Isolation and Cultivation of Bacteria from Zebrafish Embryos to Assess Infection Load

Abstract

Source: Bernut, A., et al., Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos. J. Vis. Exp. (2015).

This video demonstrates a protocol for isolating and quantifying Mycobacterium abscessus from an infected zebrafish embryo. The procedure involves embryo euthanasia, tissue lysis, and homogenization to release the bacteria, followed by centrifugation and resuspension in a surfactant buffer. Finally, the bacterial suspension is serially diluted, plated on antibiotic-supplemented agar, and incubated to enable colony formation and quantification.

Protocol

1. Preparation and Storage of the M. abscessus Inoculum
   1. M. abscessus growth conditions
      1. Plate out M. abscessus from a -80°C glycerol stock onto a Middlebrook 7H10 agar containing 10% of OADC and 0.5% glycerol (7H10ᴼᴬᴰᶜ) and supplemented with the appropriate antibiotic depending on the resistance marker carried by the vector encoding the fluorescent protein. Incubate the plates at 30 °C for 4-5 days.
      2. Pick up a fluorescence-positive mycobacterial colony and inoculate 1 ml Middlebrook 7H9 broth suppl....

Results

Figure 1. Preparation of dispersed M. abscessus inocula. Ziehl-Neelsen staining of R or S variants grown on broth medium prior to any treatment (upper panels) or after treatment (lower panels) to reduce the size .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BBL MGIT PANTA	BD Biosciences	245114
Bovine Serum Albumin	Euromedex	04-100-811-E
Catalase from Bovine Liver	Sigma-Aldrich	C40
Difco Middlebrook 7H10 Agar	BD Biosciences	262710
Difco Middlebrook 7H9 Broth	BD Biosciences	271310
Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)	Sigma-Aldrich	A5040
Oleic Acid	Sigma-Aldrich	O1008
Paraformaldehyde	Delta Microscopie	15710
Phenol Red	Sigma-Aldrich	319244
Tween 80	Sigma-Aldrich	P4780
Agar	Gibco Life Technologie	30391-023
Low melting agarose	Sigma-Aldrich
Instant Ocean Sea Salts	Aquarium Systems Inc
Borosilicate glass capillaries	Sutter instrument Inc	BF100-78-10	1 mm O.D. X 0.78 mm I.D.
Micropipette puller device	Sutter Instrument Inc		Flamming/Brown Micropipette Puller p-87
Microinjector	Tritech Research		Digital microINJECTOR, MINJ-D
Tweezers	Sciences Tools inc		Dumont # M5S
Microloader Tips	Eppendorf