A Comparison of the MPN and Fisher-Yates Estimators for the Density of Organisms

The method of moments estimator, discussed in FISHER and YATES (1970), for the density of bacteria is compared with the maximum likelihood or MPN estimator: For sample sizes used in practice the estimators are found to be very similar. Estimators which reduce bias are discussed and their use recommended.     

Stereological evaluation and Golgi study of the sexual dimorphisms in the volume,cell numbers,and cell size in the medial preoptic nucleus of the rat

The medial preoptic nucleus (MPN) and the sexually dimorphic nucleus of the preoptic area (SDN-POA) stand out as prominent sexually dimorphic cell groups of the rat brain. However, quantitative data on sex-related differences in these nuclei in the adult rat are confined to their volume. We have used stereological methods and Golgi-impregnated material to examine whether, in young adult rats, the sexual dimorphism in the volume of the MPN, including its divisions, and of the SDN-POA, reflect similar differences in the number and size of their neurons. We found that the total number of neurons in all MPN divisions is higher and the mean somatic volume larger in males than in females. In addition, the total dendritic length of MPN neurons is greater, but the dendritic spine density is smaller, in males than in females. Likewise, in the SDN-POA the total number and size of its neurons is greater in males than in females. The sex differences in all quantitative parameters evaluated accounted for the larger volume of the MPN and SDN-POA in males relative to females. In addition, the MPN neuropil also displays sex-related differences in its volume, and these differences closely match those detected for the volume of each MPN division. It deserves to be emphasised that the numerical density of neurons was the only parameter found to be significantly higher in females than in males in all MPN divisions and in the SDN-POA. Our results show that the MPN and the SDN-POA display sex differences in the volume, total number of neurons, and size of neuronal cell bodies and dendritic trees. Furthermore, they also indicate that the neuropil is critical for the establishment of sexual dimorphism in the size of the MPN.     

A quantitative method for the measurement of membrane affinity by polydiacetylene-based colorimetric assay

The measurement of membrane affinity is an important early screening step during drug discovery. However, classical methods for membrane affinity measurement are tedious and difficult to implement in high-throughput screening. This article describes a quantitative method for the measurement of membrane affinity by colorimetric assay based on polydiacetylene (PDA) sensors. Prepared lipid/PDA chromatic vesicles were used to model cell membranes. By measuring the colorimetric response of the chromatic vesicles when drug-membrane interactions occurred, membrane affinity constant K(b) could be calculated using a simple quantitative model. Under optimized preparation conditions, the calculated log(K(b)) values exhibited an in-batch relative standard deviation (RSD) of less than 4% and a between-batch RSD of less than 8% for all three reference compounds. The logarithm of K(b) of the six β-blockers exhibited excellent linear correlation with the logarithm of the liposome/water partition coefficient (K(m)) with R(2)=0.9793. For neutral compounds, the log(K(b)) of n-fatty alcohols correlated with the logarithm of the n-octanol/water partition coefficient (K(oct)) with a linear correlation coefficient R(2)=0.9833. This work provides a simple, convenient, and reproducible method for the rapid measurement of membrane affinity and presents important implications for high-throughput screening.     

Second derivative UV absorbance analysis to monitor nitrate-reduction by bacteria in most probable number determinations

Heterotrophic and autotrophic nitrate-reducing bacteria (NRB) play important roles in many environments. These bacteria are often enumerated by most probable number (MPN) methods. Measuring NO(3)(-) depletion in the MPN cultures is the definitive way to determine the presence of NRB. Media used for MPN determinations of NRB in oil field waters usually contain high Cl(-) concentrations, matching those in the water samples. Many methods for measuring NO(3)(-) concentrations, such as ion chromatography (IC), cadmium reduction and ion electrode methods, are adversely affected by high concentrations of Cl(-) and organic compounds. A second derivative UV absorbance method proved to be a fast and reliable means for measuring NO(3)(-) depletion in MPN media used for enumerating autotrophic and heterotrophic NRB, without interferences from Cl(-) or the organic components in the latter medium. The MPN results for heterotrophic NRB determined by the second derivative UV absorbance agreed well with those determined by the production of nitrous oxide, and were often higher than those determined by measuring nitrate depletion by the diphenylamine spot test.     

