Principles of Establishing and Operating a Gnotobiotic Facility

Schoeb, Trenton R.; Rahija, Richard J.; Boyd, Cassie; Orcutt, Roger P.; Eaton, Kathryn A.

doi:10.1016/b978-0-12-804561-9.00002-5

Cited by 3 publications

(9 citation statements)

References 78 publications

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“…Exception is that in cage level isolator, each of them must have its own microbiological status confirmed. In general, a mix between culture, molecular techniques and microscopy approaches is applied to define the axenic status of animals (Schoeb et al, 2017). For culture methods, feces, bedding, and food are the most frequently used samples.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…For microscopy in axenic context, wet mounts are sometime selected, but Gram strains remain the most used method for the determination of axenic status (Schoeb et al, 2017). Observations of feces and/or cecal contents of animals are performed at 1000× magnification (Fontaine et al, 2015;Schoeb et al, 2017). Readings are gathered with immersion oil by diluting the sample in sterile water or saline (Schoeb et al, 2017) or by direct observation of a swabbed feces (Fontaine et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…Observations of feces and/or cecal contents of animals are performed at 1000× magnification (Fontaine et al, 2015;Schoeb et al, 2017). Readings are gathered with immersion oil by diluting the sample in sterile water or saline (Schoeb et al, 2017) or by direct observation of a swabbed feces (Fontaine et al, 2015). However, it has been demonstrated in the literature that bacteria present in food before autoclaving or after irradiation can be detected by microscopy (Taylor et al, 1986;Midtvedt and Gustafsson, 2005;Fontaine et al, 2015;Nicklas et al, 2015;.…”

Section: Introductionmentioning

confidence: 99%

“…Consequently, this complicates the analysis when the objective is to demonstrate that an animal is free from microorganisms. It is often advisable to interpret microscopy results in parallel with culture and molecular method results (Schoeb et al, 2017). It is also recommended to use the expertise of an experienced microscopist since mastering microscopy techniques requires some practice (Packey et al, 2013;Schoeb et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Contaminants and Where to Find Them: Microbiological Quality Control in Axenic Animal Facilities

et al. 2021

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The use of axenic animal models in experimental research has exponentially grown in the past few years and the most reliable way for confirming their axenic status remains unclear. It is especially the case when using individual ventilated positive-pressure cages such as the Isocage. This type of cage are at a greater risk of contamination and expose animals to a longer handling process leading to more potential stress when opened compared to isolators. The aim of this study was to propose simple ways to detect microbial contaminants with Isocages type isolator resulting by developing, validating and optimizing three different methods (culture, microscopy, and molecular). These three approaches were also tested in situ by spiking 21 axenic mice with different microorganisms. Our results suggest that the culture method can be used for feces and surface station (IBS) swabs exclusively (in Brain Heart Infusion for 7 days at 25°C and 37°C in aerobic conditions, and at 30°C in anaerobic conditions), while microscopy (wet mounts) and molecular method (quantitative PCR) were only suitable for fecal matter analyses. In situ results suggests that the culture and molecular methods can detect up to 100% of bacterial contamination events while the microscopy approach generates many erroneous results when not performed by a skilled microscopist. In situ results also suggest that when an axenic mouse is contaminated by a microbial agent, the microorganism will colonize the mouse to such an extent that detection is obvious in 4 days, in average. This report validates simple but complimentary tests that can be used for optimal detection of contaminants in axenic animal facilities using Isocage type isolators.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Contaminants and Where to Find Them: Microbiological Quality Control in Axenic Animal Facilities

et al. 2021

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Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies

Kennedy

Goffau

Pérez-Muñoz

et al. 2023

Nature

132

112

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Whether the human fetus and the prenatal intrauterine environment (amniotic fluid, placenta) are stably colonized by microbes in a healthy pregnancy remains the subject of debate. Here, we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbiology, bioinformatics, immunology, clinical microbiology, and gnotobiology, and assess possible mechanisms by which the fetus might interact with microbes. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples, DNA extraction, and DNA sequencing. Further, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology, and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and also illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a "fetal microbiome" serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls, also incorporating biological, ecological, and mechanistic concepts.

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Managing the bacterial contamination risk in an axenic mice animal facility

et al. 2021

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A gap exists between good laboratory practises with axenic animals and procedures applied. This work aimed at choosing the appropriate disinfectant between sodium dichloroisocyanurate (MB-10) and potassium peroxymonosulfate (Virkon™) disinfectants and to adjust the soaking time of the material used with ISOcage biosafety stations. Another aim was to compare the microbial load on cage systems hosting mice since two weeks in axenic (AR) rooms and typical specific-pathogen-free (SPF) non-axenic rooms (NAR) to identify resistant microorganisms targeted for longer soaking disinfection as well as evaluate microbial concentration reduction procedures in AR. Staphylococcus was the most frequently isolated genus (AR and NAR). An average of three spore-forming microorganisms per cage were counted from AR. The disinfection time to reach 1 log reduction for Bacillus atrophaeus spores varied from 138 (100 ppm MB-10) to 290 (Virkon™) seconds and below 20 seconds for S. epidermidis (100 ppm MB-10). AR management protocols lead to 1000 times lower microorganisms burden compared to NAR. Data comparing microbial load on SPF and axenic facilities can be used as comparison for facilities aiming at improving the effectiveness of their microbial control procedures.

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Principles of Establishing and Operating a Gnotobiotic Facility

Cited by 3 publications

References 78 publications

Contaminants and Where to Find Them: Microbiological Quality Control in Axenic Animal Facilities

Contaminants and Where to Find Them: Microbiological Quality Control in Axenic Animal Facilities

Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies

Managing the bacterial contamination risk in an axenic mice animal facility

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