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Two-layer sample preparation method for MALDI mass spectrometric analysis of protein and peptide samples containing sodium dodecyl sulfate.

Sodium dodecyl sulfate (SDS) is widely used in protein sample workup. However, many mass spectrometric methods cannot tolerate the presence of this strong surfactant in a protein sample. We present a practical and robust technique based on a two-layer matrix/sample deposition method for the analysis of protein and peptide samples containing SDS by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The two-layer method involves the deposition of a mixture of sample and matrix on top of a thin layer of matrix crystals. It was found that for SDS-containing samples, the intensity of the MALDI signals can be affected by the conditions of sample preparation: on-probe washing, choice of matrix, deposition method, solvent system, and protein-to-SDS ratio. However, we found that, under appropriate conditions, the two-layer method gave reliable MALDI signals for samples with levels of SDS up to approximately 1%. The applications of this method are demonstrated for MALDI analysis of hydrophobic membrane proteins as well as bacterial extracts. We envision that this two-layer method capable of handling impure samples including those containing SDS will play an important role in protein molecular weight analysis as well as in proteome identification by MALDI-MS and MS/MS.

Bacterial Proteins↗

Miniaturized and automated sample pretreatment for determination of PCBs in environmental aqueous samples using an on-line microporous membrane liquid-liquid extraction-gas chromatography system.

A new, fast, and automated sample pretreatment technique for determination of lipophilic organic compounds in aqueous samples has been developed and applied to the determination of polychlorinated biphenyls (PCBs) in environmental river water. It is based on miniaturized microporous membrane liquid-liquid extraction coupled on-line to gas chromatography (GC) with electron capture detection. The heart of the system that simultaneously connects the sample pretreatment step to the final GC analysis has been named the extracting syringe (ESy). The ESy carries a miniaturized membrane extraction card attached to an electrically and mechanically designed installment and is mounted directly over a GC injector for fully automated injection of the extract. A method was developed to extract 10 PCB congeners from 1-mL water samples (after addition of 40% acetonitrile) with an extraction time of 10 min. The optimized methodology showed good linearity (in the dynamic concentration range of 5 ng L(-)(1)-1 microg L(-)(1)), enrichment factors of 33-40 times, repeatable extractions (RSD 2-5%, n = 4), and low detection limits (2-3 ng L(-)(1)). Acetonitrile had to be added to the samples in order to overcome the influence of PCB adsorption on the repeatability of extraction and enrichment and to minimize the overall memory effect (OME). OME and carryover depended not only on the concentration of the organic solvent added to the sample and that used in the washing procedure but also on whether the extracting card was changed or not. When an optimized washing procedure was applied, the OME was approximately 0.2% at high concentrations (i.e., 1 microg L(-)(1)). When each extraction took place in a new extraction card, no OME was detected. Additionally, no significant adsorption onto glass surfaces or a matrix effect on extraction was noticed. The main features of this methodology are good extraction repeatability, low detection limits at short extraction time, and the unsurpassed characteristic of no detectable OME in the entire system when each sample is processed in a new card. The total consumption of organic (nonchlorinated) solvents is less than 5 mL per sample.

Journal Article↗

Direct determination of lead isotopes (206Pb, 207Pb, 208Pb) in arctic ice samples at picogram per gram levels using inductively coupled plasma-sector field MS coupled with a high-efficiency sample introduction system.

Adopting strict cleanroom procedures, ice samples from the Canadian High Arctic have been analyzed for Pb concentrations and Pb isotopes (206Pb, 207Pb, 208Pb) using ICP-SMS. The detection limit for Pb (0.06 pg g(-1)) was approximately 2 orders of magnitude lower than the lowest concentration of Pb in the ice samples (range, 4.3-1660 pg g(-1); median, 45 pg g(-1)). Acidification of ice samples with high-purity HNO3 for stabilization purposes contributed only 0.004 pg of Pb g(-1), which is an insignificant source of Pb. Using a new sample introduction system consisting of a heated (140 degrees C) minicyclonic spray chamber and a Peltier cooled condenser (2 degrees C) and by replacing the conventional sample cone with a high-performance cone, signal intensities for Pb were increased by approximately 1 order of magnitude. Thus, it was possible not only to measure Pb isotope ratios directly using ICP-SMS but also to achieve reasonable precision (approximately 0.2%) at low picogram per gram concentrations of total Pb. This precision is comparable to that achievable by thermal ionization mass spectrometry at such low Pb concentrations, but the ICP-SMS requires much less sample volume (approximately 2 mL), needs no sample pretreatment, and therefore is considerably faster and less expensive than the conventional approach. Even though absolute Pb concentrations in two ice samples dating from 1974 and 1852 were very similar (9 and 6 pg g(-1)) their fundamentally different isotopic signature (206Pb/207Pb: 1.169 +/- 0.002 vs 1.147 +/- 0.003) clearly indicates different sources of Pb. The analytical procedures described here, therefore, offer great promise for fingerprinting the predominant sources of atmospheric Pb in polar snow and ice.

