Patent Description:
The skin is the body's largest organ and is part of the body's natural defense to microbial attack. The natural skin defense is made up of a multitude of components. For example, the skin's natural defense includes the skin's ability to be a physical barrier, the pH of the skin, the skin microbiome, lipids on the skin, chemical components of the skin, etc..

The skin's ability to provide a natural defense against microbial attack can be impacted by things with which it comes into contact during the day, like skin products. To this point, testing of the skin's natural defense has primarily focused on the microbiome. As noted, however, this is only one aspect of the skin's natural defense system. In addition, testing of the microbiome is often done by removing a skin sample, isolating the molecule of interest from the sample, and then quantifying the molecule of interest. Removing the target molecule from its natural environment, however, could have unintended consequences on the results and can also prevent an understanding of the impact to more than one component of the skin's natural defense and/or the potential impact of the skin's natural defense mechanism to the information being collected. In addition, the isolation of the target molecules from the original sample can be costly and time consuming.

<NPL> refers to Shampoo therapy with chlorhexidine-phytosphingosine as well as with the shampoo vehicle for reducing superficial bacteria on canine skin, which might be beneficial in the prevention of pyoderma.

<NPL> is related to Staphylococcal pyoderma that occurs commonly in atopic dogs and Adherent bacteria (Staphylococcus intermedius) were quantified by computerized image analysis on corneocytes collected from healthy or atopic dogs using double-sided adhesive tape.

<NPL> sets out optimizing an assay which could subsequently be used to compare the relative avidity of different S. aureus strains for equine corneocytes.

<NPL> relates to determine the antifungal activity in time in the stratum corneum of healthy volunteers after oral intake of R126638 at a daily dose of <NUM> or <NUM> for <NUM> week.

As such, there is a need for a new method to evaluate skin samples.

A method for testing a skin product's impact on the skin's ability to provide a natural defense against microbial attack is provided and comprises: a. applying a skin product to a target area; optionally, rinsing the skin product from the target area; b. obtaining a sample of skin cells from the target area, wherein the skin cells are obtained with the application and removal of an adhesive, wherein the adhesive comprises a tape strip, patch, or disc; c. applying a bacterial load, fungal load, viral load, or combination thereof, to the sample on the adhesive; and d. measuring a bacterial level, fungal level, viral level, or a combination thereof, of the sample.

Product testing before putting a product on the market is important. Testing of a product allows the developer to understand whether, for example, the product is performing as expected and whether there is consumer acceptance of the product. When products are made for application to the human body, the types of testing that can be done for a product can be limited and/or expensive as the need for human subjects and requirements for safety of those subjects are paramount.

Due to timing and cost constraints, it is advantageous to be able to assess efficacy of formulations in a quick and cost relevant way. Previously these quick and less costly tests included things like skin mimics or explanted human skin. A skin mimic is an artificial material made to resemble skin. However, while beneficial for initial screening of things like deposition of an ingredient onto the skin and/or lather during cleansing; it lacks the underlying biology of true skin. Thus, there can be a discrepancy in what is seen on a skin mimic and what is observed on actual human skin. Also, the inability of a skin mimic to be used for testing of any impact of a product on one or more of the skin's natural defense components or the ability to understand if the skin's natural defense components are influencing any testing are further detractors from the use of skin mimic.

Another substitute for direct testing on humans is the use of explant skin. Explant skin is skin that is removed during surgery and no longer needed. Explant skin has a few major downfalls. First, explant skin is only viable for a very short period of time making the timing of shipping, receiving, and testing a very confined window. Second, the supply of explant skin can be unpredictable. Moreover, skin explants are laborious to maintain even though they have only a short shelf life. All of these make skin explant less than ideal. Similar issues can be seen with a skin biopsy.

For direct testing on humans, product is applied directly on a target site of the skin. Depending on the type of testing being conducted, the application of the product may be followed with the inoculation of the test site with a bacteria and/or fungus. When these materials are inoculated on the skin, they are often covered with an occlusive patch to help prevent the unintended transfer of the inoculated material to other areas of the skin. There are inherent risks in inoculating a bacteria and/or fungus directly onto the skin of a user. Given this risk to the subject, there are strict requirements for these types of tests which are both costly and timely.

