Conjunctival Commensal Isolation and Identification in Mice

Summary

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

Abstract

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an “in-lab” made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Introduction

The aim of this protocol is to enhance specific isolation of viable and rare aerobic and facultative anaerobic microbes from the ocular conjunctiva to characterize the ocular microbiome. Extensive studies have profiled commensal mucosal communities on the skin, gut, respiratory and genital tracts and show that these communities influence the development of the immune system and response^1,2,3. Ocular commensal communities have been shown to change during certain disease pathologies, such as Dry eye disease⁴, Sjogren’s syndrome⁵

Protocol

All procedures involving mice follow the Institutional Animal Care and Use Committee guidelines. Follow laboratory safety guidelines (as directed by your Institutional Environmental Health and Safety department) when working with microorganisms and potentially contaminated materials. Use appropriate waste receptacles and decontamination procedures prior to disposal of potentially biohazard contaminated materials.

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 ....

Results

Representative results for an eye swab plate demonstrating different methods for plating are pictured in Figure 3A showing morphologically diverse isolates from C57BL/6 mouse. For each distinct isolate, the colonies were counted in the strip and the relative abundance, unique Colony Forming Units (CFUs) per eye swab, calculated and plotted for comparison purposes. For microbiological characterization, bacteria were picked from individual mouse eye swab plates to produce a master TSA plate (c.......

Discussion

Due to the paucibacterial state of the ocular surface, many laboratories have had difficulty isolating ocular commensals^7,20, resulting in low number of samples with growth, low abundance and low diversity⁸. This method significantly improves upon standard culture practices^4,21 by the addition of an enrichment step, as well as a redesigned eye swab and identification by MALDI-TOF M.......

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 1000µl 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
BD BBL Gram Stain kit	Fisher Scientific	B12539
Bunsen Burner	Lab equipment
Clean paper towels	Lab equipment
Cotton Batting/Sterile rolled cotton	CVS
Disposable 1 ml Pipets	Fisher Scientific	13-711-9AM	for Gram stain and catalase tests
E.coli	ATTC	ATCC 8739
Glass slides	Fisher Scientific	12-550-A3	for Gram stain and catalase tests
Ketamine (100mg/ml)	Henry Schein	9950001
Mac Conkey Agar Plates	Fisher Scientific	4321270	store at 4 °C until ready to use
Mannitol Salt Agar	Carolina Biological Supply	784641	Prepare plates according to mfr's instructions, store at 4 °C for 1 week
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 Corning Centrifuge Tube	Fisher Scientific	430829	anesthesia preparation
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
Vitek target slide	BioMerieux Inc. Durham,NC
Vitek-MS	BioMerieux Inc. Durham,NC
Vitek-MS CHCA matrix solution	BioMerieux Inc. Durham, NC	411071
Single use eye drops	CVS Pharmacy		Bausch and Lomb Soothe Lubricant Eye Drops, 28 vials, 0.02 fl oz. each