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).