Once the target site is ready for sample collection, researchers can take a skin sample by, for example, rubbing a swab over the skin, using an adhesive to lift a skin sample, or placing a cylinder over the area of the skin and utilizing special fluids and mechanical agitation. While the collection procedures are not invasive like some of the ones above, the target material is trapped in the collection implement or in the liquids used during the collection process and often needs to be removed before analysis. This is regularly done by using an extraction procedure. Extraction can be accomplished utilizing rinses, extraction buffers, etc. The need to isolate the material from the collection implement or liquid requires additional time before analysis can be performed. There can also be a partial loss of target material due to incomplete or inefficient extraction methods.

The target materials then may need to be separated from the extraction materials before they can be analyzed requiring even more time. The addition of liquids to the samples and the loss of some of the materials during the extraction and/or isolation processes can create limitations regarding analytical sensitivity. Moreover, the target materials have been removed from their natural skin environment via the extraction process. Without the intact natural skin defense components, it is unclear how / if these elements are contributing to the results. This is especially true where the researcher is investigating antimicrobial efficacy.

The present inventors have discovered that a skin sample taken with an adhesive can be utilized directly on the adhesive. This means, for example, that the skin sample on the strip can be inoculated on the strip removing the need to inoculate directly on the skin of a panelist prior to taking the skin sample. In addition, with at least some testing methods, the sample on the strip can be used without the need to remove materials from the adhesive or to remove any backing material on the adhesive prior to testing. So, the skin sample on the adhesive may be placed, for example, into a testing apparatus or even cut into pieces and placed in a testing apparatus.

To show that a skin sample on an adhesive could be utilized to similar effect as a traditional direct panelist inoculation and extraction method, the results from a traditional residual efficacy test clinical ("traditional RET method") and a new method inoculating the skin sample on the adhesive were compared ("NET method"). The residual efficacy test looks at the antimicrobial effect of a bar soap chassis (bar soap) and a liquid hand soap chassis (LHS), neither of which contain a traditional antimicrobial active.

For the traditional RET method, a randomized, placebo controlled, and double-blind study is followed utilizing a residual efficacy protocol. Panelists are subjected to a <NUM>-<NUM> day wash out period where they are asked to avoid any products with anti-bacterial efficacy until the trial is complete. After the wash out period, each panelist visits a designated site where a forearm is washed with a target product <NUM> times at one hour intervals. During the wash, the volar surface of the forearm and the target product are wetted under running tap water maintained at <NUM> to <NUM>. The bar is rubbed in an up and down motion from wrist to elbow for <NUM> seconds. For the liquid hand soap, <NUM> of the hand soap is placed on the volar surface of the arm and spread evenly for <NUM> seconds. The lather is then rubbed on the forearm using the same motion for another <NUM> seconds. The arm is then rinsed for <NUM> seconds and patted dry with a paper towel.

After the third wash on the third day of washing, one area for germ inoculation is marked on the forearm. That area is inoculated with <NUM><NUM> to <NUM><NUM> colony forming units (CFU's) of Staphylococcus aureus (ATCC <NUM>) and occluded with an occlusive chamber patch. <NUM> hours after occlusion, the patch is removed, and the bacteria harvested using a cup scrub technique. Then, each test area is disinfected using <NUM>% ethanol and antibiotics immediately and reexamined <NUM> to <NUM> days later.

For a cup scrub method of harvesting, a rigid cylinder is placed against a target area of skin, here the portion of skin that was inoculated unless it is a control area. The cylinder is pressed against the skin with enough pressure to form a liquid tight seal. A recovery liquid is placed inside the cylinder in contact with the skin. The skin is then scrubbed with an appropriate implement, like a glass or rubber rod. The liquid is then removed from the cylinder and manipulated as appropriate for the desired testing. For example, the harvested bacteria are diluted, plated, and incubated for <NUM> hours, then counted (CFU). The American Standard Test Method for a sup scrub procedure is ASTM E1874-<NUM>. A detailed description of an exemplary residual efficacy test method is included in <NPL>).

As can be seen from <FIG>, the traditional RET results showed the bar soap chassis had no measurable residual efficacy against S. Aureus whereas the liquid hand soap chassis showed a significant innate defense property against S.

The same chassis were tested utilizing the new NET method of inoculating the skin sample directly on the adhesive. For the NET method, a skin sample was taken with an adhesive after a washing and rinsing protocol as described above for the traditional RET. The skin sample on the adhesive strip was then inoculated with S. Aureus, also as noted above for the traditional RET. The adhesive is placed on a trypticase soy agar plate with the skin sample side up. The inoculated sample is then reviewed for the growth of a bacteria and/or fungus over time. This is done utilizing an optical detection method, the Soleris® system. The results are then plotted on a graph showing the difference from baseline (before treatment) and from a sample taken <NUM> hours after treatment. As can be seen in <FIG>, the new method showed the same trend as the traditional method of <FIG>. Additional details the NET method are described below in the Skin Sample Testing section.

