To be used as a model in dental research, an animal must fulfil experimental needs and information on the composition and variation of its oral flora must be available. Only limited data are available on the indigenous oral bacterial flora of BALB/c mice. In this work, a total of 671 isolates from different sites (saliva, tongue, teeth, and mucosa) of the oral cavity of BALB/c mice were identified. Only 18 different species were isolated, which indicates the relative simplicity of the flora. The predominant species of the total cultivable flora were "Lactobacillus murinus" (38%), Staphylococcus aureus (37%), Streptococcus faecalis (8%), Staphylococcus sciuri (4%), and Escherichia coli (3%). The other species each represent less than 2% of the flora. "Lactobacillus murinus" is found in greater proportion on mucosa than in the other sites, Staph. aureus predominates in saliva, and Strep. faecalis was found in greater proportion in tooth samples. Statistical analyses, using the minimum percentage of similarity, indicate that there is some variation among the microflora of different mice but that this difference is smaller for mice from the same lot. These results set the basis for the study of the variations of the indigenous oral microflora of BALB/c mice under different conditions.
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization. A bacterial culture is challenged with lytic phages, the surviving cells are screened by PCR for expansion of their CRISPR array and the newly acquired specificities are mapped to the genome of the phage. Furthermore, we offer three variants of the assay to (i) promote adaptation by challenging the system using defective viruses, (ii) challenge the system using plasmids to generate plasmid-resistant strains and (iii) bias the system to obtain natural immunity against a specifically targeted DNA sequence. The core protocol and its variants serve as a means to explore CRISPR adaptation, discover new CRISPR-Cas systems and generate bacterial strains that are resistant to phages or refractory to undesired genes or plasmids. In addition, the core protocol has served in teaching laboratories at the undergraduate level, demonstrating both its robust nature and educational value. Carrying out the core protocol takes 4 h of hands-on time over 7 d. Unlike sequence-based methods for detecting natural CRISPR adaptation, this phage-challenge-based approach results in the isolation of CRISPR-immune bacteria for downstream characterization and use.
A model of oral candidosis was developed in order to investigate histologic and microbiologic aspects of this host-parasite interaction under controlled experimental conditions. Normal adult CD-1 mice were inoculated by the topical application of 10(8) Candida albicans blastospores, and oral colonization was monitored by the quantitative culturing of saliva samples and of digested oral mucosa. Tissue sections of the mucosa were examined in a kinetic study ranging from 2 h to 13 days postinoculation. We report here that oral colonization by C. albicans can be induced in normal adult mice without the use of any compromising agent and that the animals recover from this mucosal infection following a reproducible pattern. Temporal analysis of the oral histopathology showed that distinct patterns of inflammation are associated with particular stages in the development of the infectious foci. This experimental model offers a means of further investigating the host-parasite interactions involved in the onset and development of oral candidosis.
Recent biological terrorism events have indicated that bacterial spores such as Bacillus anthracis are real threat agents. Real time detection of biological agents is possible with the use of an ultraviolet Fluorescent Aerodynamic Particle Sizer (FLAPS) that measures particles' intrinsic fluorescence. It is important to know whether intrinsic fluorescence could be used to estimate agents' viability. Two categories of Bacillus spore populations can be differentiated by the intensity of intrinsic fluorescence emitted by ultraviolet (UV) stimulation : autofluorescent and non-autofluorescent. This study was performed to determine whether intensity of autofluorescence correlates with spore viability. Spores were analyzed using flow cytometer (equipped with a cell sorter) to mimic optical properties of FLAPS. Autofluorescent and non-autofluorescent spores were sorted according to the intensity of autofluorescence emitted following UV stimulation. Culturability, membrane integrity, membrane potential and dipicolinic acid (DPA) content were assessed. Autofluorescent spores were 1.7 times more culturable than the corresponding non-autofluorescent population. Moreover, a small proportion of autofluorescent spores exhibited extracellular membrane damages. Autofluorescent spores also showed higher membrane potential activity and contained higher levels of DPA. In conclusion, this study documents that the overall viability potential of bacterial spores can be assessed by UV flow cytometry used in the FLAPS technology.
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