Estimation of microbial densities from dilution count experiments

Although dilution counts have been widely used in quantitative microbiology, their interpretation has always been widely discussed both in microbiology and in applied statistics. Maximum-likelihood (most-probable-number) methods hae generally been used to estimate densities from dilution experiments. It has not been widely recognized that these methods are intrinsically and statistically biased at the sample sizes used in microbiology. This paper presents an analysis of proposed method for correction of such biases, and the method was found to be robust for moderate deviations from Poisson behavior. For analyses at greater variance with the Poisson assumptions, the use of the Spearman-Karber method is analyzed and shown to yield an estimate of density of lesser bias than that produced by the most-probable-number method. Revised methods of constructing confidence limits proposed by Loyer and Hamilton (M.W. Loyer and M.A. Hamilton, Biometrics 40:907-916, 1984) are also discussed, and charts for the three- and four-decimal dilution series with five tubes per dilution are presented.     

Evaluation of a rapid method for the quantitative estimation of coliforms in meat by impedimetric procedures.

A 24-h instrumental procedure is described for the quantitative estimation of coliforms in ground meat. The method is simple and rapid, and it requires but a single sample dilution and four replicates. The data are recorded automatically and can be used to estimate coliforms in the range of 100 to 10,000 organisms per g. The procedure is an impedance detection time (IDT) method using a new medium, tested against 131 stock cultures, that markedly enhances the impedance response of gram-negative organisms, and it is selective for coliforms. Seventy samples of ground beef were analyzed for coliforms by the IDT method and the conventional three-dilution, two-step most-probable-number test tube procedure. Seventy-nine percent of the impedimetric estimates fell within the 95% confidence limits of the most-probable-number values. This corresponds to the criteria used to evaluate other coliform tests, with the added advantage of a single dilution and more rapid results.     

Resistance and recovery studies on ultraviolet-irradiated spores of Bacillus pumilus.

A spore suspension model and a procedure for recovering ultraviolet (UV)-irradiated spores of Bacillus pumilus were investigated. A most-probable-number tube dilution method using double-strength Trypticase soy broth was found to be superior to the agar plate method for recovering optimal numbers of spores irradiated with sublethal doses of UV energy. Aqueous suspensions of B. pumilus survived UV doses up to 108,000 ergs/mm2 as determined by a most-probable-number recovery and estimation procedure. Resistance and stability data were consistent and reproducible, indicating the dependability of this method for recovering UV-damaged spores. The procedures used to collect information concerning resistance characteristics for two strains of B. pumilus are discussed.     

Estimation of the most probable number with a programable pocket calculator.

The most-probable-number method has many potential applications, particularly if many tubes per dilution and many dilution levels are used. Increasing the number of cultures is possible with modern automatic and semiautomatic equipment. However, available tables are not sufficiently detailed to handle data from a large number of culture tubes used in an assay. This paper provides a computer program capable of handling the necessary arithmetic and written for a hand-held, advanced programable calculator.     

Comparison of direct serial dilution and most-probable-number methods for determining endotoxins in meats by the Limulus amoebocyte lysate test.

When the endotoxin content of ground beef was determined by direct serial dilution and by three-tube most-probable-number methods, the results were not significantly different, although the latter method provided more specific values for individual samples.     

14C-most-probable-number method for enumeration of active heterotrophic microorganisms in natural waters.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.     

Comparison of methods of enumerating coliforms after UV disinfection.

In view of the differences that have been found between the most-probable-number and membrane filtration methods for the recovery of coliforms from chlorinated samples, the survival of total and fecal coliforms in UV-irradiated effluent samples, as tested by the most-probable-number and standard single-step membrane filtration methods, was compared. There were no significant differences in the survival of total and fecal coliforms, as tested by the two methods. In a separate set of experiments comparing total and fecal coliform survival, as tested by the most-probable-number method, only a very small but statistically significant difference of 0.1 log survival units was found. For UV-disinfected wastewater effluents, standard one-step membrane filtration procedures are comparable to standard most-probable-number procedures.     

Redefining relativity: quantitative PCR at low template concentrations for industrial and environmental microbiology

The application of PCR techniques in environmental and industrial microbiology is complicated by innumerable organic and inorganic contaminants and enzyme inhibitors that copurify with nucleic acids. These complications are compounded in quantitative PCR (qPCR) methods, which are predicated upon subtle yet significant assumptions of amplification efficiency and the representativeness of the sample with respect to the environment or industrial process from which it was obtained. In low-biomass and/or low-template situations, additional concerns related to target gene spatial heterogeneity in the sample, differential DNA (or RNA) extraction efficiency, molecular sampling error, attenuation of PCR inhibitors and amplification bias can quickly undermine fundamental assumptions of conventional competitive PCR (cPCR) and most-probable-number PCR (MPN-PCR) formats. A critical evaluation of cPCR and MPN-PCR assumptions is therefore presented within the context of environmental microbiology and low-template enumerations. Fundamental conclusions from the analysis of qPCR assumptions are that: (a) environmental qPCR enumerations are invariably estimates, not absolute enumerations, which are relative to the PCR standard; (b) traditional cPCR assays are ill-suited for environmental applications, especially in low-biomass situations; and (c) both cPCR and traditional MPN-PCR practices insufficiently account for field-scale, process-level or experimental variations that arise and become amplified in PCR enumerations. Thus, sample representativeness and errors related to sample replication are frequently more important than errors related to the qPCR assay itself. Based upon this critique of qPCR assumptions, an alternate qPCR method for routine environmental application is described which is based upon replicative limiting dilution analysis and the pragmatic tradeoffs between analytical sensitivity and practical utility. Received 9 February 1998/ Accepted in revised form 26 June 1998     

14C-most-probable-number method for enumeration of active heterotrophic microorganisms in natural waters.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.     

A simple method to estimate insect mortality from field census data: A modification of the Kiritani-Nakasuji-Manly method

Various methods have been proposed to estimate demographic parameters such as mortality from field census data. Simple methods proposed earlier are applicable only for limited situations. For example, the Kiritani-Nakasuji-Manly method is applicable only if individuals are observable until their death. Improved methods proposed later are not subject to such limitations, but are not so widely used in the field of applied entomology, probably because of the complexity of the calculations involved. In this paper, I propose an intermediate method that requires only a pocket calculator, considering the practical convenience for field scientists. This method, which is a modification of the Kiritani-Nakasuji-Manly method, gives an estimate of the number of individuals entering a stage from the frequency of two stages when the stage duration is known.     

分形理论在微生物研究中的应用及展望

分形理论是以局部的某些特征来解释整体,微生物的生长形态可以用分形维数来定量描述。就微生物的菌落分形、菌丝分形的现象和机制开展讨论,并认为环境因素,如土壤、水、恶劣环境和抗生素环境均可对微生物的分形产生影响,为在不同土壤团聚体粒径中微生物的分布特点研究找到相关基础。同时介绍了计算机在微生物分形上的应用,指出研究中存在的问题和与土壤科学相结合的研究方向。     

A Model-Based Approach for Making Ecological Inference from Distance Sampling Data

Summary .  We consider a fully model-based approach for the analysis of distance sampling data. Distance sampling has been widely used to estimate abundance (or density) of animals or plants in a spatially explicit study area. There is, however, no readily available method of making statistical inference on the relationships between abundance and environmental covariates. Spatial Poisson process likelihoods can be used to simultaneously estimate detection and intensity parameters by modeling distance sampling data as a thinned spatial point process. A model-based spatial approach to distance sampling data has three main benefits: it allows complex and opportunistic transect designs to be employed, it allows estimation of abundance in small subregions, and it provides a framework to assess the effects of habitat or experimental manipulation on density. We demonstrate the model-based methodology with a small simulation study and analysis of the Dubbo weed data set. In addition, a simple ad hoc method for handling overdispersion is also proposed. The simulation study showed that the model-based approach compared favorably to conventional distance sampling methods for abundance estimation. In addition, the overdispersion correction performed adequately when the number of transects was high. Analysis of the Dubbo data set indicated a transect effect on abundance via Akaike's information criterion model selection. Further goodness-of-fit analysis, however, indicated some potential confounding of intensity with the detection function.     

<Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> as a template in the evaluation of automated kits for DNA extraction from bovine organs

Molecular diagnostic tests are widely implemented in animal and human health microbiology. Efficient nucleic acid extraction methods are essential in diagnostic laboratories and automation is a valuable tool for those with high throughput activity. Nucleic acid extraction protocols present variable efficiency depending on the composition of the specimen and the chemical-physical characteristics of the target pathogen. In the present study, we compared the DNA extraction performances of four automated methods (kits I, M, P, Q) adapted on a Hamilton Robotics “Microlab Starlet” extraction unit, and one manual method (kit R). Ten-fold dilutions of Mycobacterium avium subsp. paratuberculosis (MAP) were used to contaminate bovine central nervous system (CNS) and lung-spleen-liver pools (LSL). In consideration of its chemical-physical characteristics, MAP was selected as a template for pathogen DNA detection. Analytical performance and repeatability were assessed through downstream real-time PCR amplification, hands-on time (HOT), total turnaround time (TAT) and costs of all kits. MAP was detected differently depending on extraction kit and type of matrix analysed. Kits M and I showed the highest analytical performance on CNS (1 MAP/ml) and LSL (10 MAP/ml), respectively. Besides analytical results, kits I and M displayed high repeatability, the same HOT, very similar TAT, and were inexpensive. In conclusions, different standardized automated systems have been established with high throughput, sensitivity and repeatability for CNS and LSL. Our results also demonstrated that is necessary to assess the effectiveness of extraction kits in matrices not previously tested to avoid the risk of unreliable diagnostic outcomes.     

Detection of suppressor T lymphocytes and estimation of their frequency in limiting dilution assays by generalized linear regression modeling

The estimate of the frequency of suppressor T lymphocytes in unfractionated cell populations remains challenging, mainly because these regulatory cells do not display specific immunophenotypic markers. In this paper, we describe a novel theoretical approach for quantifying the frequency of suppressor cells. This method is based on limiting dilution data modeling, and allows the simultaneous estimation of the frequencies of both proliferating and suppressor cells. We used previously published biological data, characterizing the inhibiting activity of suppressor T cell clones. Starting from these data, we propose a mathematical model describing the interaction between suppressor and proliferating T cells, and applied to a Poisson process. Limiting dilution data corresponding to this non-single-hit, suppressor two-target Poisson model were artificially generated, then modeled according to a generalized linear regression procedure. Deviation from the single-hit Poisson model was revealed by a statistical slope test, and a stepwise analysis of the regression appeared to be an efficient method that strongly argued in favor of the presence of suppressor cells. By using the frequency of proliferating T cells calculated in the first step of the regression, we demonstrated the possibility to provide a reasonable estimate of the frequency of suppressor T cells. Based on these findings, a practical decision-making procedure is given to perform standard analyses of limiting dilution data.     

Computation of Most Probable Numbers

A rapid computational method for maximum likelihood estimation of most-probable-number values, incorporating a modified Newton-Raphson method, is presented. The method offers a much greater reliability for the most-probable-number estimate of total viable bacteria, i.e., those capable of growth in laboratory media.     

Computation of most probable numbers

A rapid computational method for maximum likelihood estimation of most-probable-number values, incorporating a modified Newton-Raphson method, is presented. The method offers a much greater reliability for the most-probable-number estimate of total viable bacteria, i.e., those capable of growth in laboratory media.     

Evaluation of four methods for enumeration of Vibrio parahaemolyticus.

Two membrane filter (MF) and two most-probable-number methods for enumerating Vibrio parahaemolyticus were compared. The MF methods used elevated-temperature incubations (41 and 42 degrees C) and were more specific than the most-probable-number methods (conducted at 35 degrees C). The MF method with a hydrophobic grid and a repair step was most effective.     

Microtechnique for Most-Probable-Number Analysis

A microtechnique based on the most-probable-number (MPN) method has been developed for the enumeration of the ammonium-oxidizing population in soil samples. An MPN table for a research design ([8 by 12] i.e., 12 dilutions, 8 replicates per dilution) is presented. A correlation of 0.68 was found between MPNs determined by the microtechnique and the standard tube technique. Higher MPNs were obtained with the microtechnique with increased accuracy in endpoint determinations being a possible cause. Considerable savings of time, space, equipment, and reagents are observed using this method. The microtechnique described may be adapted to other microbial populations using various types of media and endpoint determinations.     

微生物可培养性低的生态学释因与对策

纯培养技术一直是微生物学研究的基石,但其单一的营养结构和生境与自然环境中微生物多样性、协同代谢等明显矛盾,从而成为部分微生物难以复苏的主要障碍。细菌共同协作的自然生存方式的崩溃、生境的极度营养变化和生态位巨变等是微生物可培养性低的主要生态学原因。非培养技术、加富培养、混合培养、稀释培养、模拟自然培养和综合方法等是主要的研究手段和策略,可在不同程度上解决微生物可培养性低的缺陷和问题。     

Serodiagnosis of M. pneumoniae infections by enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) has been used for detection of antibodies against Mycoplasma pneumoniae. The results of ELISA and its sensitivity compared with other serological methods, such as complement fixation (CF), metabolic inhibition (MI), mycoplasmacidal test (MC), and radioimmunoprecipitation (RIP) are reported. ELISA and MC showed greater sensitivity than CF and MI, while RIP showed serum titer two- to 16-fold higher. ELISA was specific as determined using other human mycoplasma. A simplified method based on the determination of ELISA antibody end-point titer by a single serum dilution has been proposed. ELISA presented several advantages: sensitivity, rapidity, and low cost and, if adequately standardized, could become a reliable method for the serodiagnosis of M. pneumoniae infection.