Cryopreservation of Mycobacterium bovis isolates

Detalhes bibliográficos
Autor(a) principal: Ikuta, Cássia Yumi
Data de Publicação: 2016
Outros Autores: Ambrosio, Simone Rodrigues, Souza Filho, Antonio Francisco de, Grisi-Filho, José Henrique de Hildebrand, Heinemann, Marcos Bryan, Ferreira Neto, José Soares, Amaku, Marcos
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Semina. Ciências Agrárias (Online)
Texto Completo: https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/27313
Resumo: Research, development of new biotechnological methods, diagnostic tests, confirmation of results, and reinvestigations are possible because of the availability of well-preserved living organisms maintained without any changes. Cryopreservation is a simpler, more reliable and long-term stable method for culture maintenance. Storage temperature and composition of the suspending vehicle are factors that affect the viability of mycobacterial strains. Three vehicles and three storage temperatures were evaluated to define a suitable cryoprotective medium for the preservation of Mycobacterium bovis strains. Colonies of sixteen M. bovis isolates were used to prepare the suspensions, which were then added to three vehicles: sterile 0.85% saline solution (SS), Middlebrook 7H9 broth (7H9), and Middlebrook 7H9 broth with sodium pyruvate (7H9p) replacing glycerol. Aliquots of these suspensions were frozen by three different methods, directly in the -20°C freezer, directly in the -80°C freezer, and at -196°C by immersion in liquid nitrogen (LN). The frozen aliquots were thawed at room temperature after 45, 90 and 120 days. Mycobacterial viability was assessed by counting the living cells on plates of Stonebrink medium before and after the freezing procedure. Storage at -20°C exhibited a lower recovery of M. bovis compared to storage at -80°C (Dunn’s test, p=0.0018) and LN (Dunn’s test, p=0.0352). There was no statistically significant difference between storage at -80°C and in LN (Dunn’s test, p=0.1403), yet -80°C showed better results than LN. All three suspending vehicles showed no statistically significant difference in terms of viability (Friedman’s test, p=0.7765). Given the low loss proportion of 5% during storage at -20°C and the high cost equipment required for storage at -80°C and LN, we recommend storage at -20°C or -80°C, when this is available, for preservation of M. bovis field strains.
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spelling Cryopreservation of Mycobacterium bovis isolatesCriopreservação de isolados de Mycobacterium bovisCryopreservationMiddlebrook 7H9 brothMycobacterium bovis.CriopreservaçãoCaldo Middlebrook 7H9Mycobacterium bovis.Research, development of new biotechnological methods, diagnostic tests, confirmation of results, and reinvestigations are possible because of the availability of well-preserved living organisms maintained without any changes. Cryopreservation is a simpler, more reliable and long-term stable method for culture maintenance. Storage temperature and composition of the suspending vehicle are factors that affect the viability of mycobacterial strains. Three vehicles and three storage temperatures were evaluated to define a suitable cryoprotective medium for the preservation of Mycobacterium bovis strains. Colonies of sixteen M. bovis isolates were used to prepare the suspensions, which were then added to three vehicles: sterile 0.85% saline solution (SS), Middlebrook 7H9 broth (7H9), and Middlebrook 7H9 broth with sodium pyruvate (7H9p) replacing glycerol. Aliquots of these suspensions were frozen by three different methods, directly in the -20°C freezer, directly in the -80°C freezer, and at -196°C by immersion in liquid nitrogen (LN). The frozen aliquots were thawed at room temperature after 45, 90 and 120 days. Mycobacterial viability was assessed by counting the living cells on plates of Stonebrink medium before and after the freezing procedure. Storage