Skip to main content

Proposal for a method to estimate nutrient shock effects in bacteria



Plating methods are still the golden standard in microbiology; however, some studies have shown that these techniques can underestimate the microbial concentrations and diversity. A nutrient shock is one of the mechanisms proposed to explain this phenomenon. In this study, a tentative method to assess nutrient shock effects was tested.


To estimate the extent of nutrient shock effects, two strains isolated from tap water (Sphingomonas capsulata and Methylobacterium sp.) and two culture collection strains (E. coli CECT 434 and Pseudomonas fluorescens ATCC 13525) were exposed both to low and high nutrient conditions for different times and then placed in low nutrient medium (R2A) and rich nutrient medium (TSA).

The average improvement (A.I.) of recovery between R2A and TSA for the different times was calculated to more simply assess the difference obtained in culturability between each medium. As expected, A.I. was higher when cells were plated after the exposition to water than when they were recovered from high-nutrient medium showing the existence of a nutrient shock for the diverse bacteria used. S. capsulata was the species most affected by this phenomenon.


This work provides a method to consistently determine the extent of nutrient shock effects on different microorganisms and hence quantify the ability of each species to deal with sudden increases in substrate concentration.


Since the first use of culture media to grow and study bacteria, plating techniques have always been considered as the gold-standard to assess the presence of living microorganisms in a certain environment. Developments occurring during the last 30 years have shown however that these methods are actually underestimating both their numbers and diversity. For instance, application of viability-staining techniques have allowed the identification of “viable but nonculturable bacteria” (VBNC) [reviewed in [1], whereas new species of microorganisms identified by 16 S rRNA gene sequencing still remain uncultured [reviewed in [2].

The VBNC state refers to a condition where cells, once exposed to environmental stress (such as nutrient starvation, elevated or lowered osmotic concentrations, oxygen concentrations and exposure to white light), enter into a dormancy phase and fail to grow on the routine media on which they would normally grow [1, 3, 4], however, culturability for these cells could be recovered under certain conditions. Despite the lack of cultivability, the VBNC state is of great concerns because cells can display enhanced resistance to antibiotics (mainly due to the low metabolic activity), and retain the virulence properties after resuscitation [47].

By the time that the notion of VBNC was first proposed in 1982 [8], efforts to improve the recovery of microorganisms from stressful low-nutrient environments had already began. In 1983, Straškrabová [9] showed that starving aquatic bacteria died on rich media possibly due to a high nutrient shock. One of the keystone papers was published two years later by Reasoner and Geldreich [10]. In their study, a new low-nutrient medium denominated R2A, was presented and found to yield significantly higher bacterial counts than plate count agar for samples from potable water supplies. Similarly, Jensen et al.[11] concluded that culturability of specific populations of marine bacteria can be dramatically improved by the use of low-nutrient media. Alternatively, Hahn et al, developed a method - the filtration-acclimatization method - which avoids the nutrient shock by using an acclimatization procedure that provides a slow transition from the low environmental substrate concentrations to the high concentration of standard microbial media. It hence enables the isolation and cultivation (on high nutrient media) of a broad variety of previously uncultured bacteria [12, 13]. High-throughput culturing methods that rely on dilution to extinction in very-low-nutrient media or in the step-wise acclimatization to higher substrate concentrations are becoming more common for the isolation of previously uncultured microorganisms, or even novel species, from the ocean, lakes, soils and other low nutrient environments [e. g. [1418]. All these papers confirmed the earlier suggestions of substrate-accelerated death in bacteria described by Postgate and co-workers [19, 20]. More recently, insights into how bacteria adapt their metabolism to these conditions are being provided by molecular biology methods [4, 21]. Putting all this together it is acceptable to say that the correct recovery of bacteria is an essential issue on the knowledge of the true roles and function of bacteria in the specific environment.

Based on the success of the previous studies, we have designed a new low-nutrient medium with the specific purpose of a more efficient recovery of the human pathogen Helicobacter pylori exposed to water or related environments [22]. In addition, it was concluded that because the direct recovery from water to a high-nutrient medium causes nutrient shock, the bacteria could physiologically adapt to low-nutrient environments and hence be transmitted through water [23].

Even though the existence of a nutrient shock has become engrained in the scientific community, there is no proof of principle for this concept in the literature. Observations such as that R2A is only used for low nutrient environments, or the fact that some freshwater isolates that fail to grow initially in nutrient-rich media may be gradually acclimated to rich media [15], appear to further sustain that notion, but to prove it requires evidence that the ratio of recovery between low and nutrient rich media for a certain microorganism decreases as the microorganism is recovered from low to rich nutrient environments. To the author’s knowledge, this work represents the first attempt to estimate the extent of nutrient shock effects for a range of different bacteria.

Material and methods

Recovery and identification of microorganisms from tap water

Two of the microorganisms used for this study were isolated from the drinking water distribution system described by Simões et al.[24]. The bacteria were isolated in the planktonic state by plating on R2A (Oxoid, Basingstoke, U.K.) at room temperature during 15 days. Based on 16 S rRNA sequence similarity (performed at the Sequencing and Fragment Analysis Laboratory, Science Faculty of Lisbon) they have been presumptively identified as Sphingomonas capsulata (maximum identity of 99%, E value 0.0, with Sphingomonas capsulatum NR_025838.1 – has been renamed as Novosphingobium capsulatum in [25]) and Methylobacterium sp. (maximum identity of 100%, E value 0.0, with Methylobacterium sp. AB673245.1) using a Blast search.

Culture maintenance and media preparation

Culture collection E. coli (CECT 434) was maintained in Tryptone Soya Agar (TSA) whereas Pseudomonas fluorescens (ATCC 13525) was maintained in Pseudomonas medium prepared as described in Oliveira et al.[26]. The water isolates S. capsulata and Methylobacterium sp. were maintained on R2A. All strains were incubated at 23 ± 2°C and subcultured to new plates every 5 to 7 days.

To assess the existence of a nutrient shock, both a rich nutrient medium (TSA) and a lower nutrient medium (R2A) were used. The final concentration of all constituents in R2A was 18.1 g/L whereas in TSA was 45 g/L. TSA was prepared with 30 g/L of Tryptone Soya Broth (TSB; VWR International, Lisbon, Portugal) and 15 g/L of granulated agar (VWR International). For each set of experiments, both media were prepared and poured into plates seven days before the experiment, and stored at 4°C.

Exposure of bacteria to low and high-nutrient environments and subsequent recovery

For the experiment in low-nutrient environments, cells from 2 day-old cultures were harvested from R2A plates, suspended in 10 ml of autoclaved tap water, vortexed for 30s and adjusted by optical density to a concentration of 5 × 106 CFU per ml. This inoculum was transferred to a sterile bottle containing 500 ml of autoclaved tap water, to achieve a final concentration of ca. 105 CFU/ml. The bottle was maintained at room temperature (approx. 23 ± 2°C) and continuously stirred (120 rpm) using a magnetic bar. Sampling was performed at different times up to 24 h. Before serial dilution (1:10) in sterile tap water, samples were vortexed for 10s for homogenization. Cells were enumerated in quadruplicate by surface plating 100 μl of the different dilutions onto R2A or TSA. Plates were incubated at 23°C for 7 days and then colony forming units were counted.

For the experiment in high-nutrient environments, the procedure was basically the same, but the water used for the inoculum, bioreactor and dilutions, was replaced by TSB.

Analysis of data

To more simply assess the difference obtained in culturability between each medium, the average improvement (A.I.) in culturability between R2A and TSA was calculated for all species, both when they were recovered from water and from TSB. The A.I. for each bacterium and condition is defined as follows:

A . I . = t = 0 4 log C F U R 2 A log C F U TSA t log C F U TSA t 5 × 100

where t represents the different times at which the sampling was performed (0, 2, 4, 6 and 24 h); CFUR2A the colony forming units for each time point on R2A; CFUTSA the colony forming units for each time point on TSA. The formula is divided by 5 as this is the number of sampling times assessed. Based on Equation 1, a nutrient shock index can be calculated:

Nutrient shock index  =  A . I . water  A . I . TSB

where A.I.water and A.I.TSB represent the average improvement when the cells are cultured from water and TSB, respectively. The larger the nutrient shock index, the more the strains are able to adapt to low nutrient conditions.

Nutrient shock results were statistically analyzed by employing a two-way analysis of variance (ANOVA). Computations were performed using the Statistical Program for the Social Sciences (SPSS Inc., Chicago, USA). Results were considered statistically relevant if P values were ≤0.05.


Of the bacteria used for this study, S. capsulata appeared to be the one most affected by exposure to water, as after 24 h no cells could be recovered on TSA and only 100 CFU/mL could be recovered in R2A (Figure 1 and Additional file 1 Table A1). In all other bacteria a less noticeable decrease was also observed. When suspended in TSB, S. capsulata, E. coli and P. fluorescens were able to use the nutrients in the media to support growth, whereas Methylobacterium sp. lost culturability with time.

Figure 1
figure 1

Study of nutrient shock effect on 4 different microorganisms. Graphics on the left depict recovery obtained on R2A (dashed bars) and TSA (full bars) as cells are being exposed to water, whereas graphics on the right show recovery when cells are suspended in TSB.

For all cases but for P. fluorescens, R2A supported better growth for the bacteria recovered from water, a result that was found to be statistically significant for all three cases (Table 1) (P < 0,01). TSA performance improved when the bacteria were recovered from TSB, which implied that differences between recovery in TSA and R2A were no longer statistically significant (P > 0.05). On the other hand, TSA supported a statistically significant higher growth for P. fluorescens when the microorganism was recovered from TSB (P < 0.01). In this case, this difference was also attenuated when P. fluorescens was recovered from water, and differences between media were no longer statistically significant (P > 0.05).

Table 1 Preferred medium for recovering water and TSB exposed strains

Reflecting the observations described above, the A.I. was always greater when the bacteria were recovered from water, demonstrating the idea of a nutrient shock, as it can be confirmed by the positive values obtained for all strains in the nutrient shock index (Table 2). Hence, the below-expected performance of TSA for S. capsulata and of R2A for P. fluorescens is due to intrinsic characteristics of the media (such as nutrient diversity) and not to nutrient concentration differences between the two media.

Table 2 Nutrient shock index obtained for the four species used in this study

Statistical analysis also allowed to observe if the relationship between the recoveries on both media was constant with time. For all cases, the interaction term of time*media was statistically significant (P < 0.05), indicating that this relationship varied. Because the difference between the A.I. for when the cells are recovered from water or from rich-nutrient medium are not very large for three of the microorganisms tested (approx. 7%), the statistical power of the method used was essential to obtain relevant results.

Nutrient shock occurrence

This study has demonstrated for a set of waterborne microorganisms that a nutrient shock occurs. As an indirect conclusion it shows that newly developed media must not only address the type or types of microorganisms to be recovered but also from where they are being recovered from. Similarly, inferring the adaptability of a certain microorganism to water (or even other environments) based solely on the time it remains culturable might also cause incorrect interpretations as culturability clearly depends on the medium used [27].

As this is a relatively unknown term, it is important to clarify the meaning of nutrient shock and how it correlates with other similar terms such as nutrient stress and osmotic shock. Nutrient stress is a different concept in the sense that it refers to when the quantity of nutrient available decreases yield [28, 29]. However, the bacteria might still be in a nutrient rich medium that they are unable to metabolize. Nutrient shock is also different from osmotic stress, as the latter refers only to the physical pressure exerted by the transfer of water through the cell membrane to balance the osmotic pressure [3032]. Even though part of the nutrient shock effect might be explained by the damage caused by osmotic stress effects on the cells membrane, its broader concept also includes other phenomena such as the inability of the bacteria to suddenly process the large amounts of nutrient of a nutrient-rich solid medium. By the results obtained, it is not possible to understand the contribution that osmotic stress has given to the nutrient shock observed here. To understand the exact extent of this contribution, it will be necessary to develop a new experiment where the bacteria are also exposed to a salt or sugar solution.

The main reason why this study was performed with time was our lack of knowledge about whether this factor would influence the final results. Metabolomics experiments have already shown that the response time in physiological mechanisms of bacteria for an environmental change can be in the order of seconds [33]. However, physiological changes will extend for much longer [34]. In our study, the interaction term of time*media obtained from the statistical analysis was statistically significant for all cases, reflecting the continuous adaptation of the bacteria to the environment. The approach of calculating the A.I. provides therefore a more robust system, that is more representative of the nutrient shock effect for different physiological conditions and not easily affected by the transformations occurring in the population of cells in single time points.

A.I. indications

This study confirms the long suspected notion that nutrient shock hinders the recovery of low-nutrient adapted microorganisms to rich-nutrient medium [10, 11, 14, 35], and provides a method to quantify those effects in bacteria. It is important to state that the developed measure (A.I.) is dependent on the time intervals at which the samples are taken and that for comparable results to be obtained, the same intervals have to be selected. If comparable results are obtained, than the nutrient shock index can serve as a measure of the ability of different bacteria to withstand nutrient shock effects. In theory it is possible to obtain the nutrient shock index using any other combination of nutrient rich and nutrient poor media other than TSA and R2A, however, once again comparable results might not be obtained.

Future work will involve correlation of A.I. with genome analysis and protein expression in order to shed new light on the mechanisms that are affected by the nutrient shock effect. Another important study would involve the use specific substances used as resuscitation components [3, 4] prior to bacteria plating, in order to stimulate bacteria that may be at a VBNC state. This would allow to understand in what extend VBNC cells are correlated with the nutrient shock phenomenon.



Average improvement


Viable but nonculturable bacteria


Tryptone Soya Agar


Tryptone Soya Broth


Statistical Program for the Social Sciences


Colony forming units.


  1. Oliver JD: The viable but nonculturable state in bacteria. J Microbiol. 2005, 43: 93-100.

    PubMed  Google Scholar 

  2. Handelsman J: Metagenomics: Application of genomics to uncultured microorganisms. Microbiol Mol Biol R. 2004, 68: 669-685. 10.1128/MMBR.68.4.669-685.2004.

    Article  CAS  Google Scholar 

  3. Richards CL, Buchholz BJ, Ford TE, Broadaway SC, et al: Optimizing the growth of stressed Helicobacter pylori. J Microbiol Methods. 2011, 84: 174-182. 10.1016/j.mimet.2010.11.015.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  4. Oliver JD: Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev. 2010, 34: 415-425.

    Article  PubMed  CAS  Google Scholar 

  5. Du M, Chen JX, Zhang XH, Li AJ, et al: Retention of virulence in a viable but nonculturable Edwardsiella tarda isolate. Appl Environ Microbiol. 2007, 73: 1349-1354. 10.1128/AEM.02243-06.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  6. Lleo MD, Bonato B, Signoretto C, Canepari P: Vancomycin resistance is maintained in enterococci in the viable but nonculturable state and after division is resumed. Antimicrob Agents Chemother. 2003, 47: 1154-1156. 10.1128/AAC.47.3.1154-1156.2003.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Lleo M, Bonato B, Tafi MC, Caburlotto G, et al: Adhesion to medical device materials and biofilm formation capability of some species of enterococci in different physiological states. FEMS Microbiol Lett. 2007, 274: 232-237. 10.1111/j.1574-6968.2007.00836.x.

    Article  PubMed  CAS  Google Scholar 

  8. Xu HS, Roberts N, Singleton FL, Attwell RW, et al: Survival and viability of nonculturable Escherichia Coli and Vibrio Cholerae in the estuarine and marine-environment. Microbial Ecol. 1982, 8: 313-323. 10.1007/BF02010671.

    Article  CAS  Google Scholar 

  9. Straskrabova V: The Effect of Substrate Shock on Populations of Starving Aquatic Bacteria. J Appl Bacteriol. 1983, 54: 217-224. 10.1111/j.1365-2672.1983.tb02610.x.

    Article  Google Scholar 

  10. Reasoner DJ, Geldreich EE: A New Medium for the Enumeration and Subculture of Bacteria from Potable Water. Appl Environ Microb. 1985, 49: 1-7.

    CAS  Google Scholar 

  11. Jensen PR, Kauffman CA, Fenical W: High recovery of culturable bacteria from the surfaces of marine algae. Mar Biol. 1996, 126: 1-7. 10.1007/BF00571371.

    Article  Google Scholar 

  12. Hahn MW, Stadler P, Wu QL, Pockl M: The filtration-acclimatization method for isolation of an important fraction of the not readily cultivable bacteria. J Microbiol Methods. 2004, 57: 379-390. 10.1016/j.mimet.2004.02.004.

    Article  PubMed  CAS  Google Scholar 

  13. Hahn MW: Broad diversity of viable bacteria in 'sterile' (0.2 mu m) filtered water. Res Microbiol. 2004, 155: 688-691. 10.1016/j.resmic.2004.05.003.

    Article  PubMed  Google Scholar 

  14. Connon SA, Giovannoni SJ: High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates. Appl Environ Microb. 2002, 68: 3878-3885. 10.1128/AEM.68.8.3878-3885.2002.

    Article  CAS  Google Scholar 

  15. Hahn MW, Lunsdorf H, Wu QL, Schauer M, et al: Isolation of novel ultramicrobacteria classified as Actinobacteria from five freshwater habitats in Europe and Asia. Appl Environ Microb. 2003, 69: 1442-1451. 10.1128/AEM.69.3.1442-1451.2003.

    Article  CAS  Google Scholar 

  16. Page KA, Connon SA, Giovannoni SJ: Representative freshwater bacterioplankton isolated from Crater Lake. Oregon. Appl Environ Microb. 2004, 70: 6542-6550. 10.1128/AEM.70.11.6542-6550.2004.

    Article  CAS  Google Scholar 

  17. Janssen PH, Yates PS, Grinton BE, Taylor PM, Sait M: Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl Environ Microbiol. 2002, 68: 2391-2396. 10.1128/AEM.68.5.2391-2396.2002.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  18. Sait M, Hugenholtz P, Janssen PH: Cultivation of globally distributed soil bacteria from phylogenetic lineages previously only detected in cultivation-independent surveys. Environ Microbiol. 2002, 4: 654-666. 10.1046/j.1462-2920.2002.00352.x.

    Article  PubMed  CAS  Google Scholar 

  19. Calcott PH, Postgate JR: Substrate-Accelerated Death in Klebsiella-Aerogenes. J Gen Microbiol. 1972, 70: 115-117. 10.1099/00221287-70-1-115.

    Article  PubMed  CAS  Google Scholar 

  20. Calcott PH, Montague W, Postgate JR: Levels of Cyclic Amp during Substrate-Accelerated Death. J Gen Microbiol. 1972, 73: 197-201. 10.1099/00221287-73-1-197.

    Article  PubMed  CAS  Google Scholar 

  21. Weber J, Kayser A, Rinas U: Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour. Microbiology-Sgm. 2005, 151: 707-716. 10.1099/mic.0.27482-0.

    Article  CAS  Google Scholar 

  22. Azevedo NF, Vieira MJ, Keevil CW, et al: Development of an optimized technique for the recovery of Helicobacter pylori from water and drinking water biofilms. Biofilms: Persistence and Ubiquity. Edited by: McBain A, Allison D, Pratten J, Spratt D. 2005, Biofilm Club, Manchester, 221-230.

    Google Scholar 

  23. Azevedo NF, Pacheco AP, Vieira MJ, Keevil CW: Nutrient shock and incubation atmosphere influence recovery of culturable Helicobacter pylori from water. Appl Environ Microbiol. 2004, 70: 490-493. 10.1128/AEM.70.1.490-493.2004.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  24. Simões LC, Azevedo N, Pacheco A, Keevil CW, Vieira MJ: Drinking water biofilm assessment of total and culturable bacteria under different operating conditions. Biofouling. 2006, 22: 91-99. 10.1080/08927010600598603.

    Article  PubMed  Google Scholar 

  25. Takeuchi M, Hamana K, Hiraishi A: Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol. 2001, 51: 1405-1417.

    Article  PubMed  CAS  Google Scholar 

  26. Oliveira R, Melo L, Oliveira A, Salgueiro R: Polysaccharide production and biofilm formation by Pseudomonas fluorescens: effects of pH and surface material. Colloids Surf B Biointerfaces. 1994, 2: 41-46. 10.1016/0927-7765(94)80016-2.

    Article  CAS  Google Scholar 

  27. Davis KER, Joseph SJ, Janssen PH: Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria. Appl Environ Microbiol. 2005, 71: 826-834. 10.1128/AEM.71.2.826-834.2005.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  28. Malys N, Carroll K, Miyan J, Tollervey D, McCarthy JEG: The 'scavenger' m(7)GpppX pyrophosphatase activity of Dcs1 modulates nutrient-induced responses in yeast. Nucleic Acids Res. 2004, 32: 3590-3600. 10.1093/nar/gkh687.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Guinez C, Lemoine J, Michalski JC, Lefebvre T: 70-kDa-heat shock protein presents an adjustable lectinic activity towards O-linked N-acetylglucosamine. Biochem Bioph Res Co. 2004, 319: 21-26. 10.1016/j.bbrc.2004.04.144.

    Article  CAS  Google Scholar 

  30. Mager WH, Siderius M: Novel insights into the osmotic stress response of yeast. FEMS Yeast Res. 2002, 2: 251-257.

    Article  PubMed  CAS  Google Scholar 

  31. Beales N: Adaptation of microorganisms to cold temperatures, weak acid preservatives, low pH, and osmotic stress: A review. Compr Rev Food Sci F. 2004, 3: 1-20. 10.1111/j.1541-4337.2004.tb00057.x.

    Article  CAS  Google Scholar 

  32. Romantsov T, Guan ZQ, Wood JM: Cardiolipin and the osmotic stress responses of bacteria. Biochimica Et Biophysica Acta-Biomembranes. 2009, 1788: 2092-2100. 10.1016/j.bbamem.2009.06.010.

    Article  CAS  Google Scholar 

  33. Buchholz A, Hurlebaus J, Wandrey C, Takors R: Metabolomics: quantification of intracellular metabolite dynamics. Biomol Eng. 2002, 19: 5-15. 10.1016/S1389-0344(02)00003-5.

    Article  PubMed  CAS  Google Scholar 

  34. Shen D, Sharfstein ST: Genome-wide analysis of the transcriptional response of murine hybridomas to osmotic shock. Biotechnol Bioeng. 2006, 93: 132-145. 10.1002/bit.20691.

    Article  PubMed  CAS  Google Scholar 

  35. Reasoner DJ: Heterotrophic plate count methodology in the United States. Int J Food Microbiol. 2004, 92: 307-315. 10.1016/j.ijfoodmicro.2003.08.008.

    Article  PubMed  Google Scholar 

Download references


This work was supported by the Fundação para a Ciência e Tecnologia (grant SFRH/BD/38124/2007 and SFRH/BD/29297/2006).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Nuno F Azevedo.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

Conceived and designed the experiments: NFA, SMB, CA, LC, CWK, MJV. Performed the experiments: NFA, SMB, LCS. Analyzed the data: CA, NFA, SRBS, LC. Contributed reagents/materials/analysis tools: CWK MJV. Wrote the paper: NFA, SMB, CA. Revised a draft of the manuscript: CWK MJV. All authors read and approved the final manuscript.

Electronic supplementary material


Additional files 1: Table A1 Values of colony forming units (CFU) and respective standard deviation obtained for all species at different times when exposed to water and then plated on R2A and TSA. Table A2 Values of colony forming units (CFU) and respective standard deviation obtained for all species at different times when exposed to TSB and then plated on R2A and TSA. (DOC 90 KB)

Authors’ original submitted files for images

Below are the links to the authors’ original submitted files for images.

Authors’ original file for figure 1

Rights and permissions

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Azevedo, N.F., Bragança, S.M., Simões, L.C. et al. Proposal for a method to estimate nutrient shock effects in bacteria. BMC Res Notes 5, 422 (2012).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: