Preparation of inocula for experimental infection of blood with Streptococcus pneumoniae.
MethodsX
; 2: 463-8, 2015.
Article
en En
| MEDLINE
| ID: mdl-26844211
Experimental infections of either cells or animals require the preparation of good quality inocula. Unfortunately, the important pulmonary pathogen Streptococcus pneumoniae is a fastidious microorganism that suffers an autolysis process when cultured in vitro. Supplementation of Todd-Hewitt broth with a biological buffer (20 mM Tris-HCl, pH = 7.8) promotes a six hours delay in the beginning of the autolysis process. Additional improvements include washing bacteria before freezing, avoiding manipulations after thawing, and the use of glycerol (<18%) as a cryoprotectant, instead of reagents like skimmed milk that may affect cell cultures. With the proposed protocol >70% of the frozen bacteria was viable after 28 weeks at -80 °C, and aliquots were highly homogeneous. We have tested their utility in a whole blood infection model and have found that human plasma exhibits a higher microbicidal activity than whole blood, a result that we have not found previously reported. Additionally, we have also observed significant variations in the antimicrobial activity against different strains, which might be related to their virulence.â¢Media culture buffering extends S. pneumoniae viability for 6 h.â¢Washing before freezing of single use aliquots minimizes manipulation after thawing.â¢Experimental infection with the frozen inocula has shown that plasma has higher bactericidal activity than blood.
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1
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01-internacional
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MEDLINE
Idioma:
En
Revista:
MethodsX
Año:
2015
Tipo del documento:
Article
País de afiliación:
España
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Países Bajos