CLASSIFY: A group teaching exercise in microbial identification and numerical taxonomy using a Commodore 64 microcomputer

An exercise designed to teach the basic principles of numerical taxonomy is described. The exercise utilizes a computer program, CLASSIFY, written in BASIC for the Commodore 64 microcomputer, which will accept student data obtained from the identification of bacteria, calculate the similarity coefficients, and perform Single Linkage Cluster Analysis. Bacterial identification can be achieved using live cultures and rapid identification kits, or, alternatively, via the bacterial culture simulation program, BUG SIMULATOR, which would be useful for data generation by students inexperienced in handling microbes (e.g. secondary school students).     

A rapid, semi-automated SDS-PAGE identification system for oral anaerobic bacteria

SDS-polyacrylamide gel electrophoresis is a useful technique in bacterial differentiation and identification. A rapid, semi-automated SDS-PAGE system (Phast System) was assessed for identification of formate-fumarate-requiring, asaccharolytic, Gram-negative oral anaerobes. The system permitted loading, separation and staining of gels within 2 h. Percentage similarities between strains were determined using correlation coefficients and cluster analysis. The protein profiles were sufficiently reproducible provided that distorted profiles were disregarded. Strains were successfully separated into their species, with the exception of Bacteroides ureolyticus NCTC 10939, which appeared to be distinct from other strains of that species. Twenty-nine unidentified formate-fumarate-requiring, sub-gingival plaque strains were suitably clustered with the standard strains as verified by a series of physiological and biochemical tests.     

Whole-organism fingerprinting of the genus Carnobacterium using Fourier transform infrared spectroscopy (FT-IR)

Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.     

A rapid, semi-automated SDS-PAGE identification system for oral anaerobic bacteria

SDS-polyacrylamide gel electrophoresis is a useful technique in bacterial differentiation and identification. A rapid, semi-automated SDS-PAGE system (Phast System) was assessed for identification of formate-fumarate-requiring, asaccharolytic, Gram-negative oral anaerobes. The system permitted loading, separation and staining of gels within 2 h. Percentage similarities between strains were determined using correlation coefficients and cluster analysis. The protein profiles were sufficiently reproducible provided that distorted profiles were disregarded. Strains were successfully separated into their species, with the exception of Bacteroides ureolyticus NCTC 10939, which appeared to be distinct from other strains of that species. Twenty-nine unidentified formate-fumarate-requiring, sub-gingival plaque strains were suitably clustered with the standard strains as verified by a series of physiological and biochemical tests. and accepted 25 July 1989     

动物微生态制剂猪肠源乳球菌的分离与鉴定

试验的菌种是从宁夏平罗县边远农村基本自然生长的健康肉猪的小肠、大肠和盲肠中分离获得的,共分离出64株菌株。通过对这64株菌株的菌落形态观察和革兰氏染色镜检。筛选出9株进行了乳球菌属的生理生化鉴定,初步确定这9株属于乳球菌属(Lactococcus)。再通过糖醇类发酵产酸鉴定,确定2株为乳酸乳球菌乳酸亚种(L.lactissubsplactis),3株为植物乳球菌(L.plantarum),4株为棉籽糖乳球菌(L.raffi-nolactis)。各项鉴定结果均符合乳球菌属和相应种鉴定标准。     

游动分割窗技术在生态交错带定量判定中的应用:以四川巴郎山岷江冷杉林线为例

以样带上样方间的距离系数为指标 ,采用游动分割窗技术辨析了岷江冷杉 (Abiesfaxoniana)林线附近交错带的位置和宽度。结果表明 ,Bray_Curtis距离、相对欧氏距离、弦距离与平方欧氏距离的峰值和峰宽具有很好的重合性 ,上述距离系数均能作为判定林线交错带群落的边界和宽度的优良指标 ,其中 ,平方欧氏距离更能直观和准确地反映交错带植被的变异。样带上距离系数的峰值区和峰宽对生态交错带的位置和宽度有较为敏感的指示意义 ,游动分割窗技术是交错带判定和群落划分的有效方法。     