Journal Article↗

First steps in robot automation of sampling and sample management during cultivation of mammalian cells in pilot scale.

The robot automation of sampling and the subsequent treatment and storage of aliquots during mammalian cell cultivations was investigated. The complete setup, the development and testing of the sampling device, the robot arm, and the cell imaging system are described. The developed sampling device is directly coupled to a pilot bioreactor. It allows the computerized sterile filling of cell broth into 50 mL sample tubes. After each sampling the whole tubing system is steam sterilized. For further off-line treatment a robot takes the sample to the different devices. This robot is equipped with a camera and a force/torque sensor. A color-based object recognition guides the arm in a complex surrounding with different illumination situations, enabling the robot to load the sampling device with tubes and take the sample to further devices. For necessary pipetting and refilling we developed a computerized device. Cells are automatically stained and counted using an imaging system. The cell number and viability is automatically saved in a process control system together with the on-line parameters. During several cultivations in 20 and 100 L scale these main components of the automation strategy were successfully tested.

Animals↗

On the relationship between differential outcomes and differential sample responding in matching-to-sample.

Four experiments examined control over choice by differential sample responding in matching-to-sample with differential outcomes. In Experiment 1, pigeons initially learned to match with food versus no-food outcomes. Their performances later transferred to other samples to which responding versus not responding had been explicitly reinforced with a single outcome (food). In Experiment 2, pigeons initially learned to produce the comparisons by pecking one sample but not the other. Transfer was then observed to new samples associated with food versus no food (and thus often vs. seldomly pecked). Experiments 3 and 4 showed that transfer of matching required differential behavior to each sample set and did not depend on explicit conditioning of that behavior prior to acquisition. Together, these results show that differential sample behaviour provides a redundant cue for choice in differential outcome matching-to-sample.

Animals↗

Sampling in qualitative research. Purposeful and theoretical sampling; merging or clear boundaries?

Sampling is a very complex issue in qualitative research as there are many variations of qualitative sampling described in the literature and much confusion and overlapping of types of sampling, particularly in the case of purposeful and theoretical sampling. The terms purposeful and theoretical are viewed synonomously and used interchangeably in the literature. Many of the most frequent misinterpretations relate to the disparate meanings and usage of the terminology. It is important that the terminology is examined so that underlying assumptions be made more explicit. Lack of shared meanings and terminology in the nursing discourse creates confusion for the neophyte researcher and increases the production of studies with weak methodologies. This paper analyses critically purposeful and theoretical sampling and offers clarification on the use of theoretical sampling for nursing research. The aim is not to make prescriptive statements on sampling; rather, to enhance understanding of the differences between purposeful and theoretical sampling for nursing research.

Data Collection↗

Comparison of tear sampling techniques for pharmacokinetics analysis: ofloxacin concentrations in rabbit tears after sampling with schirmer tear strips, capillary tubes, or surgical sponges.

