Application of flow cytometry and PMA-qPCR to distinguish between membrane intact and membrane compromised bacteria cells in an aquatic milieu

• Published in: International journal of hygiene and environmental health 2015-11, Vol.218 (8), p.714-722
• Main Authors: Zacharias, Nicole, Kistemann, Thomas, Schreiber, Christiane
• Format: Article
• Language: English
• Subjects: Azides; Cell Membrane; Disinfection - methods; DNA, Bacterial; Escherichia coli; Flow cytometry; Flow Cytometry - methods; Hot Temperature; Humans; Legionella; Legionella pneumophila; LIVE/DEAD; Microbial Viability; PMA; Polymerase Chain Reaction - methods; Propidium - analogs & derivatives; qPCR; Water Microbiology
• ISSN: 1438-4639; 1618-131X
• DOI: 10.1016/j.ijheh.2015.04.001

The paper compares two methods of distinguishing between alive and dead cells by differentiation on the basis of their membrane structure: LIVE/DEAD flow cytometry and PMA-qPCR. LIVE/DEAD flow cytometry was established using the LIVE/DEAD® BacLight™ Bacterial Viability Kit with different ratios of Legionella pneumophila and Escherichia coli cells with intact and compromised membranes (heat treated). The PMA-qPCR method was tested and modified, and results were compared with those from LIVE/DEAD flow cytometry using L. pneumophila cells. Ratios of membrane intact to membrane compromised cells were well shown by LIVE/DEAD flow cytometry in all combinations. PMA-qPCR seems to work best in even mixed ratios (1:1) of intact and compromised cells. In other respects, we noticed an overestimation of intact cells in the samples which contained a high percentage of membrane compromised cells, and an underestimation of intact cells in samples with a small percentage of membrane compromised cells. However, looking at total counts instead of ratios, the results were within an order of magnitude. This implies that the use of PMA-qPCR is appropriate only for a qualitative analysis to monitor the success of a process such as disinfection. Furthermore, we were able to assess that both methods have advantages and disadvantages: LIVE/DEAD flow cytometry as applied in this study works well on some bacteria monocultures, but does not distinguish between bacteria species. The PMA-qPCR method allows the possibility of distinguishing between membrane intact cells and membrane compromised cells and can be used to screen for specific bacteria.