Alan J. Hedges

Estimating the precision of serial dilutions and viable bacterial counts.

The propagation of error in serial dilutions was investigated theoretically and by means of computer simulations. The principal aim of the study was, given only the pipette manufacturers specification, to estimate the variance of any step in a dilution series both of pure solutions and of homogeneous bacterial suspensions by means of simple formulae. The study was extended to include bacterial plate counts by both the standard and the Miles and Misra methods. It was found that such estimation was possible and that the distributions approximated the normal sufficiently for the construction of confidence intervals (Cls) by the usual method. Such intervals can be regarded as minima which could be inflated by other, possibly undetermined, factors. It is suggested that laboratories could construct tables such as that reported here for pipettes and methods in common use to facilitate estimation. While replication of the final sampling step of a plate count increases the precision of estimation, averaging across dilutions may decrease precision and is not recommended for the standard pour-plate count.

Human CD8 Responses to a Complete Epitope Set from Preproinsulin: Implications for Approaches to Epitope Discovery

PurposeIn this study, we explored the breadth of CD8 T cell reactivity to preproinsulin (PPI) in type 1 diabetes.Materials and MethodsWe tested a complete peptide set in pools covering all 406 potential 8–11mer epitopes of PPI and 61 algorithm-predicted human leukocyte antigen (HLA)-A2-specific epitopes (15 pools) from islet-specific glucose-6-phophatase catalytic subunit-related protein (IGRP), using a CD8-specific granzyme B enzyme-linked immunosorbent spot assay.ResultsResponses were seen to 64 of the 102 PPI pools in two or more newly diagnosed patients (63%) compared to 11 pools in the control subjects (11%, p < 0.0001, Fisher’s exact test). We identified five pools containing 20 peptides, which distinguished patients from control subjects, most of which had predicted low-affinity binding to HLA class I molecules. In contrast, fewer (5 of 15 = 33%) IGRP peptide pools, selected by higher binding affinity for HLA-A2 (present in seven of eight patients and five of seven control subjects), stimulated responses in two or more patients, and none stimulated responses in more than two control subjects (p = 0.042, Fisher’s exact test).ConclusionThus, we conclude that CD8 T cell reactivity to PPI in patients with type 1 diabetes can be much broader than shown previously and more diverse than seen in control subjects. Furthermore, responses were often stimulated by peptides with low predicted HLA-binding affinities.

The killing of sensitive cells by colicin D

Abstract 1. At low multiplicities, the large molecular weight protein antibiotic colicin D inhibits protein synthesis in sensitive cells without markedly affecting the synthesis of DNA or RNA. In this respect it is very much like colicin E3 but unlike those colicins which appear to inhibit oxidative phosphorylation. It was previously suggested that the lethal action of colicin D was the inhibition of oxidative phosphorylation. 2. Chloramphenicol causes an immediate decrease in the sensitivity of cells to the action of colicin D, but does not similarly alter the sensitivity of cells to colicin E3. This may indicate that these two colicins are not completely identical in their modes of action. 3. Although the lethal adsorption of colicin D to sensitive cells follows single-hit kinetics, on average 100 molecules are required to kill a cell. Thus, only 1 % of adsorbed colicin molecules actually effects the lethal action.

Estimates of measurement uncertainty from proficiency testing schemes, internal laboratory quality monitoring and during routine enforcement examination of foods

Aims:  To determine the levels of measurement uncertainty (MU) obtained in proficiency testing and routine microbiological analyses of foods and to compare these with estimates of MU obtained for results of analyses obtained in collaborative interlaboratory studies of microbiological methods.

Minimising the between-sample variance in colony counts on foods

The objective of this study was to assess whether it is possible to minimise the variance in colony counts on replicate target samples of foods by aseptic compositing of the samples or by increasing the quantity of sample examined. The results show that compositing reduces the overall variance, and hence the standard deviation, to very low levels, although in some cases the overall variance remains relatively high, reflecting the heterogeneous distribution of microorganisms in the foods. Increasing the weight of target sample examined (e.g. from 10 g to 100g) had a pronounced effect on the mean log(10) colony count and significantly reduced the variance of the mean. The results are discussed in relation to the quantity of sample that is recommended for examination in international and other standards.

The contribution of sampling uncertainty to total measurement uncertainty in the enumeration of microorganisms in foods.

Random samples of each of several food products were obtained from defined lots during processing or from retail outlets. The foods included raw milk (sampled on farm and from a bulk-milk tanker), sprouted seeds, raw minced meat, frozen de-shelled raw prawns, neck-flaps from raw chicken carcasses and ready-to-eat sandwiches. Duplicate sub-samples, generally of 100 g, were examined for aerobic colony counts; some were examined also for counts of presumptive Enterobacteriaceae and campylobacters. After log(10)-transformation, all sets of colony count data were evaluated for conformity with the normal distribution (ND) and analysed by standard ANOVA and a robust ANOVA to determine the relative contributions of the variance between and within samples to the overall variance. Sampling variance accounted for >50% of the reproducibility variance for the majority of foods examined; in many cases it exceeded 85%. We also used an iterative procedure of re-sampling without replacement to determine the effects of sample size (i.e. the number of samples) on the precision of the estimate of variance for one of the larger data sets. The variance of the repeatability and reproducibility variances depended on the number of replicate samples tested (n) in a manner that was characteristic of the underlying distribution. The results are discussed in relation to the use of measurement uncertainty in assessing compliance of results with microbiological criteria for foods.

A method to apply the robust estimator of dispersion, Qn, to fully-nested designs in the analysis of variance of microbiological count data.

This note outlines a method for the application of Rousseeuws robust estimator of scale, Q(n), to fully-nested designs such as those used to estimate the components of variance in collaborative trials employed in quantitative food microbiology. A computer program is freely available on-line.

Transient μm scale protein accumulation at the center of the T cell antigen presenting cell interface drives efficient IL-2 secretion

A µm scale complex, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen presenting cells. It is of interest as µm scale protein complexes have unique biophysical and signaling properties. To causally determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC recruitment varied between the adaptors and was consistently diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of adaptor cSMAC localization by fusion with domains with a strong interface localization preference restored IL-2 secretion as a key T cell effector function as dependent on the precise reconstitution dynamics. Our data establish a model where the cSMAC enhances early signaling by facilitating extensive signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates.