RESUME AND JOURNAL REVIEW

1. Identity

Name :

Npm :

Journal Title : A comparison of fluorescent stains for the assessment of

viability and metabolic activity of lactic acid bacteria

Number of pages : 10

DOI : 10.1007/s11274-011-0889-x

2. Background

Lactic acid bacteria (LAB) play a fundamental role in the production of many
fermented and functional foods (Klaenhammer et al. 2002; Corcoran et al. 2008;
Prado et al. 2008). During their use as starter and/or probiotic cultures, the strains
suffer physical and chemical stresses which may affect their performance in food
biotechnology- and healthrelated applications. The use of rapid and efficient
methods to detect cell viability is, therefore, of practical relevance for commercial
and technological purposes. Plate counts, which are commonly used to detect
viable cells, are timeconsuming and often provide an underestimate of microbial
numbers due to the unequal distribution of cells in the medium or to the presence
of sub-lethally damaged or viable but non-culturable (VNC) cells (Giraffa 2004;
Temmerman et al. 2004; Bjergbaek and Roslev 2005; Zotta et al. 2009). For this
reason, different fluorescent techniques based on the detection of membrane
integrity, enzymatic activity, transmembrane potential gradients, ion level shifts,
intracellular pH, protein conformational changes and gene expression.
 http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The
Handbook.html) have been recently proposed as alternative tools. Among
fluorescent probes the dual staining (Syto 9 and Propidium Iodide, PI) provided in
the Live/Dead Bacterial BacLightTM Viability kit (Molecular Probes), which
discriminates viable from dead cells on the basis of the membrane integrity, and
the hydrolysis of fluorogenic substrate 5,(6)-carboxyfluorescein diacetate (cFDA)
by bacterial esterases (with production of fluorescent carboxyfluorescein),
combined with nucleic acid stains (such as PI and/or DAPI), are frequently used in
food and environmental applications (Hoefel et al. 2003; Papadimitriou et al. 2006;
Berney et al. 2007; Kramer et al. 2009; Zotta et al. 2009). The selection of the most
appropriate staining procedure is made complicated by several factors, including
cell physiology, and interpretation of results by operators. Part of the problem is
related to the ambiguous use of the terms ‘‘active’’ and ‘‘live’’ or ‘‘inactive’’ and
‘‘dead’’ as related to the staining results: for example a cell stained by PI can be
unambiguosly defined as ‘‘dead’’, because PI only permeates cells with a damaged
membrane. On the other hand, staining by Syto 9 only indicates that the cell has an
intact membrane; staining by cFDA indicates that cell esterases are active and the
membrane is intact, but neither stain can indicate if the cell will be able to grow in
a given environment.

Additionally, acquiring high quality images by digital cameras is important for

accurate analysis of data. Visual enumeration and estimation of cell dimensions is
laborious and time-consuming and the development of rapid and automated
measurements is important. Today, epifluorescent microscopes are generally
equipped with advanced image acquisition and analysis systems, but commercially
available software packages are expensive and sometimes complicated to use,
especially for inexperienced operators, and several studies are still done with
simple manual counting and measurement procedures. In this study, we
investigated the use of four fluorochromes and fluorogenic substrates (Syto 9, PI,
DAPI, cFDA) to evaluate the viability of 10 LAB strains belonging to several
industrially important species of the genera Enterococcus, Lactococcus,
Leuconostoc, Lactobacillus, Streptococcus and Weissella, subjected to oxidative
and heat stresses, in order to develop an efficient assay providing a valid indication
of cell viability status. Additionally, we optimized protocols for specimen
preparation and staining procedure to obtain high quality epifluorescence images to
avoid manipulation after acquisition step and we used a public domain software
package (ImageJ, National Institute of Health, USA; http://rsbweb.nih.gov/ij/) to
develop a useful automated cell counts system.

3. Problem

In this study 10 LAB strains, belonging to the genera Enterococcus,

Lactococcus, Leuconostoc, Lactobacillus, Streptococcus and Weissella, were
subjected to heat and oxidative stresses and cell injury or death was assessed
comparing different fluorescent probes (Syto 9; Propidium Iodide, PI; 4,6-
diamidino-2-phenylindole, DAPI; 5,(6)-carboxyfluorescein diacetate, cFDA) to
identify the stain combination which most reliably allowed the detection of
live/metabolically active and dead cells. Protocols for specimen preparation and
staining were optimized and a simple procedure for automated cell counts was
developed using NIH ImageJ macros. Cysteine and semi-solid agar solution were
efficiently used as anti-fading agent and mounting medium, respectively. The
double staining cFDA-PI apparently offered the best and most versatile indication
of both cell metabolic activity and membrane integrity. An excellent correlation
between manual and automated cell counts for the majority of strain/stain
combinations was found. This work provides a simple protocol for specimen
preparation and staining based on the use of safe, easy to prepare and inexpensive
reagents as compared to other methods. Additionally, the automated cell count
procedure developed can be applied to several bacterial species and allows an
increase in the number of experimental trials and the reproducibility and sensitivity
of the analysis

4. Materials and Methods

Enterococcus faecalis ATCC27274, Lactococcus lactis ATCC11454 (obtained

from American Type Culture Collection, Rockville, MD, USA), Leuconostoc
mesenteroides subsp. mesenteroides DSM20343, Lactobacillus curvatus
DSM20019, Lb. paracasei DSM4905, Lb. sakei subsp. sakei DSM20017 (Lb.
delbrueckii subsp. bulgaricus NCFB2772, Lb. plantarum NCIMB8826
(Streptococcus thermophilus Sfi39 and Weissella cibaria were used in this study
with a few steps;

 Strains and culture conditions

 Stress treatments
 Optimization of specimen preparation before staining and image acquisition
Reduction of fluorochrome fading
 Probes
 Live/Dead dual staining
 cFDA/PI dual staining
 DAPI/cFDA and DAPI/PI dual staining
 Microscopy image acquisition
 Image analysis and cell counts
 Statistical analysis
 5. Result and Discussion
 Optimization of cell mounting medium and staining procedur
 Staining and image acquisition
 Image analysis and cell counts
 Comparison between direct microscopy counts and plate counts

6. Conclusions

Our study confirms that no single staining protocol can provide optimal results
with all species, because some fluorochromes are not always appropriate to detect
cell viability and that the use of more than one viability indicator seems to be
useful. Therefore, caution should be used in the choice of the best stain
combination, which should keep into account the properties of the microorganism
tested, and should reflect the efficiency of the stain in truly discriminating live and
dead cells. This work provides a simple protocol for specimen preparation based
on the use of safe, easy to prepare and inexpensive reagents and, additionally,
when appropriate procedures are selected, automated analysis of the images can be
performed in a simple and straightforward fashion.

7. Advantages of Journals

a. The advantage of this scientific article is that it raises a trivial problem but
the impact is very large if it occurs. As the background that has been shown.
b. The contents of the discussion have been clearly explained from the problem
to the solution, making it easier for readers to understand this scientific
article
 8. Weakness of Journals

The drawback of this article is that it is not mentioned in detail in the name of
the area that is the object of the research carried out. This can be seen from the
object points and research locations. For the reader, it raises doubts about the shape
of the facts of the object of research carried out. Is it really a research or just an
essay.