at -20°C exhibited a lower recovery of M. bovis compared to storage at -80°C (Dunn’s test, p=0.0018) and LN (Dunn’s test, p=0.0352). There was no statistically significant difference between storage at -80°C and in LN (Dunn’s test, p=0.1403), yet -80°C showed better results than LN. All three suspending vehicles showed no statistically significant difference in terms of viability (Friedman’s test, p=0.7765). Given the low loss proportion of 5% during storage at -20°C and the high cost equipment required for storage at -80°C and LN, we recommend storage at -20°C or -80°C, when this is available, for preservation of M. bovis field strains.Pesquisa, desenvolvimento de novos métodos biotecnológicos, testes diagnósticos, confirmação de resultados e reinvestigações são possíveis por causa da disponibilidade de organismos vivos bem preservados e mantidos inalterados. A criopreservação tem mostrado ser um método de manutenção de cultura mais simples, mais confiável e estável em longo prazo. Temperatura de armazenamento e composição do veículo de suspensão são fatores que afetam a viabilidade de cepas micobacterianas. Três veículos e três temperaturas de armazenamento foram avaliados para definir um meio crioprotetor adequado para preservação de estirpes de Mycobacterium bovis. Colônias de 16 isolados de M. bovis foram usadas na preparação de suspensões, as quais foram adicionadas a três veículos: solução salina a 0,85% estéril, caldo Middlebrook 7H9 (7H9) e caldo Middlebrook 7H9 com piruvato de sódio (7H9p) substituindo o glicerol. Alíquotas dessas suspensões foram congeladas por três métodos diferentes, direto em freezer a -20°C, direto em freezer a -80°C e a -196°C por imersão em nitrogênio líquido (LN). As amostras foram descongeladas a temperatura ambiente após 45, 90 e 120 dias. A viabilidade das micobactérias foi avaliada pela contagem de células vivas em placas com meio Stonebrink, antes e depois dos processos de congelamento. Armazenamento a -20°C exibiu menor recuperação de M. bovis comparado a -80°C (Teste de Dunn, p=0.0018) e LN (Teste de Dunn, p=0.0352). Não houve diferença entre armazenamento a -80°C e LN (Teste de Dunn, p=0.1403), mas -80°C apresentou melhores resultados do que LN. Os três veículos de suspensão não apresentaram diferença estatística (Teste de Friedman, p=0.7765). Dada à baixa proporção de perda (5%) durante o armazenamento a -20°C e ao alto custo do equipamento necessário para o armazenamento a -80°C, nós recomendamos o armazenamento a -20°C ou -80°C, quando disponível, para a preservação das estirpes de campo de M. bovis.UEL2016-11-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/2731310.5433/1679-0359.2016v37n5Supl2p3701Semina: Ciências Agrárias; Vol. 37 No. 5Supl2 (2016); 3701-3708Semina: Ciências Agrárias; v. 37 n. 5Supl2 (2016); 3701-37081679-03591676-546Xreponame:Semina. Ciências Agrárias (Online)instname:Universidade Estadual de Londrina (UEL)instacron:UELenghttps://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/27313/19936Copyright (c) 2016 Semina: Ciências Agráriashttp://creativecommons.org/licenses/by-nc/4.0info:eu-repo/semantics/openAccessIkuta, Cássia YumiAmbrosio, Simone RodriguesSouza Filho, Antonio Francisco deGrisi-Filho, José Henrique de HildebrandHeinemann, Marcos BryanFerreira Neto, José SoaresAmaku, Marcos2022-11-29T16:57:13Zoai:ojs.pkp.sfu.ca:article/27313Revistahttp://www.uel.br/revistas/uel/index.php/semagrariasPUBhttps://ojs.uel.br/revistas/uel/index.php/semagrarias/oaisemina.agrarias@uel.br1679-03591676-546Xopendoar:2022-11-29T16:57:13Semina. Ciências Agrárias (Online) - Universidade Estadual de Londrina (UEL)false
dc.title.none.fl_str_mv Cryopreservation of Mycobacterium bovis isolates
Criopreservação de isolados de Mycobacterium bovis
title Cryopreservation of Mycobacterium bovis isolates
spellingShingle Cryopreservation of Mycobacterium bovis isolates
Ikuta, Cássia Yumi
Cryopreservation
Middlebrook 7H9 broth
Mycobacterium bovis.
