**Acknowledgements**

*Nanoemulsions - Properties, Fabrications and Applications*

bacteria. After, overnight incubation, the tubes or wells are inspected for growth. The lowest concentration of nanoparticles that results in no-growth is the MIC

After adding the test formulation to a broth culture, antimicrobial activity can be assessed *in vitro* by collecting sequential samples to count survivors. Time-kill allows the assessment of *in-vitro* synergy or antagonism between

For the time-kill experiments, Mueller Hinton broth is prepared with serial dilutions of the test antimicrobial, alone or in combination. The nano-antimicrobial concentrations may span a range above and below the formulation's MIC, previously obtained from agar dilution tests. Broths are then inoculated with 106 cfu/mL and incubated overnight at 37°C. From time 0 when bacteria are first exposed to the test antimicrobial, samples are obtained at 30 min intervals for up to 6 h. The samples are then plated on nutrient agar. After incubation overnight at 37°C, survivor counts

Taking into account the future development and applications of the metal oxides nanoparticles in medicine, a constant search as emergent antimicrobial agents is required, due to the increase of diseases caused by microorganisms resistant to the

Implementation of the metal oxides nanoparticles as an alternative to combat bacterial resistance due to increased findings in the mechanisms by which they act, have been the key to a better understanding and approach about the effect and

For this reason it is necessary to establish guidelines and quality standards to research nano-antimicrobials given the fact that there are many alternative methods *in vitro* testing to achieve this objective, but some of them present

kinetics that metal oxides nanoparticles have on microbial strains.

**24**

[68] (**Figure 4**).

**Figure 4.**

*4.2.4 Time-kill method*

*Broth dilution method with NPs.*

nano-antimicrobials.

**5. Conclusions**

are plotted to obtain a 'time-kill curve' [69].

action of antimicrobial agents as antibiotics.

This work was financially supported by Facultad de Odontología, División de Estudios de Posgrado e Investigación and Instituto de Ciencias Aplicadas y Tecnología in Universidad Nacional Autónoma de México.