This study compared the precision and accuracy of 4 tear sampling methods. In vivo, albino rabbits were treated with single bilateral eye drops ofofloxacin 0.3% solution, 3 hr after which tear samples were collected using capillary tubes (CT), surgical sponges (SS), or tear strips for 15 sec (15sTS) or 60 sec (60sTS). In vitro, CT, SS, and tear strips were spiked with known volumes of ofloxacin solution in order to assess the bioanalytical accuracy of each technique. Ofloxacin levels were quantified by HPLC in all samples. Results showed that tear volumes and ofloxacin masses sampled in vivo depended on sampling method. Tear volume followed the rank order 60sTS > 15sTS > SS > CT. The volume collected by 60sTS exceeded precorneal tear volume. Ofloxacin mass followed the order 60sTS approximately 15sTS > SS > CT. Tear concentrations (mean +/- SD; N = 12) were 3.28 +/- 3.76 microg/g for 60sTS, 10.3 +/- 10.0 microg/g for 15sTS, 9.75 +/- 8.04 microg/g for SS, and 5.83 +/- 3.35 microg/g for CT. In vitro, SS, 15sTS, and 60sTS yielded accuracies of 103-107% and coefficients of variation (CV) < 9%. CT was only 85% accurate with a CV of 31%, indicating incomplete extraction during analysis. We concluded from this study that: 1) rabbit tear sampling by SS was rapid, easy, accurate, precise, and easily analyzable; 2) sampling by CT or 15sTS was accurate, but may require aggressive extraction (for CT) or be affected by tear flow rate (for 15sTS); and 3) tear sampling by 60sTS underestimated actual tear concentrations.

Animals↗

A novel method for isolation of Campylobacter spp. from environmental samples, involving sample processing, and blood- and antibiotic-free medium.

AIMS: To develop a method that involves sample processing, and blood- and antibiotic-free medium for isolation and enumeration of Campylobacter spp. from environmental samples. METHODS AND RESULTS: The sample processing (preT) was standardized to minimize the population of competing bacteria. A blood- and antibiotic-free differential, Kapadnis-Baseri medium (KB medium) was formulated and tested for isolation of Campylobacter spp. in comparison with CAT medium. PreT-KB method was evaluated in comparison with the conventional viable count method and with the conventional most probable number (C. MPN) method for enumeration of Campylobcater from environmental samples. The results indicated that sample processing significantly reduced population of competing bacteria. The KB medium selected Gram-negative bacteria and differentiated Campylobacter from lactose-fermenting competing bacteria. The population of Campylobacter detected by preT-KB method was similar to that by conventional viable count method. While, the population of Campylobacter spp. determined by preT-KB method was higher than that by C. MPN method. In addition, the preT-KB method detected antibiotic sensitive campylobacters. CONCLUSION: The preT minimizes population of competing bacteria and the KB medium selects Gram-negative bacteria and differentiates Campylobacter from them. Therefore, Campylobacter can be isolated from environmental samples without using antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The preT-KB method is simple and facilitates isolation of antibiotic sensitive and enumeration of Campylobacter in the environmental samples. Therefore, the new method will be useful for isolation and enumeration of Campylobacter from water, food and sewage samples. Besides, it would also detect antibiotic-sensitive campylobacters, which are not detected by conventional viable count and MPN methods.

Animals↗

Efficiency of Salmonella isolation from meat-and-bone meal of one 300-g sample versus ten 30-g samples.

Twenty-five meat-and-bone meal samples were analyzed for salmonellae, comparing a single 300-g to ten 30-g samples. Seventeen were positive using the larger sample; eighteen were positive with the smaller. The 300-g sample showed a significantly higher (P < 0.01) percentage of confirmed salmonellae at 2 days of incubation than at 1 day. The ten 30-g samples did not show changes at 2 days. At 2 days, the 30-g samples showed significantly fewer confirmed salmonellae than the 300-g sample; however, there was no difference at 1 day. Of 1,417 presumptive colonies picked, 1,215 (85.7%) were lysine decarboxylase-positive and 1,152 (81.3%) were agglutinated by one of the somatic antisera. There were no significant differences in diversity or total numbers of different somatic groups between the large and small samples.

Bacteriological Techniques↗

Efficiency of the ortho VITROS assay for detection of hepatitis C virus-specific antibodies increased by elimination of supplemental testing of samples with very low sample-to-cutoff ratios.