游动分割窗技术在生态交错带定量判定中的应用*-以四川巴郎山岷江冷杉林线为例

 以样带上样方间的距离系数为指标,采用游动分割窗技术辨析了岷江冷杉(Abies faxoniana)林线附近交错带的位置和宽度。结果表明,Bray-Curtis距离、相对欧氏距离、弦距离与平方欧氏距离的峰值和峰宽具有很好的重合性,上述距离系数均能作为判定林线交错带群落的边界和宽度的优良指标,其中,平方欧氏距离更能直观和准确地反映交错带植被的变异。样带上距离系数的峰值区和峰宽对生态交错带的位置和宽度有较为敏感的指示意义,游动分割窗技术是交错带判定和群落划分的有效方法。以样带上样方间的距离系数为指标,采用游动分割窗技术辨析了岷江冷杉(Abies faxoniana)林线附近交错带的位置和宽度。结果表明,Bray-Curtis距离、相对欧氏距离、弦距离与平方欧氏距离的峰值和峰宽具有很好的重合性,上述距离系数均能作为判定林线交错带群落的边界和宽度的优良指标,其中,平方欧氏距离更能直观和准确地反映交错带植被的变异。样带上距离系数的峰值区和峰宽对生态交错带的位置和宽度有较为敏感的指示意义,游动分割窗技术是交错带判定和群落划分的有效方法。     

Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry

A protocol has been developed that allows protein identifications using available DNA-based or protein sequences from a reference strain of a bacterial species to be extended to bacterial strains for which no prior DNA-based or protein sequence information exists. The protocol is predicated on careful isolation of a specific sub-cellular group of proteins. In this study, ribosomal proteins were chosen due to their high relative abundance and similarity in copy number per cell. After isolation of ribosomal proteins, MALDI-MS is used to acquire accurate protein molecular weights. An iterative comparison of reference protein molecular weights and identities is made to the resulting data, allowing for the straightforward identification of ribosomal proteins from any non-reference strains. This approach can reveal differences between proteins at the amino acid or post-translational level. The protocol was developed, validated and applied to ribosomal proteins from three strains of the extreme thermophile Thermus thermophilus. This approach revealed that nearly 60% of the ribosomal proteins from all three strains are identical. The extension of protein identification to additional bacterial strains can be useful in phylogenetic studies as well as in biomarker identification.     

群体遗传学研究中的数据处理方法I．RAPD数据的AMOVA分析

近年来,RAPD数据和AMOVA分析广泛地应用于群体遗传学和保护遗传学研究。然而,由于RAPD标记具显性特点。加上目前进行AMOVA分析所依赖的RAPDistance软件不完善,使得对RAPD数据进行AMOVA分析时存在许多不足。本文介绍了AMOVA分析的基本过程,同时引入一个新的程序DCFA用以替代RADistance并详述了将DCFA与WINAMOVA联用,对RAPD数据进行AMOVA分析的具体步骤与注意事项,最后,以产自中国和巴西8个普通野生稻（Oryza furipogon)天然群体为例,演示了对RAPD表型数据进行AMOVA分析的过程,讨论了AMOVA分析结果在群体遗传结构上的意义。通过对AMOVA算法的分析,同时比较4种距离系数所得AMOVA结果,我们认为在进行AMOVA分析时选择NEI－LI距离和欧氏距离平方较为合适,而目前国内使用较多的JACCARD系数不适合AMOVA分析。     

Evaluation of the Biolog Substrate Utilization System To Identify and Assess Metabolic Variation among Strains of Xanthomonas campestris pv. Citri

Metabolic fingerprints of 148 strains of Xanthomonas campestris pv. citri originating from 24 countries and associated with various forms of citrus bacterial canker disease (CBCD) were obtained by using the Biolog substrate utilization system. Metabolic profiles were used to attempt strain identification. Only 6.8% of the studied strains were correctly identified when the commercial Microlog 2N data base was used alone. When the data base was supplemented with data from 54 strains of X. campestris pv. citri (40 CBCD-A strains, 8 CBCD-B strains, and 6 CBCD-C strains) and data from 43 strains of X. campestris associated with citrus bacterial spot disease, the percentage of correct identifications was 70%. Thus, it is recommended that users supplement the commercial data base with additional data prior to using the program for identification purposes. The utilization of Tween 40 in conjunction with other tests can help to differentiate strains associated with CBCD and citrus bacterial spot disease. These results confirmed the separation of X. campestris pv. citri into different subgroups (strains associated with Asiatic citrus canker [CBCD-A], cancrosis B [CBCD-B], and Mexican lime canker [CBCD-C]). The utilization of l-fucose, d-galactose, and alaninamide can be used as markers to differentiate strains associated with these groups. A single strain associated with bacteriosis of Mexican lime in Mexico (CBCD-D) was closely similar to CBCD-B strains.     

The occurrence of peptaibols and structurally related peptaibiotics in fungi and their mass spectrometric identification via diagnostic fragment ions.

Peptaibols and related peptide antibiotics (peptaibiotics) display diagnostically useful fragmentation patterns during mass spectrometry (FAB-MS, ESI-CID-MS/MS and CID-MSn]. The paper compiles fragmentation data of pseudo-molecular ions reported in the literature as a guide to the rational identification of recurrently isolated and new peptaibols and peptaibiotics. Taxonomic and ecological aspects of microorganisms producing peptaibols and peptaibiotics are discussed.     

Importance of immunoelectrophoresis in agar and disc electrophoresis in polyacrylamide gel for characterizing different strains of Bordetella pertussis

The antigenic composition of typical and atypical B. pertussis strains obtained in the foci of pertussis infection, as well as experimentally obtained antibiotic-resistant B. pertussis strains, has been studied by the methods of immunoelectrophoresis in agar and electrophoresis in polyacrylamide gel (PAAG). Immunoelectrophoresis in agar has been found capable of differentiating B. pertussis culture from a group of unidentified morphologically similar Gram-negative bacilli by their antigenic composition and thus suitable for use as an additional criterion in the identification of atypical B. pertussis strains. PAAG electrophoresis has permitted finding differences in the set of protein antigens in the control strain and in its clones obtained by multiple subculturing in media with antibiotics added.     

Characterization of Paracoccidioides brasiliensis atypical isolates by random amplified polymorphic DNA analysis

Two atypical Paracoccidioides brasiliensis strains (yeast form at room temperature) have been isolated from chronically infected patients living in Brazil. Different random primers were used to characterize these isolates and compare them to typical strains. The RAPD patterns allowed the differentiation of all the selected isolates. Their genetic distance ranged from 5% to 80% of non-shared bands depending on the strains and the primer used. The RAPD data were used to build a Wagner phenogram, which showed two major branched with more than 56% of genetic distance separating them. No significant difference was observed between the atypical isolates and the others suggesting that specific genes are involved in the dimorphism phenomenon.     

Genomic variations in mosquitocidal strains of Bacillus sphaericus detected by M13 DNA fingerprinting.

The genomic variation of Bacillus sphaericus reference and local strains belonging to different serotypes was examined by DNA fingerprinting. A phage M13 DNA probe detected a number of variable fragments in the restriction digests of total strain DNAs. The patterns of band distribution showed a certain homology among mosquitocidal strains, expressed by similarity index D and might be a reliable criterion for assessing the level of genomic similarity between closely related strains. An important advantage of DNA fingerprinting is the differentiation of one bacterial strain from another, both expressing common phenotype and possessing highly similar genomic portions. The strain variation revealed by the M13 probe will be useful for characterization of individual strains within a serotype. It could help as well to solve some uncertain cases based on the results obtained by other methods of identification.     

