*4.1.1 Disinfectants*

When evaluating chemical disinfectants, bacterial endospores are considered the microbial life-form hardest to kill, followed in descendent order by mycobacteria, bacteria in vegetative form, and viruses.

In the United States, the Food and Drug Administration (FDA) regulates chemical sterilants and high level disinfectants (HLD) that are used to reprocess medical instruments [59]. The AOAC International sporicidal and tuberculocidal tests are the accepted methods for evaluation. For a liquid chemical sterilant, the FDA standard tolerates no failures in the AOAC sporicidal test 966.04, and accepts no survivors in simulated-use testing with a challenge inoculum of six logs of spores. The FDA defines HLD as sterilants used under the same contact conditions but for only the contact time needed to reduce *Mycobacterium bovis* in 6 log10 in the tuberculocidal test 965.12 [60]. Moreover, to be approved, the disinfectants should be subjected to worse case scenarios, such as the presence of organic or inorganic contamination, and under simulated use conditions.

In Europe, the CEN/TC 216 technical committee produces current and future disinfectant testing standards [61]. Standard EN-14885-2006 indicates test methods to be used to substantiate claims for products intended for instrument disinfection [62], including mycobacterial/tuberculocidal (EN-14348, EN 14563), bactericidal (EN-13727, EN-14561) and fungicidal (EN-13624, EN-14562) activity tests but the terms "sterility, sterile, sterilization, sterilant" fall outside the scope of CEN/TC 216.

In the US, high, intermediate and low level disinfectants, are regulated by the Environment Protection Agency (except HLD intended to reprocess medical instruments, which fall under FDA's jurisdiction). Intermediate level

#### *Nanoemulsions - Properties, Fabrications and Applications*

disinfectants must be tuberculocidal. Products effective only against vegetative bacteria and viruses are regarded as low level disinfectants [63].

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 living tissues.

#### *4.1.2 Antiseptics*

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, antiseptics are regulated by the FDA.

#### **4.2 Antimicrobial activity tests**

A relevant test microorganism is chosen: preferably a strain from the American 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 differentiate between kill and inhibition of growth.

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 antimicrobial activity test, such as a disc diffusion test.

#### *4.2.1 Disk-diffusion method*

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

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 will be examined to assess inhibition rings around the disc.

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 center (**Figure 2**).

#### *4.2.2 Agar dilution method*

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 is plated into Petri dishes. An aliquot containing 104 cfu of the test microorganism

**23**

**Figure 3.**

*Agar dilution method with NPs.*

**Figure 2.**

*Disk-diffusion method with NPs.*

*In vitro Antimicrobial Activity Evaluation of Metal Oxide Nanoparticles*

is placed onto the agar's surface, and incubated overnight. Then, the plates will be examined for growth to determine the last effective concentration to inhibit growth

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,

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

*DOI: http://dx.doi.org/10.5772/intechopen.84369*

(**Figure 3**).

*4.2.3 Broth dilution method*

and can be automated.

*In vitro Antimicrobial Activity Evaluation of Metal Oxide Nanoparticles DOI: http://dx.doi.org/10.5772/intechopen.84369*

is placed onto the agar's surface, and incubated overnight. Then, the plates will be examined for growth to determine the last effective concentration to inhibit growth (**Figure 3**).