The clinical significance of specimens with low sample-to-cutoff (S/Co) ratios in the Ortho VITROS chemiluminescence assay (CIA) for detection of antibodies to hepatitis C virus (HCV) was evaluated. In one study of 482 CIA-reactive samples, none of the 83 samples with S/Co ratios of < 5 was HCV RNA positive. In a subsequent study, 332 samples with S/Co ratios of between 1 and 20 were tested with the recombinant immunoblot assay (RIBA). None of the 163 samples with S/Co ratios of < 5 was RIBA positive, 83% were RIBA negative, and 28 samples (18%) were RIBA indeterminate. HCV RNA and/or clinical evidence of hepatitis was not found in the 27 indeterminate cases examined. These results show that over 99% of samples with very low S/Co ratios (< or = 5) have no evidence of HCV infection. Therefore, we suggest that the HCV antibody testing algorithm for the VITROS assay might be modified to eliminate supplemental testing of samples with very low S/Co ratios.

Hepacivirus↗

Split-sample analysis of discarded cells from liquid-based Pap smear sampling devices.

OBJECTIVE: To examine cells that were retained on sampling devices used to collect ThinPrep (Cytyc Corp., Boxborough, Massachusetts, U.S.A) Pap smears in order to evaluate both the number and significance of cells that are routinely discarded with these devices after liquid-based specimens are collected. STUDY DESIGN: One hundred Pap smears from 100 women were prospectively procured after gynecologic Pap smears were collected for the ThinPrep Pap test. The sampling end of the collection devices was cut off and placed in a vial that contained SUREPATH preservative fluid (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A). The residual cell samples were processed using the SurePath PREPSTAIN slide processor (TriPath). A single liquid-based slide was prepared from the sampling devices from each of the 100 specimens collected. The slides produced from the discarded devices were reviewed for the following: squamous cells, endocervical component, epithelial cell abnormalities and miscellaneous findings. The slides prepared from the "throw-away" (TA) material were subsequently compared with the primary ThinPrep Pap smear slide. RESULTS: Twenty-five percent of the TA samples had an equal or greater number of squamous cells per high-power microscopic field when compared to the primary ThinPrep slide, with 8% of the TA slides demonstrating greater overall cellularity. An endocervical component was present on 27 of 66 cervical samples (40.9%). Three of five cases (60%) interpreted as atypical squamous cells of undetermined significance had similar cells on the TA slides. Two cases of atypical glandular cells of undetermined significance had no abnormal cells on the TA slides. Twelve of 14 cases (85.71%) of low grade squamous intraepithelial lesion contained similar cells on the TA slides. Two of four cases (50%) of high grade squamous intraepithelial lesion also had similar abnormal cells on the TA slides. Miscellaneous findings included 1 case of benign endometrial cells and 4 Candida infections present on both preparations, along with 1 case of Trichomonas vaginalis organisms present on the ThinPrep slide only. In 1 specimen, several multinucleated histiocytic giant cells were present only on the TA slide. CONCLUSIONS: Specimens prepared from TA collecting devices used for the ThinPrep Pap test are less sensitive than the primary specimen for the detection of cervical lesions. This is in contrast to split-sample studies involving ThinPrep and conventional smears. Our study documented the presence of normal and abnormal cells discarded from ThinPrep sampling devices in a high percentage of cases. Discarded abnormal cells on the TA slides were, however, few when compared to the primary specimen, with only 1 exception involving a high grade lesion.

Aged↗

Improving the sampling technique of arterialized capillary samples to obtain more accurate PaO2 measurements.

Arterialized earlobe capillary blood samples (ELCS) have been used as a measurement of blood gas status for over 20 years. There is general acceptance that there is a strong correlation and limits of agreement between arterial and arterialized blood samples with respect to pH and PaCO2. Although the correlation between the arterial and arterialized PaO2 is good, the limits of agreement poor. Our aim was to improve the accuracy of this technique in the measurement of PaO2 by simultaneously monitoring the oxygen saturation by pulse oximetry whilst taking an ELCS. We hypothesize that significant discrepancies between the SaO2 and SpO2 highlight either a poorly arterialized sample or an over aerated sample from air bubbles. We compared the SpO2 with the SaO2 of an arterial sample from 27 inpatients. We used the limits of agreement between these samples to define the degree of discordance we would accept between SaO2 and SpO2 before repeat ELCS. Subsequently, 252 consecutive patients attending our respiratory physiology unit over a six-month period had an ELCS and simultaneous SpO2. If there was a discrepancy between SaO2 and SpO2 of > 2% the ELCS was repeated. There was a good correlation and limits of agreement between the SpO2 and arterial SaO2 (r = 0.97, mean difference +/- 95% limits of agreement: 0.34 +/- 2.68). A difference of more than 2% between arterialized SaO2 and SpO2 was identified in 21 patients out of 252 (8.3%) with SaO2 higher in two and lower in 19 (r = 0.96, mean difference +/- 95% limits of agreement: 0.66 +/- 3.1). Repeat ELCS of these 21 samples reduced this discrepancy improving the concordance of the measurements (r = 0.98, mean difference +/- 95% limits of agreement: 0.47 +/- 1.0). In one case a difference of 3% remained between the saturations. We conclude that the addition of simultaneous pulse oximetry with ELCS will identify rogue measurements in about 8% of cases highlighting the need for repeat samples and thus increasing the accuracy of the measurement of PaO2 by ELCS.

