Variableregions of the 16s ribosomal RNA have been frequently used as the target for DNA probes to identify microorganisms. In some situations, however, there is very little sequence variation observed between the 16s rRNA genes of closely related microorganisms. This study presents a general method to obtain species-specific probes using the spacer (intergenic) region between the 16s and 23s rRNA genes. The overall strategy is analogous to that which has previously been developed for the variable regions of the 16s rRNA genes. Sequence analysis of the 16s rRNA and 23s rRNA coding sequences flanking the spacer regions resulted in the design of PCR primers that can be used to amplify the spacer regions of a wide range of eubacterla.Sequencing the amplified spacer region then gives rise to the information that can be used to select specific DNA sequences for use as a DNA probe or for the generation of specific PCR primers to a microorganism of interest. In this study the approach to develop specific DNA markers for members of the genus Clostridium is described in detail. A specific DNA oligonucleotide probe and PCR primers have been designed for Clostridium perfringens that distinguish it from other organisms in the genus. Theapplication of DNA probe methodology to the identification and detection of microorganisms is becoming well established in the field of diagnostic bacteriology. Fundamental to such a technology is the ability to define suitable nucleic acid sequences that identify a particular microorganism or group of related microorganisms. DNA probes have been used successfully in the identification and detection of microorganisms./1,21 In general, nucleic acid sequences that are used as DNA probe targets for microorganisms fall into five main categories: (1) DNA sequences that code for antigens, (2) DNA sequences that code for toxins, (3) DNA sequences identified by differential hybridization using total DNA probes from related species against a DNA bank made from the microorganism of interest, (4) unique plasmid-borne DNA sequences, and (5) ribosomal RNA (rRNA) sequences.In the case of the first four categories, the means by which the DNA targets are selected involve laborious cloning methodologies and frequently a signficant amount of biochemical or immunological data. Subsequently, cross-reactivity, nonspecificity, and the possibility of the loss of the target sequences due to recombination and deletion events, especially with relation to plasmid-borne sequences, results, in some cases, in a limited usefulness of these probes, rRNAs in general have been the main targets for the generation of DNA markers for microorganisms, and we have used this region as the target for DNA probes for a number of microorganisms. (2) The major disadvantage these sequences have as candidates for DNA markers is that the "variable" regions can almost be identical when closely related microorganisms are examined, resulting in the need for finely defined stringency conditions when using such probes to detect a microorganism of...

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In a study of starch hydrolysis by strains of Streptococcus equinus, 52 isolates were obtained and their amylolytic abilities determined. It was found that all the strains could hydrolyze starch to some extent when grown in the presence of an easily fermentable carbohydrate, viz., glucose. Without this carbohydrate the organisms did not hydrolyze starch. The hydrolysis of starch was inhibited when the organisms were grown in an atmosphere of 5% CO2 and 95% N2, even if grown in the presence of a fermentable monosaccharide. S. bovis, which was used as a reference organism, readily hydrolyzed starch in the absence of monosaccharides and in atmospheres containing CO2. In no instance did S. equinus hydrolyze the starch to the level of reducing sugars. Negligible amounts of reducing sugars were recovered when the cell-free filtrates of S. equinus were incubated with starch. With S. bovis, the yield of reducing sugars under such conditions was almost quantitative. These facts extend further the differences between these related orgaiiisms. The ability to synthesize an internal starchlike polysaccharide was noted in most of the strains of S. equinus. Synthesis was found when the organisms were grown on maltose or on a starch medium containing a small amount of fermentable monosaccharide. The systematic application of the starch hydrolysis test in the classification of streptococci was first used by Andrewes (1930) to distinguish between hemolytic and nonhemolytic

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