Isolation of Antibiotic-Resistant Bacteria from a Water Sample for Molecular Analysis

Samenvatting

Source: Ghadigaonkar, D. and Rath, A. Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes. J. Vis. Exp. (2023)

This video demonstrates the isolation and preparation of antibiotic-resistant bacteria from a water sample using selective media. The DNA is extracted from antibiotic-resistant bacteria and amplified to identify the bacterial antibiotic-resistant genes.

Protocol

1. Sample collection and processing

1. Sample collection
   1. Collect the appropriate volume of the water sample in sterile sample containers, ensuring that not more than 3/4 of the container is filled.
   2. Transport the samples to the laboratory under aseptic conditions as soon as possible after collection and immediately process them.
2. Sample processing
   1. Aseptically filter the water sample through a sterile muslin cloth to remove any particulate matter.
   2. Carry out appropriate serial dilutions of the filtered water for further analysis.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
2x PCR Taq mastermix	HiMedia	MBT061-50R	For making PCR reaction mixture
37 °C Incubator	GS-192, Gayatri Scientific	NA	For incubation of bacteria
Autoclave	Equitron	NA	Required for sterilization of media, glass plates, test tubes, etc
Bisafety B2 Cabinet	IMSET	IMSET BSC-Class II Type B2	Used for microbiological work like bacterial culturing, AST etc.
Cefotaxime antibiotic powder	HiMedia	TC352-5G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Centrifuge minispin	Eppendorf	Minispin Plus-5453	Used to pellet the debris during crude DNA preparation
Ciprofloxacin antibiotic powder	HiMedia	TC447-5G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Colorimeter	Quest	NA	For checking the OD of culture suspensions
Cooling shaker incubator	BTL41 Allied Scientific	NA	For incubation of media plates for culturing bacteria
Erythromycin antibiotic powder	HiMedia	CMS528-1G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Erythromycin antibiotic powder	HiMedia	TC024-5G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Kanamycin antibiotic powder	HiMedia	MB105-5G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Luria Bertani broth	Himedia	M1245-500G	For enrichment of cultures
R2A Agar, modified	HiMedia	M1743	For preparation of media plates for isolation of total and antibiotic resistant (AR) bacterial load
Sodium chloride	HiMedia	TC046-500G	For preparation of 0.85% saline for serially diluting the water sample
Staphylococcus aureus subsp. aureus ATCC 29213	HiMedia	0365P	Used as a control while performing AST
Vancomycin antibiotic powder	HiMedia	CMS217	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Weighing balance	Mettler Toledo	ME204 Mettler Toledo	Used for weighing media powders, reagent powders etc.