Arteries↗

Manure sampling for nutrient analysis: variability and sampling efficacy.

Reliable estimation of nutrient concentrations is required to manage animal manure for protecting waters while sustaining crop production. This study was conducted to investigate sample variability and reliable nutrient analysis for several manure types and handling systems. Serial samples were collected from dairy, swine, and broiler poultry operations while manure was being loaded onto hauler tanks or spreaders for field application. Samples were analyzed for total solids (TS), total nitrogen (N), ammoniacal nitrogen (NH4-N), total phosphorus (P), and potassium (K). The least number of samples needed for reliable testing of total N and P, defined as +/- 10% of the experimental means with 99% probability, was obtained for each farm using a computer-intensive random resampling technique. Sample variability within farms, expressed as the coefficient of variation (CV), was mostly 6 to 8% for farms that used agitation of manure storages but several times higher (20-30%) on farms where no agitation was applied during the sampling period. Results from the random resampling procedure indicated that for farms that used agitation, three to five samples were adequate for a representative composite for reliable testing of total N and P; whereas for farms without agitation, at least 40 samples would be required. Data also suggest that using book values for manure nutrient estimations could be problematic because the discrepancies between book standards and measured farm data varied widely from a small amount to several fold.

Agriculture↗

Microcurettage sampling of the endometrium for histopathological examination--simpler but not safe? Comparison of endometrial histopathology in samples obtained by a disposable mechanical curette and by traditional curettage.

The routine use of dilatation and curettage (D&C) is questioned as a standard procedure for endometrial sampling. Instruments have been developed in order to simplify the procedure. The aim of the present study was to estimate the sensitivity to detect malignancy/premalignancy by histological examination of endometrial samples obtained by microcurettage using a disposable plastic curette, Gynoscann. The histological examination of the combined microcurettage and D&C material was defined as the true diagnosis. All patients undergoing diagnostic D&C at the two participating hospitals were included in the study. The endometrial samples were evaluated at the Department of Pathology as part of their routine work. The microcurettage samples were re-examined independently by two pathologists (observer 1 and observer 2) who did not have access to the rest of the tissue material nor the diagnosis given previously. A total of 902 patients were enrolled in the study. Endometrial cancer was found in four women below 50 years of age. None of these were detected by histological examination of the Gynoscann obtained sample. Among women aged 50 and above, samples from 16 women turned out to have endometrial cancer. The microcurettage samples were correctly classified as malignant/premalignant by observer 1 in 13 cases and by observer 2 in 14 cases. We conclude that diagnostic D&C among women 50 years and below cannot be replaced by histological examination of Gynoscann samples without the risk of overlooking significant pathology.

Adult↗

The comparison between capillary blood sampling and arterial blood sampling in an NICU.

To study the correlation between the results of blood examination from arterial blood sampling via arterial puncture and capillary blood sampling via heel-stick, 33 premature infants with birthweights ranging from 635 grams to 2500 grams were enrolled in this study. The 33 paired samples of capillary blood sampling via heel-stick and simultaneous arterial blood sampling via arterial puncture were analysed within 3 minutes after collection for hemoglobin, hematocrit, sodium, potassium, chloride, calcium, glucose, lactate, osmolality, and blood gas. The average correlations between capillary and arterial samples were 0.92 for pH, 0.93 for PCO2, 0.87 for hematocrit, 0.88 for hemoglobin, 0.86 for sodium, 1.16 for calcium, 1.14 for glucose, 1.01 for lactate, 1.03 for base excess (BE), 1.05 for HCO3-, 0.91 for osmolality, 1.35 for PO2, 0.28 for O2 saturation, 0.67 for potassium, and 0.78 for chloride. We concluded that capillary blood sampling via heel-stick is a rapid, simple, less invasive, and reliable alternative to arterial blood sampling for the evaluation of hemoglobin, hematocrit, sodium, calcium, glucose, lactate, osmolality, and blood gas (pH, pCO2, HCO3-, BE) in premature infants.

Blood Specimen Collection↗

Effect of sample preparation and pH-mediated sample stacking on the analysis of multiplexed short tandem repeats by capillary electrophoresis.

An amplification system was used in this study to evaluate the effect of sample preparation and its injection on the electrophoretic separation by capillary electrophoresis using a commercially available capillary electrophoresis instrument. The effect of dilution of amplified samples in water and in different sources of formamide (with conductivity values ranging from 47 to 1000 microS) was evaluated, as was contamination of the sample with high DNA concentrations or buffer salts. Although resolution remained constant in the different solvents tested, the sensitivity increased in samples diluted in water and high-purity formamide. An on-column sample preconcentration method for capillary-based DNA analysis was evaluated to increase the sensitivity of low-quality samples. This technique, pH-mediated sample stacking, is based on the injection of NaOH immediately before sample injection: A neutralization reaction occurs between OH- and tris+ ions so that a low-conductivity zone is created at the head of the capillary. DNA fragments are concentrated at the front of this zone. Using coated capillaries with hydroxycellulose 2% (MW 250,000) as a separation matrix, an improvement in sensitivity was detected in all the solvents studied. The gain in sensitivity was higher for more conductive solvents, and was not correlated with the size of the DNA fragments.

Electrophoresis, Capillary↗

Evaluation of urine sampling technique: bacterial contamination of samples from women students.

A urine specimen is traditionally sampled using the clean-catch midstream technique, but this technique is poorly documented. The aim of this study was to evaluate the bacterial content of urine samples taken using individual steps of the clean-catch midstream technique, or combinations of steps. One hundred and ten women students provided a total of 864 early morning urine samples, using a different sampling technique each morning. Holding the labia apart during sampling more than halved the percentage of samples containing 10(5) colony-forming units per ml or more, and increased the percentage of uncontaminated samples, compared with passing urine without employing any of the precautions. Cleaning the perineum or using the midstream technique resulted in percentages no different from urination without employing any precautions. It is therefore recommended that women should be instructed to carry out the simplest effective urine sampling technique: holding the labia apart.

Adult↗

Sampling--how big a sample?

It is thought that, in a consignment of discrete units, a certain proportion of the units contain illegal material. A sample of the consignment is to be inspected. Various methods for the determination of the sample size are compared. The consignment will be considered as a random sample from some super-population of units, a certain proportion of which contain drugs. For large consignments, a probability distribution, known as the beta distribution, for the proportion of the consignment which contains illegal material is obtained. This distribution is based on prior beliefs about the proportion. Under certain specific conditions the beta distribution gives the same numerical results as an approach based on the binomial distribution. The binomial distribution provides a probability for the number of units in a sample which contain illegal material, conditional on knowing the proportion of the consignment which contains illegal material. This is in contrast to the beta distribution which provides probabilities for the proportion of a consignment which contains illegal material, conditional on knowing the number of units in the sample which contain illegal material. The interpretation when the beta distribution is used is much more intuitively satisfactory. It is also much more flexible in its ability to cater for prior beliefs which may vary given the different circumstances of different crimes. For small consignments, a distribution, known as the beta-binomial distribution, for the number of units in the consignment which are found to contain illegal material, is obtained, based on prior beliefs about the number of units in the consignment which are thought to contain illegal material. As with the beta and binomial distributions for large samples, it is shown that, in certain specific conditions, the beta-binomial and hypergeometric distributions give the same numerical results. However, the beta-binomial distribution, as with the beta distribution, has a more intuitively satisfactory interpretation and greater flexibility. The beta and the beta-binomial distributions provide methods for the determination of the minimum sample size to be taken from a consignment in order to satisfy a certain criterion. The criterion requires the specification of a proportion and a probability.

Bayes Theorem↗