Characterization of the human ortholog of Mov34 reveals eight N-terminal residues important for MPN domain stability

Eukaryotic MPN domain proteins are components of the complexes proteasome lid, COP9-signalosome (CSN), and translation initiation factor 3 (eIF3). The proteasome lid Rpn11 and COP9-signalosome Csn5 subunits, which contain the conserved JAMM motif involved in zinc ion coordination, show catalytic isopeptidase activity. Homology modeling indicates that the MPN domain of Mov34 cannot coordinate a zinc ion in the same manner as catalytically active MPN domains. In this work, we show that the MPN domain of Mov34 is highly resistant to proteolysis and the major product comprises residues 9-186, which includes the conserved MPN domain. Two clones containing the MPN domain region (MPN1-177 and MPN1-186) including the eight N-terminal residues show a less pronounced band in the 220 nm region of the CD, indicating lower alpha-helical content relative to the clones lacking these residues (MPN9-177 and MPN9-186). However, clones lacking residues 1-8 show lower expression levels and thermal stability, indicating that residues 1-8 are required for proper folding and stability of this particular MPN domain.     

A Most-Probable-Number Assay for Enumeration of Infectious Cryptosporidium parvum Oocysts

Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed P value (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log₁₀ inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Development of a real-time PCR assay for detection and quantification of Rhizobium leguminosarum bacteria and discrimination between different biovars in zinc-contaminated soil

Primers were designed to target 16S rRNA and nodD genes of Rhizobium leguminosarum from DNA extracted from two different soil types contaminated with Zn applied in sewage sludge. Numbers of rhizobia estimated using 16S rRNA gene copy number showed higher abundance than those estimated by both nodD and the most-probable-number (MPN) enumeration method using a plant trap host. Both 16S rRNA gene copies and the MPN rhizobia declined with increased levels of Zn contamination, as did the abundance of the functional gene nodD, providing compelling evidence of a toxic effect of Zn on R. leguminosarum populations in the soil. Regression analysis suggested the total Zn concentration in soil as a better predictor of rhizobial numbers than both NH4NO3-extractable and soil solution Zn. R. leguminosarum bv. viciae nodD gene copies were generally less sensitive to Zn than R. leguminosarum bv. trifolii nodD. The latter were generally below detection limits at Zn levels of >250 mg kg(-1). Although there were differences in the actual numbers estimated by each approach, the response to Zn was broadly similar across all methods. These differences were likely to result from the fact that the molecular approaches assess the potential for nodulation while the MPN approach assesses actual nodulation. The results demonstrate that the use of targeted gene probes for assessing environmental perturbations of indigenous soil rhizobial populations may be more sensitive than the conventional plant bioassay and MPN methods.     

Release of orphanin FQ/nociceptin in the medial preoptic nucleus and ventromedial nucleus of the hypothalamus facilitates lordosis

Opioid regulation of reproduction has been widely studied. However, the role of opioid receptor-like 1 receptor (NOP; also referred to as ORL-1 and OP4) and its endogenous ligand orphanin FQ/nociceptin (OFQ/N) have received less attention despite their extensive distribution throughout nuclei of the limbic-hypothalamic system, a circuit that regulates reproductive behavior in the female rat. Significantly, the expression of both receptor and ligand is regulated in a number of these nuclei by estradiol and progesterone. Activation of NOP in the ventromedial nucleus of the hypothalamus (VMH) of estradiol-primed nonreceptive female rats facilitates lordosis. NOPs are also expressed in the medial preoptic nucleus (MPN), however, their roles in reproductive behavior have not been studied. The present experiments examined the role of NOP in the regulation of lordosis in the MPN and tested whether endogenous OFQ/N in the MPN and VMH mediates reproductive behavior. Activation of NOP by microinfusion of OFQ/N in the MPN facilitated lordosis in estradiol-primed sexually nonreceptive female rats. Passive immunoneutralization of OFQ/N in either the MPN or the VMH reduced lordosis in estradiol-primed females, but had no effect on lordosis in estradiol+progesterone-primed sexually receptive rats. These studies suggest that OFQ/N has a central role in estradiol-only induced sexual receptivity, and that progesterone appears to involve additional circuits that mediate estradiol+progesterone sexual receptivity.     

A comparison of several point estimators of the odds ratio in a single 2 x 2 contingency table.

The relative performance of the unconditioned maximum likelihood estimators (UMLEs), conditional MLEs (CMLEs), and Jewell-type estimators of the odds ratio (OR) and its logarithm were investigated in sets of single 2 x 2 contingency tables. The tables were generated by complete enumeration of all possible cell frequencies consistent with a single fixed margin. The bias, mean squared error (MSE), and average absolute error (AAE) were computed for all estimators using the individual table probabilities as weights. The results showed that, for the OR, Jewell's estimator usually had smaller bias, MSE, and AAE than either of the MLEs. While the differences were often slight for MSE and AAE, for bias it was sometimes substantial. For the log(OR), the UMLE usually had the lowest bias, and its MSE and AAE were only slightly greater than those for the other estimators. Overall, we recommend estimation on the log scale using the UMLE. If OR is to be estimated, Jewell's method had strong merit, although it is nonsymmetric with respect to the table orientation. In view of this, the UMLE may again be favoured in some situations.     

The crystal structure of the human Mov34 MPN domain reveals a metal-free dimer

The 26S proteasome is a large protein complex involved in protein degradation. We have shown previously that the PSMD7/Mov34 subunit of the human proteasome contains a proteolytically resistant MPN domain. MPN domain family members comprise subunits of the proteasome, COP9-signalosome and translation initiation factor 3 complexes. Here, the crystal structure of two C-terminally truncated proteins, MPN 1-186 and MPN 1-177, were solved to 1.96 and 3.0 A resolution, respectively. MPN 1-186 is formed by nine beta-strands surrounded by three alpha-helices plus a fourth alpha-helix at the C terminus. This final alpha-helix emerges from the domain core and folds along with a symmetrically related subunit, typical of a domain swap. The crystallographic dimer is consistent with size-exclusion chromatography and DLS analysis showing that MPN 1-186 is a dimer in solution. MPN 1-186 shows an overall architecture highly similar to the previously reported crystal structure of the Archaeal MPN domain AfJAMM of Archaeoglobus fulgidus. However, previous structural and biophysical analyses have shown that neither MPN 1-186 nor full-length human Mov34 bind metal, in opposition to the zinc-binding AfJAMM structures. The zinc ligand residues observed in AfJAMM are conserved in the yeast Rpn11 proteasome and Csn5 COP-signalosome subunits, which is consistent with the isopeptidase activity described for these proteins. The results presented here show that, although the MPN domain of Mov34 shows a typical metalloprotease fold, it is unable to coordinate a metal ion. This finding and amino acid sequence comparisons can explain why the MPN-containing proteins Mov34/PSMD7, RPN8, Csn6, Prp8p and the translation initiation factor 3 subunits f and h do not show catalytic isopeptidase activity, allowing us to propose the hypothesis that in these proteins the MPN domain has a primarily structural function.     

Passive smoking and lung cancer: a publication bias?

To assess the likelihood of publication bias in a recent review of the effect of passive smoking on lung cancer the evidence from the reviewed papers was visualised on a “funnel” plot. In such a plot if the relative risks from various studies are plotted according to sample size they should scatter round some underlying true value, the scatter being greatest where the studies have the lowest statistical power—thus showing a “funnel” pattern. If there is publication bias and studies with non-significant results are not being published there should be a “gap” in the plot. The logarithm of the relative risks was plotted against the standard error of the logarithm of the relative risk (which was used instead of sample size as a measure of statistical uncertainty). The resulting plot was compatible with a publication bias but only in studies on men.Further studies of passive smoking and lung cancer in men seem to be warranted.     

Rapid Quantitative Detection of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> in Seafood by MPN-PCR

This study aimed to adopt MPN-PCR (most probable number-polymerase chain reaction) for rapid detection of the quantity of Vibrio parahaemolyticus in seafood. V. parahaemolyticus in seafood could be quantitated by MPN statistics according to PCR products. The sensitivity of MPN-PCR was 100 times higher than that of direct PCR. Of 225 seafood samples from Qingdao, 165 were positive for the presence of V. parahaemolyticus, with an MPN value of >719 per gram, and about 41.5% of samples were positive for tdh gene-possessing cells. Eighty muscle tissues from the 225 seafood samples were investigated by direct PCR and MPN-PCR, but no V. parahaemolyticus was detected. The MPN-PCR test could be completed in less than 16 h from the time of sample preparation. It was rapid, sensitive, and reliable for comprehensive detection and quick quantitative determination of V. parahaemolyticus in seafood and it revealed the potential risk of illness associated with their consumption.     

RAPID ENUMERATION OF CLOSTRIDIAL SPORES IN RAW MILK SAMPLES USING AN IMPEDIMETRIC METHOD

Late spoilage of cheese is due to gas formation during lactic acid fermentation by spore-forming, gram-positive, anaerobic clostridia of the species Ciostridium tyrobutyricum, Clostridium butyricum and Clostridium sporogenes. Since small numbers of such clostridial spores readily cause considerable losses in cheese production, spore numbers of fewer than 100 spores/liter must be determined reliably. Until recently, the only reliable method available was the time-consuming (7 days) and cumbersome Most Probable Number Method (MPN). The objective of this study was to examine the feasibility of using impedance technology as an alternative method for the enumeration of clostridial spores. Three to fifteen replicates of 7.5–12.0 mL samples were tested using an impedimetric method with and without the addition of Oxyrase to generate anaerobic conditions within the impedance measurement cells. Results were obtained in less than 48 h. Data derived from the rapid impedance method were statistically comparable to those obtained using the reference method (MPN).     

