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

1. 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.
   1. Autoclave appropriate amount of cotton batting and toothpicks for number of mice to be swabbed.
   2. Pinch off a half a centimeter-long piece of cotton batting with t....

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 i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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