Criopreservação
Caldo Middlebrook 7H9
Mycobacterium bovis.
title_short Cryopreservation of Mycobacterium bovis isolates
title_full Cryopreservation of Mycobacterium bovis isolates
title_fullStr Cryopreservation of Mycobacterium bovis isolates
title_full_unstemmed Cryopreservation of Mycobacterium bovis isolates
title_sort Cryopreservation of Mycobacterium bovis isolates
author Ikuta, Cássia Yumi
author_facet Ikuta, Cássia Yumi
Ambrosio, Simone Rodrigues
Souza Filho, Antonio Francisco de
Grisi-Filho, José Henrique de Hildebrand
Heinemann, Marcos Bryan
Ferreira Neto, José Soares
Amaku, Marcos
author_role author
author2 Ambrosio, Simone Rodrigues
Souza Filho, Antonio Francisco de
Grisi-Filho, José Henrique de Hildebrand
Heinemann, Marcos Bryan
Ferreira Neto, José Soares
Amaku, Marcos
author2_role author
author
author
author
author
author
dc.contributor.author.fl_str_mv Ikuta, Cássia Yumi
Ambrosio, Simone Rodrigues
Souza Filho, Antonio Francisco de
Grisi-Filho, José Henrique de Hildebrand
Heinemann, Marcos Bryan
Ferreira Neto, José Soares
Amaku, Marcos
dc.subject.por.fl_str_mv Cryopreservation
Middlebrook 7H9 broth
Mycobacterium bovis.
Criopreservação
Caldo Middlebrook 7H9
Mycobacterium bovis.
topic Cryopreservation
Middlebrook 7H9 broth
Mycobacterium bovis.
Criopreservação
Caldo Middlebrook 7H9
Mycobacterium bovis.
description Research, development of new biotechnological methods, diagnostic tests, confirmation of results, and reinvestigations are possible because of the availability of well-preserved living organisms maintained without any changes. Cryopreservation is a simpler, more reliable and long-term stable method for culture maintenance. Storage temperature and composition of the suspending vehicle are factors that affect the viability of mycobacterial strains. Three vehicles and three storage temperatures were evaluated to define a suitable cryoprotective medium for the preservation of Mycobacterium bovis strains. Colonies of sixteen M. bovis isolates were used to prepare the suspensions, which were then added to three vehicles: sterile 0.85% saline solution (SS), Middlebrook 7H9 broth (7H9), and Middlebrook 7H9 broth with sodium pyruvate (7H9p) replacing glycerol. Aliquots of these suspensions were frozen by three different methods, directly in the -20°C freezer, directly in the -80°C freezer, and at -196°C by immersion in liquid nitrogen (LN). The frozen aliquots were thawed at room temperature after 45, 90 and 120 days. Mycobacterial viability was assessed by counting the living cells on plates of Stonebrink medium before and after the freezing procedure. Storage at -20°C exhibited a lower recovery of M. bovis compared to storage at -80°C (Dunn’s test, p=0.0018) and LN (Dunn’s test, p=0.0352). There was no statistically significant difference between storage at -80°C and in LN (Dunn’s test, p=0.1403), yet -80°C showed better results than LN. All three suspending vehicles showed no statistically significant difference in terms of viability (Friedman’s test, p=0.7765). Given the low loss proportion of 5% during storage at -20°C and the high cost equipment required for storage at -80°C and LN, we recommend storage at -20°C or -80°C, when this is available, for preservation of M. bovis field strains.
publishDate 2016
dc.date.none.fl_str_mv 2016-11-09
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/27313
10.5433/1679-0359.2016v37n5Supl2p3701
url https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/27313
identifier_str_mv 10.5433/1679-0359.2016v37n5Supl2p3701
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/27313/19936
dc.rights.driver.fl_str_mv Copyright (c) 2016 Semina: Ciências Agrárias
http://creativecommons.org/licenses/by-nc/4.0
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Copyright (c) 2016 Semina: Ciências Agrárias
http://creativecommons.org/licenses/by-nc/4.0
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv UEL
publisher.none.fl_str_mv UEL
dc.source.none.fl_str_mv Semina: Ciências Agrárias; Vol. 37 No. 5Supl2 (2016); 3701-3708
Semina: Ciências Agrárias; v. 37 n. 5Supl2 (2016); 3701-3708
1679-0359
1676-546X
reponame:Semina. Ciências Agrárias (Online)
instname:Universidade Estadual de Londrina (UEL)
instacron:UEL
instname_str Universidade Estadual de Londrina (UEL)
instacron_str UEL
institution UEL
reponame_str Semina. Ciências Agrárias (Online)
collection Semina. Ciências Agrárias (Online)
repository.name.fl_str_mv Semina. Ciências Agrárias (Online) - Universidade Estadual de Londrina (UEL)
repository.mail.fl_str_mv semina.agrarias@uel.br
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