2-Fold serial dilution method

Diffusion tests are used to determine the susceptibility of microorganisms but have their limitations when equivocal results are obtained or in the case of prolonged serious infection, e.g., bacterial endocarditis, the elucidation of antibiotic action in relation to the pathogen needs to be more precise. Also the terms susceptible and resistant are open to interpretation. Thus, when in douht, a precise assessment is needed and involves determining the MIC of the antimicrobial compounds to the organisms using the 2-fold serial dilution method, which shown in Figure 11.2 [20,24]. 

Table A3.1 Summary of methods for preparing a 3-fold, 2-fold, and 1.5-fold serial dilution set for inhibitor (or substrate) titration... 

Animal infectivity methods Some viruses do not cause recognizable effects in cell cultures but cause death in the whole animal. In such cases, quantification can only be done by some sort of titration in infected animals. The general procedure is to carry out a serial dilution of the unknown sample, generally at ten-fold dilutions, and samples of each dilution are injected into numbers of sensitive animals. After a suitable incubation period, the fraction of dead and live animals at each dilution is tabulated and an end point dilution is calculated. This is the dilution at which, for example, half of the injected animals die. Although such serial dilution methods are much more cumbersome and much less accurate than cell culture methods, they may be essential for the study of certain types of viruses. 

The serial dilution method of assay referred to in Section 1,4 is readily adapted for use in the estimation of relatively low antibiotic concentrations such as are found in the human (or other animal) body following administration of one or more therapeutic dosage forms. In order to achieve the sensitivity not available with other methods, the serial dilution procedure employs a smaller amount of test organism inoculum and a longer period of incubation. However, the precision that can be achieved with the other methods is lost here because of the rather wide potency ranges not covered by two-fold dilution increments. As mentioned earlier, the precision of the usual serial dilution assay may be improved by setting up graded dilution series (30) at the same time. 

Serial dilution is less a standardization method as it is a method of generating solutions to be used for standardizations. Nevertheless, its importance and utility, as well as the popularity of its application, warrants mention in this section. A serial dilution is the stepwise dilution of a substance, observant of a specified, constant progression, usually geometric (or logarithmic). One first prepares a known volume of stock solution of a known concentration, followed by withdrawing some small fraction of it to another container or vial. This subsequent container is then filled to the same volume as the stock solution with the same solvent or buffer. The process is then repeated for as many standard solutions as are desired. A ten-fold serial dilution could be 1 M,... 

The analytical sensitivities of the different quantitation methods have been compared using serial dilutions of patients specimens (Butterworth et al., 1996) and the Eurohep HBV DNA standards (Zaaijer et al., 1994). In both cases, bDNA was shown to be about 10-fold more sensitive than the liquid hybridization (Abbott) and the hybrid capture (Digene) assays. Using the Eurohep HB V standards, the detection limits were 2.5 X 106 genomes/ml for bDNA and 2.5 X 107 genomes/ml for both liquid hybridization (LH) and hybrid capture (HC) assays. 

In either approach, if the goal is to compare close titer values (such as eightfold difference between titers of two ADA samples), it is important to apply the interpolation method to determine the titers, or if a noninterpolation method is used, the serial dilutions should be narrowly spaced (ideally twofold or less). In addition, an objective method is needed for determining the ability of the assay to differentiate titers (titer precision). This titer precision is defined by the minimum significant ratio (MSR), which is the smallest fold change between the titers of any two ADA-positive samples that can be considered as significant. For example, MSR = 5 means that at least a fivefold difference in the titer results between any two positive samples will be required to conclude that they are statistically different. Evaluation of MSR is mostly relevant when comparison of samples with close titers is of interest. 

DIA is produced by transient expression in COS cells using the method described in ref. 21. Serial dilutions of the medium are tested on ES cells plated in 24-well plates. A 100-fold higher dilution than the minimal dilution required to keep ES cells undifferentiated is typically used. Serum batches are tested for their ability to sustain the growth, differentiation, and viability of ES cells grown at clonal density in the presence and absence of DIA. 

Titration method The antiserum to be tested is diluted serially to establish the limit at which enzymic reaction can no longer be detected. Visual inspection is possible (about similar detectability as absorptiometry). The concept is simple and the test is quantitative for the antibody activity. The definition of the titre, however, may be confusing since with one method the titre may be much larger than with another for the same serum. Titrations generally are workintensive and costly. It is difficult to establish by visual inspection the dilution (e.g. 1 512, or reciprocals, e.g., 512) at which the real end-point occurs. Thus, reproducibility may easily be one two-fold dilution difference from the accurate value. Therefore, a titre of, e.g. 1 4096 implies a false sense of accuracy (Section 1.3). Titration by visual inspection yields a discontinuous scale of results. 

In order to determine nitrofuran activity in vitro, the minimum inhibitory concentration (MIC) in bacterial cultures must be found by using the serial two fold dilution technique with broth as a medium. Incubation time is 24 hours at 37°C. The cup method, which is usually employed for the assay... 

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