The new method is faster and cheaper due, at least in part, to the elimination of the need to inoculate bacteria and/or fungus directly on the skin. It also allows, if desired, the sample to be used on the adhesive without extraction. Moreover, the ability to inoculate the skin sample on the adhesive also presents a safety advantage. Previously, bacteria and/or fungal samples were placed directly on the skin of a panelist. By removing the need for application of the bacteria and/or fungus to the skin of the panelist, any risk with having to do this is removed. It also allows for testing against a wider range bacteria and/or fungus as some of these materials were not able to be placed directly on the skin. Moreover, it allows panelists to leave after removal of the skin sample with the adhesive where they previous would have been required to stay an additional <NUM> hours to complete the inoculation and collection period. This change could make it easier to recruit panelists and could require less payment to panelists since much less of their time is required.

The ability to inoculate samples directly on an adhesive also contributes to the robustness of the test. Normally, only <NUM> samples are taken from one arm test site due at least in part to the need to adequately space samples for inoculation and occlusive coverage. Inoculating the skin samples directly on the adhesive allows for more samples to be taken from a sampling site. This increases the robustness of the testing. The ability to take more samples from a site also allows for a higher throughput and, thus, faster cycling of testing for products.

Additional advantages come from the ability to test the sample directly on the adhesive. As noted above, there are many drawbacks from the procedures utilized to extract the skin sample or at least the target material from the adhesive. These are eliminated or at least minimized when the entire adhesive including the skin sample is used for testing.

Moreover, testing on a skin sample while still on the adhesive showed the skin sample is in a more natural state. For example, the skin corneocytes on the adhesive tape strips maintain the same pH as the skin surface sampled (see <FIG>). Thus, one can use an adhesive sampling method, like a tape strip, after application of a product to the skin to determine whether the application of the product impacted the pH of the treated area of skin. This allows for the determination of whether the product is impacting the skin's pH, and thus, impacting the skin's natural defense.

In addition to being able to understand the impact from a pH perspective, it is believed the skin sample lifted on an adhesive has a representative sample of at least some of the components of the skin's natural defense system as well. This allows for any product application to be evaluated in a more natural skin environment. This can be important, for example, when trying to understand whether a product impacts the skin's natural ability to protect itself or whether a product has an antimicrobial effect on skin. The test can also be used to understand the differences in natural skin defense from one person to another. Once a skin sample is taken and inoculated with a bacteria and/or fungus the growth rate of the bacteria and/or fungus can be measured. This will give the researcher an understand of the skin's natural ability to repel an invasion by the selected bacteria and/or fungus.

Going further, the researcher can apply a product to the same area of skin, take a sample of the skin with an adhesive, inoculate the sample with the same bacteria and/or fungus, and measure the growth of the bacteria and/or fungus. The results can then be compared to those of the original skin sample to determine the impact of the product on the skin's natural defense. The results can also be compared at different time points to understand the impact over time and over different numbers of applications. In addition, the results can be used to determine if a tested product provides any antimicrobial and/or antifungal benefit to the skin beyond that which is supplied by the skin's natural defense components. This process could also be used to compare the impacts from multiple products against one another as well as compare one or more of the products to an untreated area of skin.

As noted above, a skin sample may be taken with an adhesive. The adhesive is in the form of a strip, patch, or disc. The adhesive can have a backing. The adhesive can also be double sided to allow, for example, the adhesive with a skin sample to be stuck to another surface for testing. For example, the side of the adhesive not containing the skin sample could be adhered to a slide. Such adhesive or tape materials can include, without limitation, adhesive tapes such as D-SQUAME®, SEBUTAPE® (CuDerm Corporation, Dallas, Tex. ), BLENDERM®, SCOTCHTAPE® (<NUM>® Company, St. Paul, Minn. ), patchProtect™ skin patch, hydrogels such as HYPAN® (Hymedix International, Inc. , Dayton, N. ), and other types of materials with adhesive properties or appropriate stickiness, such as glues, gums, and resins, and combinations thereof.

