VIABILITY ASSESSMENT OF BACTERIA STORED IN BHI-GLYCEROL BROTH AT -20ºC FOUR YEARS AGO

Abstract

With the biotechnological and scientific advancements, the need for preserving microorganisms in laboratories for diagnostic, research, or educational purposes arises. Thus, this study aims to analyze the viability of bacteria stored for four years using the cryopreservation technique. The viability of 70 bacterial isolates was evaluated, including 17 species of Gram-positive bacteria and 53 species of Gram-negative bacteria stored in cryogenic tubes containing Brain Heart Infusion (BHI) broth and glycerol, maintained in a -20ºC freezer since 2019. To reactivate the isolates, bacterial suspensions were thawed and plated on Mueller Hinton Agar plates, while the remaining material was added to tubes containing BHI broth and incubated in a bacteriological incubator at 35ºC for 24 hours. The results demonstrated that 84.3% (59) of the samples showed satisfactory growth without contamination, with Gram-negative bacteria achieving a higher viability rate (84.9% - 45) compared to Gram-positive bacteria (82.3% - 14). Therefore, this study demonstrates that freezing bacteria in BHI-glycerol broth at -20ºC is an effective technique for the long-term conservation of Gram-negative bacteria.