Comparison of Two Methods for Enumeration of Anaerobe Numbers on Forages and Evaluation of Ethylene Oxide Treatment for Forage Sterilization

Experiments were conducted to (i) compare most-probable-number (MPN) procedures with roll tube procedures for enumeration of forage anaerobic bacteria and (ii) evaluate the efficacy of using ethylene oxide to sterilize wet herbage. Alfalfa, corn, and alfalfa-orchardgrass silages and alfalfa and orchardgrass herbages were analyzed for total anaerobic bacteria (medium pH, 6.8) and acid-tolerant anaerobic bacteria (medium pH, 4.5) by both roll tube and MPN procedures. No difference was found between the roll tube and MPN procedures for total bacteria; however, higher counts were obtained for acid-tolerant bacteria when the MPN procedure was used. Although MPN procedures require less time to obtain an estimate of bacterial numbers, isolation and identification of the microbial population is not possible. Alfalfa herbage was treated with ethylene oxide for 12, 24, or 36 h, incubated for 7 days at 37°C with or without addition of a bacterial inoculant, and analyzed for total bacteria by MPN procedures. Microbial growth after inoculation of ethylene oxide-treated herbage indicated that there was insufficient residual ethylene oxide to inhibit subsequent microbial growth. The results also indicated that 24 h was required to adequately sterilize fresh herbage. Thus, ethylene oxide can be used to sterilize wet herbage for use as a substrate for pure cultures of silage bacteria.     

Hypertrophy of the ageing rat medial preoptic nucleus

The medial preoptic nucleus (MPN) plays an essential role in the coordination of behaviours and physiological responses necessary for reproduction. Since ageing is associated with a progressive deterioration of reproductive functions we have explored the possibility that changes in the structural organization of the MPN might be implicated in this process. Thus, we have estimated the volume of the MPN, and the total number and size of its neurons, using stereological methods, and quantitatively evaluated the dendritic trees of MPN neurons in Golgi-impregnated material. Male and female rats, aged 6, 24 and 30 months, were independently analysed. No cell loss was observed in aged rats of both sexes. However, the volume of the MPN and the somatic size of its neurons were remarkably enlarged in aged rats. No significant age-related changes in the size or shape of the dendritic trees or in dendritic spine density were found. To evaluate whether the changes observed in aged rats could be ascribed to an altered interaction between gonadal steroids and steroid-sensitive neurons, we have additionally estimated the to tal number of MPN neurons immunoreactive for the estrogen receptor-α. No significant age-related variations were detected. The age effects upon the MPN were more marked in females than in males and, consequently, the sexual dimorphisms in neuronal size and in the number of estrogen receptor-immunoreactive neurons were blunted in aged rats.     

Rapid estimation of Escherichia coli in live marine bivalve shellfish using automated conductance measurement

Conductance measurement for quantitative estimations of Escherichia coli in live bivalve shellfish was evaluated as an alternative to the conventional most probable number (MPN) method used in France. The sensitivity of the two techniques was comparable. A single regression line ( r =-0·968, P < 10^-6) between log₁₀ MPN and detection time (DT) was used to estimate E. coli concentrations for all shellfish examined. Estimation accuracy was ± 0·92 log unit. Repeatability was better for DT than the log₁₀ of MPN estimations (average coefficients of variation 2·7 and 9·3%, respectively). The conductance signal was attributable to E. coli in 96% of cases, and only 0·7% of E. coli cultures failed to exhibit a signal within 20 h. The conductance method reduces analysis handling time and is much easier to use than the MPN method. Moreover, results can be obtained within 5–9 h compared to 3 d for the MPN method.     

Nitrification of swine waste

Complete oxidation of ammonia nitrogen (approximately 1000 mg/L) to nitrite was observed in stabilized swine waste after 49 days in incubation at 400 rpm and 29 degrees C, only if 10% (v/v) activated sludge from a wastewater treatment unit and 1.5% (w/v) CaCO3, were added. Stabilized swine waste contains less than 0.09 most probable number (MPN) per millilitre of nitrosobacteria and 2.3 MPN/mL of nitrobacteria. In activated sludge, the concentrations of these bacteria were 2.4 MPN/mL for nitrosobacteria and 4.2 x 10(5) MPN/mL for nitrobacteria. In the swine waste where ammonia was oxidized to nitrite, the nitrosobacteria growth increased to 5.5 x 10(5) MPN/mL, while the nitrobacteria growth decreased to 2.3 MPN/mL. Inoculation of a freshly stabilized swine waste with 10% (v/v) of the active nitrifying waste and addition of 1.5% (w/v) CaCO3, accelerated the oxidation of ammonia nitrogen to nitrite; the reaction was completed after only 5 days of incubation. Increasing the incubation period to 10 days resulted in the complete oxidation of the accumulated nitrite to nitrate. In the stabilized swine waste, complete nitrification without accumulation of nitrite was obtained in only 5 days of incubation when the waste was inoculated with both enriched nitrifying populations (10(6)-10(7) MPN/mL).