Fluorescence in situ hybridisation in Carnoy's fixed tonsil tissue

Area of Science:

• Microbiology
• Molecular Biology
• Pathology

Background:

• Few studies have established Fluorescence in situ hybridisation (FISH) protocols for tonsil tissue fixed in Carnoy's fixative.
• This limitation hinders the investigation into the pathogenesis of tonsillar hyperplasia.

Purpose of the Study:

• To establish and validate a robust FISH protocol for identifying multiple bacterial species in Carnoy's fixed tonsil tissue.
• To investigate the prevalence and organization of specific bacterial taxa in pediatric tonsil tissue.

Main Methods:

• Optimized specificity of five FISH probes (Fusobacterium spp., Bacteroides spp., Streptococcus spp., Haemophilus influenzae, and Pseudomonas spp.) using pure and mixed bacterial isolates.
• Applied the optimized FISH protocol to Carnoy's fixed tonsil tissue samples from 24 children.

Main Results:

• Successfully visualized and identified multiple aerobic and anaerobic bacteria in Carnoy's fixed tonsil tissue.
• Bacteroides spp. were detected in 100% of patients with microcolonies, followed by Fusobacterium spp. (93.8%), Streptococcus spp. (85.7%), H. influenzae (82.35%), and Pseudomonas spp. (76.5%).
• Observed variations in the organization of bacterial taxa within microcolonies.

Conclusions:

• This study presents the first robust FISH protocol for identifying diverse bacteria in Carnoy's fixed tonsil tissue.
• The protocol facilitates combined histological and microbiological analyses of tonsil samples.
• This method has potential implications for analyzing microorganisms in other human tissues prepared similarly.