*4.2.3 Broth dilution method*

*Nanoemulsions - Properties, Fabrications and Applications*

living tissues.

*4.1.2 Antiseptics*

antiseptics are regulated by the FDA.

entiate between kill and inhibition of growth.

antimicrobial activity test, such as a disc diffusion test.

will be examined to assess inhibition rings around the disc.

is plated into Petri dishes. An aliquot containing 104

**4.2 Antimicrobial activity tests**

*4.2.1 Disk-diffusion method*

center (**Figure 2**).

*4.2.2 Agar dilution method*

disinfectants must be tuberculocidal. Products effective only against vegetative

Special testing procedures may be applicable to some pathogens of epidemiological interest, such as *Clostridium difficile* [64]. Disinfectants are intended for use on inanimate surfaces. In general, their high concentration precludes their use on

Antiseptics are antimicrobials intended for use on skin and mucous membranes. The same as with low level disinfectants, antiseptics are tested against *Escherichia coli*, *Staphylococcus aureus* and *Pseudomonas aeruginosa* in vegetative form. In the US,

A relevant test microorganism is chosen: preferably a strain from the American

The antimicrobial capability of nanoparticles has been explored by this techniques due studies have suggested that NPs are excellent microbicidal activity [16, 65]. The *in vitro* tests described below are the ones that the most have been used and the regulatory agencies recommend to determine antimicrobial activity of chemical formulations and can be used in the studies of nano-antimicrobials. The use of such tests depends on the objectives and the type of information it want to obtain. A first approach if nanoparticle has antimicrobial activity is to conduct an

Mueller-Hinton agar (pH 7.2–7.4) is the culture medium of choice. To standardize disc diffusion, the agar is poured into either Petri dish to only 4 mm in depth, as

The bacteria are suspended to a 0.5 McFarland turbidity standard equivalent to 150 × 106 cfu/mL. From this suspension, 100 μL are uniformly spread onto the agar. Filter-paper discs 6 mm diameter, containing the test nano-antimicrobial, will be placed over the seeded agar (alternatively, a 50–100 μL well, punched into the agar, will contain the test antimicrobial). After overnight incubation at 37°C, the plates

The size of the nanoparticle, its rate of diffusion, the agar's porosity, and possible charge interactions between the antimicrobial and the agar may affect diffusion and the final size of the inhibition zone. In theory, the highest concentrations will be near the antimicrobial-containing disc and will be diluted away from the

This method is the gold standard for assessing the minimal inhibitory concentration (MIC) [67]. In this method, the melted agar is mixed to contain serial dilutions of the nano-antimicrobial. The resulting antimicrobial containing medium

cfu of the test microorganism

indicated in the Clinical and Laboratory Standards Institute method [66].

Type Culture Collection (ATCC) or a similar repository. Although, wild-type bacteria from clinical samples also have been used. All necessary controls must be included to assess test reliability and reproducibility. Also, it is important to differ-

bacteria and viruses are regarded as low level disinfectants [63].

**22**

This method is often used because is more versatile and less laborious than the agar dilution method. Its microtiter plate version (broth microdilution), allows for testing more microorganisms against diverse concentrations of nano-antimicrobials, and can be automated.

Test tubes or wells in a microtiter plate, are prepared with bacteriological broth containing serial dilutions of the test nano-antimicrobial, and seeded with

**Figure 2.** *Disk-diffusion method with NPs.*

**Figure 3.** *Agar dilution method with NPs.*

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

#### *4.2.4 Time-kill method*

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 nano-antimicrobials.

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