 ml of water in the second for another hundredfold dilution. Portions from this dilution bottle are pipetted into the fifth and sixth tube sets. After incubation (48 h at 35 C), the tubes are examined for gas production and the number of positive reactions for each of the serial dilutions is recorded. 

First, let us consider batch mixing processes, as exemplified by ordinaiy laboratory practice in solution kinetics. A portion of one solution (say, of the substrate) is added by pipet to a second solution (containing the reagent) in a flask, the flask is shaken to achieve homogeneity, and then samples are withdrawn at known times for analysis, or the solution is subjected to continuous observation as a function of time, for example, by spectrophotometry. For reactions on a time scale (measured by the half-life) of hours or even several minutes, the time consumed in these operations is a negligible portion of the reaction time, but as the half-life of the reaction decreases, it becomes necessary to consider these preliminary steps. Let us distinguish three stages ... 

Face wipe samples are treated similarly. The face wipe is placed in an appropriate jar and wet with the appropriate amount of wash solution. The sample is then spiked using a 1-mL volumetric pipet and immediately capped, processed, and frozen. 

One alternative method for preparing field fortifications solutions/suspensions is to prepare each fortification sample of each matrix in a separate mini-vial in the analytical laboratory and ship the vials to the field for use. This procedure precludes the use of pipets in the field and may be useful when Field Scientists not experienced in the use of pipets are involved in the field fortification process. One disadvantage of this procedure is that the mini-vials, if not designed correctly, will be hard to handle in the field, and surface tension of the suspension or fortification solution will tend to leave unacceptable amounts of the solution/suspension in the vial or at the lip of the vial and not on the matrix in question. This procedure may lead to cross-contamination of samples as the field fortification liquid is forced from the top... 

Spread the mix by using a medicine dropper. Do not use disposable pipets The disposable pipets have extremely narrow openings at the end and they clog up easily. There exists a dipping method for preparing TLC slides, but since the usual solvents, methanol and chloroform (Caution Toxic ) do not activate the binder, the powder falls off the plate. Because the layers formed by this process are very thin, they are very fragile. 

Disposable serological pipets are available. They are termed disposable because the calibration lines are not necessarily permanently affixed to the outside wall of the pipet. The calibration process is thus less expensive, resulting in a less expensive product that can be discarded after use. 

Some pipets are calibrated TC. Such pipets are used to transfer unusually viscous solutions such as syrups, blood, etc. With such solutions, the wetness remaining inside after delivery is a portion of the sample and would represent a significant nontransferred volume, which translates into a significant error by normal TD standards. With TC pipets, the calibration line is affixed at the factory so that every trace of solution contained within is transferred by flushing the solution out with a suitable solvent. Thus, the pipetted volume is contained within and then quantitatively flushed out. Such a procedure would actually be acceptable with any TC glassware, including the volumetric flask. Obviously, diluting the solution in the transfer process must not adversely affect the experiment. 

After the flasks have cooled, swirl the solutions and add four or five drops of bromcresol green or methyl red indicator mix. If the solution is green, all the base has not been neutralized. In this case, add 25.00 mL of 0.2 M HC1 with a volumetric pipet and boil again for 10 min. Repeat this process until the solution remains red. 

After calibration the probe was inserted into the flask shown in Figure 2. A concentrated solution of NH3-H2S-CO2-H2O of measured density was then pipeted into the flask and after temperature and pH equilibration the pH was read. This normally took a period of five minutes for the equilibration process. 

It is possible to check the calibration of a pipet, flask, or buret. The process involves weighing with a calibrated analytical balance. The volume of water (temperature noted) delivered or contained by the glassware is weighed. Then the analyst converts this weight to volume (using the density of water at the temperature noted), corrects the result to 20°C (the usual temperature of the factory calibration), and compares it to the factory calibration. If the difference is not tolerable, the piece of glassware is either not used for accurate work or a correction factor is applied. It should be pointed out that the thermometers used must be properly calibrated and that the timer used to measure the delivery time for the burets and pipets must also be calibrated. 

System Suitability System suitability refers to the validation of all components of an analysis system taken as a unit, a "system." For example, the analysis of an environmental water for pesticide residue involves a "method," which includes sampling (must represent the water in question), sample handling (e.g., what container is appropriate), sample preparation (perhaps an extraction process that includes the glassware, technique, timing, etc.), standards preparation (pipets, flasks, technique, etc.), injection technique, the instrument, and data handling (computer hardware and soft-... 

A 500-pl aliquot of sample, 100 pi trichloroacetic acid, and 400 pi HC1 30% are mixed by vortexing and then kept on ice for at least 1 h, centrifuged at 14,000 rpm (16,100 xg), and filtered (AcrodiscLC 13, Millipore) into brown HPLC autosampler tubes. Aliquots (20 pi) of standards and samples are injected into the HPLC. Samples with high concentrations of porphyrins should be diluted with 3 M HC11 2 initially. To measure the erythrocytes they are pretreated as follows 100 pi of 3 M acetic acid is pipetted into a tube, 50 pi of erythrocytes is added drop by drop under constant vortexing, followed by 1 min further mixing by vortex. This solution is further processed as described for plasma. 

Each instrument that we use has a scale of some sort to measure a quantity such as mass, volume, force, or electric current. Manufacturers usually certify that the indicated quantity lies within a certain tolerance from the true quantity. For example, a Class A transfer pipet is certified to deliver 10.00 0.02 mL when you use it properly. Your individual pipet might always deliver 10.016 0.004 mL in a series of trials. That is, your pipet delivers an average of 0.016 mL more than the indicated volume in repeated trials. Calibration is the process of measuring the actual quantity of mass, volume, force, electric current, and so on, that corresponds to an indicated quantity on the scale of an instrument. 

The BF3 method takes 40 min to complete. If a large number of samples must be processed, the time will be longer because of the time needed between the experimental steps, such as transferring tubes and pipetting solvents, and space limitations of the heating block and evaporator. 

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