Swab-based Conjunctival Commensal Bacteria Isolation: A Technique to Isolate Commensal Bacteria from Conjunctiva of Murine Model

Published: April 30, 2023

Abstract

Source: Smith-Page, K. et al. Conjunctival Commensal Isolation and Identification in Mice. J. Vis. Exp. (2021)

This video describes a swab-based technique to isolate viable commensal bacteria from the ocular conjunctiva. The technique can be used to profile the commensal bacterial microbiome of the conjunctiva, which plays a defensive role against pathogens.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Eye Swab Preparation, Work Field Set-up, Mouse Eye Swabbing and Sample Enrichment Prepare Sterile Eye Swabs NOTE: Sterile eye swabs are thinly coated wooden toothpicks with a fiber thin layer of cotton batting and made in house. Autoclave appropriate …

Representative Results

Figure 1: In-house eye swab. (A) A thin, approximately 1 cm, piece of pulled cotton batting was held between the sterile toothpick point and index finger and the toothpick was rolled while maintaining slight pressure against the index finger until the cotton was completely rolled onto the toothpick tip. (B) Example of an eye swab. <p cla…

Offenlegungen

The authors have nothing to disclose.

Materials

0.1 to 10 μl pipet tip  USA Scientific  1110-300  Autoclave before use
0.5 to 10 μl Eppendorf pipet  Fisher Scientific  13-690-026
1 ml syringe  Fisher Scientific  BD309623  1 syringe for each eye swab group
1.5 ml Eppendorf tubes  USA Scientific  1615-5500  Autoclave before use
1000 μ ml pipet tip  USA Scientific  1111-2021  Autoclave before use
200 to 1000 μl Gilson pipetman (P1000) Fisher Scientific  F123602G
25 G needle  Fisher Scientific  14-826AA  1 needle per eye swab group
3 % Hydrogen Peroxide  Fisher Scientific  S25359
37 ° C Incubator  Lab equipment
70 % Isopropanol Fisher Scientific  PX1840-4
Ana-Sed Injection (Xylazine 100 mg/ml) Santa Cruz Animal Health  SC-362949Rx
Bunsen Burner  Lab equipment
Clean paper towels  Lab equipment
Cotton Batting/Sterile rolled cotton  CVS
Ketamine (100 mg/ml)  Henry Schein 9950001
Mice  Jackson Labs C57/BL6J
Petri Dishes  Fisher Scientific  08-757-12  For Mannitol Salt agar plates
RPI Brain Heart Infusion Media  Fisher Scientific  50-488525  Prepare according to directions and autoclave
SteriFlip (0.22 μm pore size polyester sulfone) EMD/Millipore, Fisher Scientifc SCGP00525  To sterilize anesthesia
Sterile mouse cage Lab equipment
Tooth picks (round bamboo)  Kitchen Essentials  Autoclave before use and swab preparation
Trypticase Soy Agar II with 5% Sheep's Blood Plates Fisher Scientific  4321261 Store at 4 °C until ready to use
Single use eye drops  CVS Pharmacy  Bausch and Lomb Soothe Lubricant Eye Drops, 28 vials, 0.02 fl oz. each

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Swab-based Conjunctival Commensal Bacteria Isolation: A Technique to Isolate Commensal Bacteria from Conjunctiva of Murine Model. J. Vis. Exp. (Pending Publication), e20745, doi: (2023).

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