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. 2023 Sep 15;4(3):102492.
doi: 10.1016/j.xpro.2023.102492. Epub 2023 Aug 13.

Detection of viable commensal bacteria in murine melanoma tumors by culturomics

Catherine M Phelps  1 , Jake H Shapira  2 , Colin R Laughlin  2 , Marlies Meisel  3
Affiliations

Detection of viable commensal bacteria in murine melanoma tumors by culturomics

Catherine M Phelps et al. STAR Protoc. .

Abstract

Emerging evidence suggests the tumor microbiome at gut-distal sites can modulate tumor immunity and response to cancer immunotherapy. However, detection of commensal bacteria at gut-distal tumor sites is challenging given their low abundance. Here, we present a culturomics approach to facilitate recovery of phylogenetically diverse live commensal bacteria within gut-distal melanoma tumors. We describe steps for media preparation, tissue isolation, tissue homogenization, and host cell lysis. We then detail broth expansion culture followed by agar culture and single-colony 16S rRNA sequencing. For complete details on the use and execution of this protocol, please refer to Bender and McPherson et al. (2023).1.

Keywords: Cancer; Microbiology; Molecular Biology; Sequencing.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Expansion broths and corresponding agars (A) Expansion broth cultures will be plated onto corresponding agar plates as follows: CMM broth plated on CMM agar; RCL broth plated on RCL agar; MRS broth plated on MRS agar; BHI+5% broth plated on BHI+5% agar, CHA agar, TSB agar, and BBE agar. The bacterial phyla supported by each broth and agar combination and the unique properties of each agar are indicated.
Figure 2
Figure 2
Preparation of colony selection tubes (A) Gather a 250 μL pipette tip. (B) Cut the wide end of a pipette tip so the tip fits within a closed 1.5 mL flip-cap tube. (C) Place the shortened pipette tip into a previously autoclaved 1.5 mL tube. (D) Close the tube and sterilize it by autoclaving at 121°C at 15 Psi for 20 min.
Figure 3
Figure 3
Sterile resection of subcutaneously engrafted murine melanoma tumor (A) Thoroughly wet euthanized mouse with 70% ethanol. (B) Make a single incision through the skin 1–2 cm above the tumor. (C) Pull skin back far enough to reveal tumor. Pin down skin and back legs. (D) Remove all tumors sterilely.
See this image and copyright information in PMC

References

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