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

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…

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