Validation of Alternative Microbiological Method in Non-sterile Pharmaceutical Product Through the Reference Strains and Productive Environment Bioburden

  • Michelle Andrade LemosEmail author
  • Daniela Dal Molim Ghisleni
  • Terezinha de Jesus Andreoli Pinto
Original Article



The traditional methods described in pharmacopeias most commonly used by the pharmaceutical industry are easy to perform and the costs are affordable, but they require long periods of time to obtain the results and often do not present sensitivity for recovering microorganisms in vulnerable physiological states known as viable but nonculturable. Thus, the objectives of this study were to evaluate the potential applicability of flow cytometry in non-sterile pharmaceutical products.


This paper implemented the analytical validation steps, through the reference strains and productive environment bioburden, by analyzing the traditional method in parallel to the alternative method with flow cytometry.


The results indicated, with a 95% probability of detection, that there were no significant differences between the methods in relation to the ability to detect microbial contamination; however, the detection was faster with the flow cytometry method than the traditional method, which indicates that this technology is a viable alternative to be implemented.


The study demonstrated that the alternative microbiological method presents greater sensitivity in the analyses carried out, guaranteeing greater patient safety, besides allowing results to be obtained in a short period of time, thus enabling anticipation of investigations on possible failures that may occur during the process. Additionally, the study contributes to the environment by reducing waste generation and saving energy.


Flow cytometry Non-sterile pharmaceutical product Alternative microbiological method Viable but nonculturable Rapid microbiology method 



The authors are grateful to the leaders of the Pharmaceutical Industry for providing the flow cytometry technology, the experimental batches of the product, the consumables, and all the support necessary for performing this study.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. 1.
    FARMACOPEIA BRASILEIRA, 5ª edição. Métodos Microbiológicos Alternativos. Primeiro Suplemento. Brasília, 36–39, 2016Google Scholar
  2. 2.
    Breeuwer p, Abee t. Assessment of viability of microorganisms employing fluorescence techniques. Int J Food Microbiol. 2000;55(1–3, 193):–200.CrossRefGoogle Scholar
  3. 3.
    Bugno A, et al. Application of the BacT/ALERTR 3D system for sterility testing of injectable products. Braz J Microbiol São Paulo. 2015;46(3):743–7.CrossRefGoogle Scholar
  4. 4.
    Bugno, Adriana, Almodóvar, Adriana Aparecida Buzzo, & Pereira, Tatiana Caldas. Enumeration of heterotrophic bacteria in water for dialysis: comparison of the efficiency of Reasoner'2 agar and plate count agar. Braz J Microbiol. 2010;41(1):15–18. Scholar
  5. 5.
    Díaz M, et al. Application of flow cytometry to industrial microbial bioprocesses. Biochem Eng J. 2010;48:385–407.CrossRefGoogle Scholar
  6. 6.
    European Pharmacopoeia. 9.0 ed. 5.1.6 – Alternative methods for control of microbiological quality. Strasbourg: European Directorate for the Quality Medicines; 2017.Google Scholar
  7. 7.
    Japanese Pharmacopoeia 17th ed. [internet]. Tokyo: Ministry of Health, Labour and Welfare; 2016. Available from: [cited 2019 Mai 09];
  8. 8.
    Li L, et al. The importance of the viable but non-culturable state in human bacterial pathogens. Front Microbiol. 2014;5(JUN):1–1.Google Scholar
  9. 9.
    Miller M. The implementation of rapid microbiological methods. Eur Pharm Rev. 2010:24–6.Google Scholar
  10. 10.
    Parenteral Drug Association. PDA Technical Report No. 33 (Revised 2013). Bethesda: Evaluation, validation and implementation of alternative and rapid microbiological methods; 2013.Google Scholar
  11. 11.
    Pinto T d JA, Kaneco TM, Pinto AF. Controle Biológico de Qualidade de Produtos Farmacêutico, Correlatos e Cosméticos. Quartaedição. São Paulo: Manole; 2015. p. 129–35.Google Scholar
  12. 12.
    Ratajczak M. Microbiological quality of non-sterile pharmaceutical products. Saudi Pharm J. July 2015;23:303–7.CrossRefGoogle Scholar
  13. 13.
    Sage, A. et al. European Journal of Parenteral & Pharmaceutical Sciences for automated rapid microbiology EM methods determining incubation regime and time to results for automated rapid microbiology EM methods. 19(2), p. 45–55, 2014;Google Scholar
  14. 14.
    Sandle, Tim. Introduction to pharmaceutical microbiology. In: Pharmaceutical microbiology: essentials for quality assurance and quality. Wood head Publishing, 2016. Accessed 02 Apr 2019.
  15. 15.
    Santos A, Doria M, Soares L, Almeida A, Menezes J. A QRM discussion of microbial contamination of non-sterile drug products, using FDA and EMA warning letters recorded between 2008 and 2016. PDA J Pharm Sci Technol. 2018;72(1):62–72. Scholar
  16. 16.
    UNITED STATES PHARMACOPEIA, USP 40. Validation of alternative microbiological methods. The National Formulary, NF 35. Rockville: The United States Pharmacopeial Convention; 2017.Google Scholar
  17. 17.
    Wilkinson MG. Flow cytometry as a potential method of measuring bacterial viability in probiotic products: a review. Trends Food Sci Technol. 2018;78(May):1–10.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Michelle Andrade Lemos
    • 1
    Email author
  • Daniela Dal Molim Ghisleni
    • 1
  • Terezinha de Jesus Andreoli Pinto
    • 1
  1. 1.Department of Pharmacy, Faculty of Pharmaceutical SciencesUniversity of São PauloSão PauloBrazil

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