The adhesive is placed on the target skin surface. This can be, for example, the forearm, shin, forehead, and/or back, but any desired skin surface can be sampled. The adhesive is left in place for a sufficient amount of time to allow for attachment to the target skin surface. This can be, for example <NUM> seconds, <NUM> seconds, <NUM> seconds, <NUM> second, <NUM> seconds, <NUM> seconds, etc. This can be adjusted as needed for a particular test. In addition, pressure can be applied to the adhesive while on the skin to allow for better and/or more uniform adhesion. This can be done, for example, by pushing down on the adhesive while in contact with the skin, utilizing a roller to press the adhesive onto the skin, etc..

Once a sufficient amount of time has passed, the adhesive is peeled away from the skin. This can be done, for example, with fingers, tweezers, tongs, etc. The adhesive can be peeled, for example, starting at one edge and gently pulling the adhesive back and away from the skin surface.

Once the adhesive has been removed from the skin, it contains a skin sample. The skin sample on the adhesive can be placed on another surface, for example, a slide or an agar plate. The portion of the adhesive with the skin sample can be applied face up or face down depending on the desire of the tester. For example, a skin sample taken on an adhesive that is double sided can be adhered to a glass slide so that the skin sample containing portion is face up and the slide can be viewed under a microscope. In another example, the adhesive is placed on a trypticase soy agar plate with the skin sample side up. In addition, a skin sample can be quantified by absorption spectrometry, like D-Squame® Scan 850A.

The skin sample can then be tested. For example, a pH of the skin sample can be taken. This can be done, for example, by pH flat surface electrodes being placed on the skin.

A sample can also be treated while on the adhesive. This can include, for example, inoculation of the skin sample with a bacteria, fungus, or combination thereof. The materials for inoculation can be chosen based on the type of antimicrobial and/or antifungal properties of interest. These can include, for example, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Candida albicans, Malassezia furfur, or combinations thereof.

The sample can be treated by placing the desired material onto the skin sample containing side of the adhesive. For example, an adhesive can be inoculated by placing an aliquot of Staphylococcus aureus onto the skin sample. The inoculated adhesive can then be further processed or evaluated. In the case of inoculation, the inoculated sample may be spread on the strip utilizing an inoculation loop, allowed to air dry, placed in a humidifier, etc. until time to collect data.

An inoculated sample may be reviewed for the growth of a bacteria and/or fungus over time. This can be done, for example, utilizing an optical detection method, like the Soleris® system. In this type of system, the skin sample containing adhesive is placed into a liquid broth. As the microorganisms grow and metabolize nutrients, the optical assay monitors pH and other biochemical reactions that are taking place. The reagents used change their spectral patterns as the microorganisms grow. The changes can be detected optically, photometrically, by the instrument, at predetermined time intervals.

For each sample, the detection time (DT) will be recorded by the instrument. The DT is inversely proportional to the number and/or metabolic health of bacteria carried over on the adhesive. The longer DTs are indicative of a strong antibacterial effect while shorter DTs suggest a weak or no antibacterial effect. One can load germs with known loads (CFUs) onto an adhesive to generate a correlation curve with DTs. Then, germ load (CFU) on test tapes can be calculated by the standard curves. Further, germ efficacy comparisons like LOG reduction and germ inhibition rate (%) can also be calculated.

An inoculated sample can also be evaluated utilizing isothermal microcalorimetry. This is a method for looking at real time data of biological processes. For isothermal microcalorimetry, the skin sample containing adhesive is inoculated with the desired material. The inoculated adhesive is then placed into an ampule. The ampule is sealed, for example, with a crimper, and the ampule can be wiped to remove any potential contaminates from the exterior of the ampule. The ampule is then placed in an apparatus which can keep it at a constant predetermined temperature, chosen as appropriate for the materials being tested. For microorganism testing, the principle used to select temperature can be to utilize the temperature which will produce the fastest growth rate for the targeted microorganism. The temperature can be set, for example, in the range of <NUM> to <NUM>. The set temperature can be, for example, <NUM>, <NUM>, or <NUM>. The apparatus is capable of measuring and recording vs. elapsed time the net rate of heat flow (µJ/sec = µW) to or from the specimen ampule, and the cumulative amount of heat (J) consumed or produced. From this information, microbial growth parameters within the sample can be determined. This measurement method can allow an increased sensitivity in quantification of commensal microorganisms and exogenous microorganisms present on the adhesive.

A skin sample on an adhesive can be utilized for product testing. A skin sample on an adhesive can be taken as noted above. The sample may be utilized to determine the health of the skin and/or skin microbiome prior to any product application. This can be done utilizing the above test methods or other known test methods for determining such properties. These properties can be used as a control in product testing. In addition, multiple products can be tested against one another and/or compared to a control with respect to a particular attribute.

When testing a product, the target area of skin for testing can first be subjected to a wash out period. This is where a subject or subjects are given a specific cleanser to use on the skin for a specified amount of time prior to beginning a study. A wash out is generally utilized so that the skin of each subject has been subjected to the same treatment prior to product testing and thus, effects from other products can be ruled out as contributing to the results. A wash out period can also include a non-washing period meaning a subject may be asked to not cleanse the skin for a certain period of time. Also, during a wash out period, a subject may be asked to not use any products on the skin other than what has been supplied for the test. A wash out period will typically be at least <NUM> day and can last up to <NUM> days.

Once any requested wash out period is complete, a baseline skin sample can be taken from a target area. After any desired baseline sample is taken, a subject will start using a target product. The product may be a rinse-off product like, for example, a bar soap, liquid hand soap, facial cleanser, body wash, etc. The product may also be a leave-on product like, for example, a hand sanitizer, facial moisturizer, body lotion, etc. For a rinse-off product the protocol can include, for example, a water temperature, water hardness, flow rate, target area of skin to be washed, handling of the product sample, how to apply the sample to the skin, how to wash the skin with the sample, how much of the product to use, how long to wash with the product, how long to rinse after use of the product, if/how to dry the treated are of the arm after rinsing, how many times to repeat the process, how long to wait after application before a skin sample is taken, or varying combinations thereof. Once this process is complete, a skin sample can be taken with an adhesive as described above. This sample can then be treated (or not) and utilized to determine the target attribute, as also described above.

For a leave-on product a protocol can include, for example, a target area of skin on which the product is to be applied, handling of the product sample, how to apply the sample to the skin, how much of the product to use, how long to apply the product to the skin, how many times to repeat the process, how long to wait after application for taking a skin sample, or varying combinations thereof. Once this process is complete, a skin sample can be taken with an adhesive as described above. This sample can then be treated (or not) and utilized to determine the target attribute, as also described above.

This protocol provides a non-invasive method for evaluating the residual antimicrobe efficacy of a rinse-off product e.g. liquid hand soap, body wash product, bar soap, etc. against designated bacteria and fungi. The method includes in vivo product application, skin stratum corneum sampling via tape stripping, germ challenge test on skin samples on the tape strip, and germ load quantification via Soleris® Detection time (DT).

The test organism choice is based on the intended use of the results, for example, for advertising claim support. Two examples of bacteria that can be tested include S. aureus (ATCC® <NUM>/ATCC® <NUM>) and E. coli (ATCC® <NUM>). Ideally, the strain is within <NUM> generations and has a routine identification at a frequency as makes sense for the bacteria and the test.

The test organism is prepared for the study. aureus and E. coli, if needed, refresh the test organism by streaking on a trypticase soy agar (TSA) plate and growing <NUM>-<NUM> hrs. On the second day, inoculate <NUM> colony of organism in a <NUM> tube containing <NUM> trypticase soy broth (TSB), and grow at <NUM>±<NUM> for <NUM> hours ±<NUM>. One the test day, dilute the above bacteria culture by <NUM>:<NUM> or other concentrations to new TSB (e.g. <NUM> culture to <NUM> TSB media). The test organism can also be prepared with nutrient-limited mediums or buffers (e.g. <NUM>:<NUM> diluted TSB, nutrient buffer, and/or glycerol). The test organism culture may be used within half an hour for inoculating tape strips for all samples tested.

A target demographic is selected for testing, for example healthy Chinese individuals ages <NUM>-<NUM> inclusive with an even age distribution between <NUM>-<NUM> and <NUM>-<NUM>. The subjects can be instructed to have a wash out period. For example, the wash out period can be to wash with a prescribed soap for days <NUM>-<NUM>, shower on day <NUM> with city water only, and to refrain from taking a shower on day <NUM>.

After the wash out period, a baseline skin sample may be taken on the target site prior to any product application. Then, the subjects are either instructed on how to do the following steps or a trained professional may do the steps on the subject. Utilizing tap water with a temperature of <NUM> +/- <NUM> and a water flow rate of <NUM>/min +/- <NUM>/min wet the volar surface of the forearm under the running water. If utilizing a solid soap, like a bar soap, briefly wet the soap under the running water. Then, rub the bar on the forearm, using an up-and-down motion from the wrist to the elbow, for <NUM> seconds. For liquid products, add <NUM> on forearm and spread evenly for <NUM> seconds. If necessary, set the bar down, continue to rub the lather on the forearm using the same up-and-down motion from the wrist to the elbow, for an additional <NUM> seconds. Rinse the forearm for <NUM> seconds by holding the arm under the running water. Do not rub the arm during rinsing. Remove the arm from the running water. Pat the arm dry with a paper towel without rubbing. The site is now ready for skin sampling with an adhesive. In the alternative, the above steps can be repeated so that the area is washed multiple times before sampling.

Once the target site, here the arm, is ready for sampling, which can be after the third wash on the third day of washing, a strip of adhesive tape is adhered to the skin site of interest avoiding folds. The skin site of interest can be marked in advance for consistency. To keep uniform pressure and reach optimal adhesive bond, a roller can be used to press the tape onto the skin surface (ex. twice on each site). Then, the tapes were peeled off from the skin and placed on the surface of a TSA agar plate with the skin sample side up. The tape strips can also be inoculated prior to being placed on the TSA agar plate.

Inoculate 10µl of the tested microorganism culture on the skin sample side of each tape strip. Spread evenly over the tape surface, for example, with a sterile inoculation loop or pipette tip. Use one inoculation loop or pipette tip for each tape strip, and discard inoculation loop or pipette tip after use. Allow the inoculum to visually dry on the surface of the tape strip (approximately ~<NUM>-<NUM> minutes).

The inoculated test strips can then be prepped for sampling measurement depending on what is intended to be measured. For example, if the inoculated test strips are going to be run through an optical analysis for detection of microbial growth and/or pH, then the inoculated tape strip residing on the TSA agar plate is placed into a humidified incubator at <NUM> and <NUM>% ± <NUM>% relative humidity until time of collection.

At each sampling time, aseptically transfer each tape into one NF-TVC (non-fermenting total viable count) vial for continuous monitoring for <NUM> hours to determine the detection times (DTs). Soleris parameters can be set as: Temperature <NUM>; Threshold: <NUM>; Shuteye: <NUM>; Skip <NUM>.

A tryptic soy broth (TSB) / Tween®<NUM> (polyoxyethylenated sorbitan ester) solution is made. Tween®<NUM> is added to generate a <NUM>% solution. If only the commensals (bacteria already present on skin) are of interest, the inoculate is applied to the strip and carefully spread using, for example, an inoculation loop to cover the strip. Various volumes can be used, for example, between <NUM> and <NUM>µL.

aureus and/or E. coli is desired as part of testing on the strip, first dilutions should occur to generate the correct strength solution. aureus and/or E. coli is diluted <NUM>:<NUM> into TSB, the first dilution generates a solution of <NUM><NUM> cfu. This solution is then spread onto the strips, as above. The strips are then placed on a TSA agar plate with the skin sample side up. The TSA agar plates with strips on the surface were left to dry in a laminar flow cabinet, generally between <NUM> minutes and <NUM> hours depending on the amount of solution applied to each strip.

Once the strip is dry it can be tested. Agar from a TSA plate is cut into small cubes and <NUM> or <NUM> of these are placed into the bottom of the ampule that will contain a strip. The agar is used as a source of moisture within the ampule, as without it the bacteria may not grow. The strip is then put into the ampule. If the full strip is being used it can be carefully pushed into the ampule using tweezers, a funnel shape is often the end shape of the strip. If a thin section of the strip is being used, it can be dropped into the neck of the ampule using tweezers. The ampule is sealed with a crimper and the ampule is wiped with a paper towel to remove any potential moisture or oils on the external surface. The crimped ampule is loaded into an isothermal microcalorimetry machine (IMC).

The isothermal microcalorimetry machine is set to the temperature which produces the fastest growth rate for the target organism, for example, <NUM>. A baseline reading is taken before the ampules are placed into the IMC. The IMC performs an equilibration phase before recording data. A baseline is recorded at the end of the run. The data is collected. The heat flow of the ampules can be plotted and used to determine bacterial growth. Runs can take <NUM> or more days.

Claim 1:
A method for testing a skin product's impact on the skin's ability to provide a natural defense against microbial attack, comprising:
a. applying a skin product to a target area; optionally, rinsing the skin product from the target area;
b. obtaining a sample of skin cells from the target area, wherein the skin cells are obtained with the application and removal of an adhesive, wherein the adhesive comprises a tape strip, patch, or disc;
c. applying a bacterial load, fungal load, viral load, or combination thereof, to the sample on the adhesive; and
d. measuring a bacterial level, fungal level, viral level, or a combination thereof, of the sample.