Introduction

In this era of fusion and intra and inter cultural exchange, science is no longer streamlined into respective disciplines, interdisciplinary research has emerged tall and strong. Nanotechnology is an area that embraces diverse researchers from varied disciplines. Nanotechnology has made progress in leaps and bounds in the recent decade; there was certainly ‘Plenty of Room at the Bottom’ [1]. Today, physicist, biologist, doctors, pharmacists, chemists, engineers have all harnessed more than imagined from the nano resources; this is still an unsaturated reservoir, with scope for more [2,3,4,5,6,7,8,9]. With the discovery of newer nanoparticles, newer visions are realized and higher dreams envisioned. Jack Uldrich and Deb Newberry’s, ‘the next big thing is really small’, summarizes the expectation and enthusiasm from these nano beings [10].

Nanotechnology is an ever expanding, never saturating field making outstanding impact in all spheres of human life [11]. Nanomaterials can be synthesized by different methods including chemical, physical, and biological methods. These synthesis methods result in environmental contaminations, since the procedures involved in the synthesis of nanomaterials generate a large amount of hazardous byproducts [12]. Thus, there arose a need for “green nanotechnology” that includes clean, safe and environmentally friendly methods of nanoparticle synthesis [13, 14]. The biological methods of green synthesis of nanoparticles include synthesis of nanomaterials using extracts from plant, bacteria and fungi [15]. Nanomaterials synthesized via green synthesis have been well accomplished for their significant bactericidal, parasiticidal, insecticidal, mosquitocidal activities apart from their antibacterial activities [16,17,18,19,20,21,22]. Across decades the unseen bugs have been efficiently handled by the miniature particles, antimicrobial therapy offered by nanotechnology is of course one of the largest offers. The antibacterial [23, 24], antifungal [25], antiviral [26] and antialgal [27,28,29,30,31] activities of nanoparticles are well documented. While reviewing the statistics of manuscripts published in this area of research, we could see from pubmed that there is a lot of interest in this area (Fig. 1). Nanoparticle interactions and their inhibitory influence on microbes have a publication record in the order of antibacterial (2951 pubmed hits) > antifungal (795) > antiviral (257) > antialgal (13) research publications, with obvious increase with time.

Fig. 1
figure 1

Histogram showing the statistics of research published in nanomaterial based antimicrobial activity based a pubmed database search

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These are the days when biologist, physicist, chemists, engineers have expanded their territories, embracing cross culture research. However when fusion occurs, although encouraging results appear, there is scope for stepping aside from the hard core fundamentals of the embraced discipline. Citing an example, for a botanist to work on a live animal specimen, to conduct an animal study, is a huge challenge, since, one skipped bachelors, masters in zoology, where all the fundamentals are taught and directly he arrives at researching. A non-biologist researching with bacteria, would completely underestimate its potentials and do’s and dont’s. A biologist working with nanomaterials will on the other hand underestimate its destructive abilities. Numerous misconceptions, myths, pre notions, erratum can occur as we traverse cross disciplines. We highlight one such crucial flaw occurring during microbiology and nanotechnology integrated testing in the current communication.

Evaluating the antimicrobial activity of a nanomaterial is usually done following standard microbiological techniques. The most widely used method is the disc diffusion and well diffusion methods; the other method is the plate count method, or the total viable count method. The disc diffusion method is carried out by dipping a sterile paper disc into the nanoparticle suspension and then placing it on a plate inoculated with microbes, an inhibition zone appears around the disc be it the nanomaterial is bactericidal. The measure of the inhibition zone is a measure of the antibacterial activity of the test material. The well diffusion method is almost similar, except for the fact that instead of a disc, a well is dug into an inoculated agar plate using a cork borer. The nanomaterial is loaded into these wells and the inhibition studied based on the inhibition zone. Almost 50% of the papers demonstrating microbicidal activity of nanomaterials use either of these two methods. The reason is for the fact, that these methods require less microbiological expertise, required less microbial training and handling knowledge and required simple protocols and preparation time. As microbiologists working on nanotechnology integrated research, we have always been intrigued with the fact: how can the nanoparticles possibly interact with the bacteria in the agar. If the nanoparticles are diffusible compounds like the antibiotics and solvents, then it makes sense that they would diffuse into the porous agar base. Here the case is different, we just load the nanoparticles on a paper disc or into a well and how can we study its effect on the bacteria embedded in the agar? Is this adequate? Disc and well diffusion assays were originally designed for diffusible compounds like antibiotics, organic extracts that needed no interaction strategies. Are we overstepping the original design and extrapolating it for a different system which operates under a different strategy?

Experimental Methods

In a recent set of routine experiments, we have confirmed this methodology based variation using three antibacterial assays. The nanoparticle synthesis flow with procedures detailed in our previous publications as follows: C dots [32], curcumin NPs [33], graphene [34], CdS QDs [35], Ag NPs [36]. TiO2 NPs [37] and ZnO NPs [38]. The disc assay [39,40,41], well diffusion assay [42, 43] and plate count methods [44,45,46] were carried out following standard microbiological methods.

Results and Discussion

The results from the plate count method following incubation with various nanomaterials are shown in Fig. 2a–g. These results were correlated with disc and well assays. As observed from Fig. 3, the disc diffusion and well diffusion results were similar, but both these were very different from the plate count results (Fig. 2). Following the plate count method allows for the nanomaterial and the bacteria/fungi to interact with each other in a growth medium, after a particular interaction time, the bacteria/fungi are grown on agar plates. The survivors of the nanomaterial damage (resistant cells) grow on the plates following incubation to form colonies. Each colony forming unit (cfu) represents one viable bacterium/fungi. The final counts are expressed as cfu/mL depicting the number of survivors. Even for nanomaterials that showed no activity in the well diffusion and disc diffusion methods, marked activity was exhibited whilst using the plate count method. The sensitivity of the disc diffusion and well assays was very limited, only those that showed very high activity registered an inhibition zone. Moreover, only those that was capable of diffusion, showed positive responses.

Fig. 2
figure 2

Results of plate count method showing antimicrobial activity of different nanoparticles

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Fig. 3
figure 3

Comparison of disc, well diffusion methods and plate count methods of antibacterial activity of various nanomaterials against E. coli

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The mechanism behind antibacterial activity of nanomaterials is well worked out [47,48,49,50,51,52,53,54,55,56] and the interactions of nanomaterials with the microbial cells, differs within organism, genus and even at species level. Based on the ability of the nanomaterials to be able to control a wider group of bacteria, they are classified to possess broad spectrum or narrow spectrum antibacterial activity. In all these interactions between nanomaterials and microbes, mode of action is though contact of the nanomaterial with the bacterial systems to result in damage of cell wall, or cell organelles or degradation of protective slime layer or nucleic acid damage.

Fundamental Flaw in the Testing Method

As observed from our results a wide difference in results within the same nanoparticles effect on the same bacteria was observed. Thinking about the plausible reason for this difference, the most logical existence of a physical barrier is the answer. How could nanopacticle interaction with bacteria and fungi be studied without allowing them to interact? How can effect nanoparticles on microbes be studied without allowing the nanoparticles to act on the microbial cells? Loading the nanomaterial on a disc and placing the bacteria on agar, putting up a huge physical barrier between the two, how can one orchestrate the silver nanomaterial interactions which can bring about cell membrane oxidation or the titanium nanoparticles effect which can cause cell wall lysis and the quantum dot effect that can enter the cell and lead to a cascade of destructive effects? (Fig.  4).

Fig. 4
figure 4

Schematic representation demonstrating the physical barrier between the microbes and nanoparticles while using a disc/well diffusion method and b plate count method

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This is owing to the gap in the understanding of the microbes by non-biologists from chemistry, physics, engineering backgrounds working on real time living specimens. We feel many a nanomaterial which have true potential have been given up or laid off, due to testing using the inappropriate assay. This article calls the audience to check on their assay method and adopt the more realistic and logical plate count method for studying nanomaterial/microbial interactions. We do not say that plate count methods are not practiced or that well and disc methods are not useful. We emphasize the need that all three methods or at least well.disc and plate count methods are tested before conclusive decisions are made. The plate count method has its own problems as well, like the well known ‘plate count anomaly’ reported. Where the number of bacteria which grow may not exactly represent the actual number of bacteria in the medium and also the bacteria that suffered damages, may revive when plated on the nutrient rich agar environment (hence not reflecting the degree of damage) [57, 58]. Hence, it is also suggested that a direct microscopy based live/dead cell fluorescence imaging based quantification could also go hand in hand with the plate counts to doubly cross check the results [57, 58]. Whatever be the case, the actual concern is the need for allowing the nanoparticles to interact with the microbes, before quantifying the degree of damage.

Conclusion

Dealing with solution/compounds or chemicals is absolutely different from dealing with living cells/microbes. Interdisciplinary scientists need to never lose foresight of the fundamentals of the systems they are dealing with. This curtain raiser calls for an alert among those studying antimicrobial activity of nanoparticles to adopt the right procedure that will allow for maximizing the action of nanoparticles on microbes, in order to see accurate and reliable results.