Comparison of API 20NE and Biolog GN identification systems assessed by techniques of multivariate analyses.

The increasing use of commercial multitest systems for identification of environmental bacteria creates the problem of how to compare the identification results obtained from different systems. The limited use of species designations in such comparisons is caused by low usage of environmental bacteria in the development of commercial identification schemes. Two multivariate statistical methods, the Mantel's test and the co-inertia analysis, were applied to analyze data derived from the Biolog GN and the API 20NE systems of identification for 50 environmental bacterial strains. We found these two methods to be useful for revealing the relationship between the two sets of numerical taxonomic traits. Both of these methods showed that the distances according to the Biolog GN results between the studied strains were related to those derived from the API 20NE results, despite the differences in the test sets of the two systems. In addition, the co-inertia analysis allowed us to visualise the relationships between classifications of strains derived from the two identification systems and, simultaneously, to estimate the contribution of particular tests to the differentiation of bacterial strains.     

EZ-FIT: a practical curve-fitting microcomputer program for the analysis of enzyme kinetic data on IBM-PC compatible computers

EZ-FIT, an interactive microcomputer software package, has been developed for the analysis of enzyme kinetic and equilibrium binding data. EZ-FIT was designed as a user-friendly menu-driven package that has the facility for data entry, editing, and filing. Data input permits the conversion of cpm, dpm, or optical density to molar per minute per milligram protein. Data can be fit to any of 14 model equations including Michaelis-Menten, Hill, isoenzyme, inhibition, dual substrate, agonist, antagonist, and modified integrated Michaelis-Menten. The program uses the Nelder-Mead simplex and Marquardt nonlinear regression algorithms sequentially. A report of the results includes the parameter estimates with standard errors, a Student t test to determine the accuracy of the parameter values, a Runs statistic test of the residuals, identification of outlying data, an Akaike information criterion test for goodness-of-fit, and, when the experimental variance is included, a chi 2 statistic test for goodness-of-fit. Several different graphs can be displayed: an X-Y, a Scatchard, an Eadie-Hofstee, a Lineweaver-Burk, a semilogarithmic, and a residual plot. A data analysis report and graphs are designed to evaluate the goodness-of-fit of the data to a particular model.     

A numerical taxonomic study of anaerobic gram-negative bacilli classified as Bacteroides ureolyticus isolated from patients with non-gonococcal urethritis

A numerical taxonomic study of 64 strains of anaerobic Gram-negative bacilli isolated from men with non-gonococcal urethritis, two unclassified laboratory strains of 'corroding bacilli', and 12 other strains of anaerobic Gram-negative bacilli, including nine received as anaerobic curved rods and three as 'Bacteroides corrodens' (B. ureolyticus), isolated from women with bacterial vaginosis, was undertaken. Seventeen reference anaerobic strains belonging to the genera Bacteroides, Fusobacterium, Mobiluncus, Mitsuokella and Wolinella were included. Morphological, biochemical and physiological characteristics were examined in 103 tests. The resemblance between the 95 strains was calculated using the SSM, SJ and DP coefficients for cluster analyses based on the UPGMA method. All three approaches gave similar groupings, and the estimated average probability of test error was 2.46%. The strains fell into 10 phenons. The unclassified strains from men and three from women with lower genital-tract infections, and the laboratory strains of 'corroding bacilli' clustered in one phenon with the reference strains of B. ureolyticus, indicating that they correspond to B. ureolyticus. The other unclassified strains of anaerobic curved rods clustered as a distinct phenon. They correspond to species of the newly described genus Mobiluncus. The taxonomic data and the compilation of diagnostic tables serve as a useful guide for the laboratory identification of clinical isolates regarded as B. ureolyticus.     

Bacterial Species Determination from DNA-DNA Hybridization by Using Genome Fragments and DNA Microarrays

Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.     

Bacterial species determination from DNA-DNA hybridization by using genome fragments and DNA microarrays

Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.