Faster Evaluation of Contaminated Surfaces for Mould Inspections by Tape Sampling

Taking a tape-lift sample is one of the main practices used by indoor environmental quality investigators for detecting whether mould structures (for example, spores and hyphae) have either settled onto or colonized the surface. Despite the popularity of the method, there can be signi ﬁ cant inconsistency in how tape lifts are collected and evaluated. The common ASTM standard D7910-14: Practice for the Collection of Fungal Material from Surfaces by Tape Lift, describes the correct way to collect a tape-lift sample. Using ASTM D7658-17: Standard Test Method for Direct Microscopy of Fungal Structures from Tape, semi-quantitative results in percentage of infested area in a scale from 0 up to 5 are available only. In case histories or for mould removal control, the total cell count is needed. This cannot be realized by the ASTM method. Therefore, an innovative method is asked to combine the quickness of taping and the precision of total cell count. Our research team developed two methods to quickly and fully quantify the tape samples. Regarding the assessment criteria, the user can decide to operate with the 3-LINE method to achieve the highest precision or use the faster 3-STEP method for even better results. Therefore, an innovative method is asked to combine the quickness of taping and the precision of total cell count. The aim of the work is to develop two strategies to quickly and comprehensive quantify the tape samples. ABSTRACT


INTRODUCTION
In case of moist damage and for measuring the success of a mould remediation, a diagnosing method is needed for a fast, simple, and yet an accurate determination of the mycelia growth and contamination by spores, hyphae, and other fungal structures [1]. The method should determine the concentration of cells and cell structures to evaluate case histories or whether the biomass has been suffi ciently reduced.
Cultivating samples can deliver results with suffi cient precision concerning diff erent species and genus. However, this process also has its limitations, as it requires a lengthy standard incubation and the only organisms that will grow are the ones that are ready to germinate. These factors are further infl uenced by the types of culture medium and the incubation environment.
The question is: how to analyze the mould biomass within a shorter timeframe and independently of cultivation and cell vitality?. Using the standard method, ASTM standard D7658-17, it is not possible to get a result conforming to the WTA or other guidelines demanding a total cell count value.
Therefore, the quantifying microscopic analysis of surface tape samples is brought into a standardization process [3].
However, in order to obtain reproducible results for the evaluation according to table 1, an appropriate approach and standardization of the method is required. For the past two years, the team associated with this paper has been working on the validation of the adhesive fi lm method. The results show several methods that allow a reliable assessment of adhesive fi lms [4].
The starting point of the investigation was a defi nition of the task ahead that allows us to derive diff erent procedures having the necessary quality [5]. In this paper we want to examine that this task can be carried out with a microscopy method using adhesive tape preparation. Hence the aim of the work is to develop two strategies to quickly and comprehensive quantify the tape samples.

Qualitative and quantitative detection of biomass
Basically, adhesive fi lm analysis concerns the detection of biomass. The detection is diff erentiated into quantify and qualify the biomass. The fi rst option means to determine an amount or a partial amount, using reference basis e.g. cm 2 .
The second option is the detection of the composition, but also of the condition or properties (distinctions, infestation/ contamination/ intact, defective). The two diff erent types of analysis enabling equally wide-ranging grades of compatible measurement. The information from these methods can range from yes/no to type of species and number of species per cm 2 ( Figure 1).
To evaluate a new method of analysis, 37 commercial tape samples from case histories were counted completely in 24,000 fi elds of view, and later in 100, 300 and 900 fi elds of view by 3 analysts of two independent laboratories. In the end, up to 200 tape sample enumerations were used for a statistical analysis. To evaluate the quality of the method it was analysed fi rst the deviation of cell counts by one analyst in 24,000 fi elds of view in comparison to 900, 300 and 100 at least. In a second evaluation the deviation of cell counts by all analysts were compared. As statistical method for evaluation the excellence the least squares linear regression was used.
In applying the quantifying adhesive tape analysis, the aim is to record the biomass per cm 2 . Therefore it was defi ned a procedure that yields to comparable results, using the same requirements regardless of the type of microscope. For evaluation and statistical analysis of this method fi rst it had no relevance of counting mould growing or a contamination.
The analysts had to count it as one hit without a qualifying comment. Further, the qualitative method was simplifi ed, and the countable fractions were specifi ed for counting typical spores and fractured hyphae.
In addition, a cancellation criterion was defi ned to avoid 'meaningless counting' especially when there are clearly interpretable results. One must always remember, especially in the case of 'non-microscopic' readers, that if a semiquantitative adhesive tape is clearly evaluable, trying to achieve a full quantifi cation is clearly a waste of time. The problem could be solved with microtome sections, but this is rarely feasible in practice in case of a mould investigation.
The invention of the adhesive fi lm preparations solved this problem, at least in part. In this context, examining an adhesive tape is not a 'real' direct microscopy [5].
The adhesive fi lm covers the biomass when the tape is pressed onto the substrate and then removed again. In this way, the examined biomass can be assigned to a sampling location.
Limitations can arise due to the reproducibility of the biomass fi xed to the fi lm. When it is compared with the total biomass, which may also be present in deeper layers, this could lead to inaccurate conclusions. Depending on the task, a diff erent biomass recording method may have to be used [6].  The samples were evaluated by two methods: the 3-LINE process and the 3-STAGE method. In the 3-LINE process, the adhesive tape was counted in three horizontal rows, each having a length of over 3cm. In the 3-STAGE process, the adhesive fi lm was also counted in three 3 rows. However, with this method, the 3cm was divided among the 3 rows

Cancellation criteria
For a fast analysis, especially in the 3-line process,

RESULTS AND DISCUSSION
The evaluation of the analytical results has clearly shown how well the results of the individual test counters are consistent with each other and how well the values correspond with a fully counted sample (Figure 4).
Furthermore, it was proved that even the simplifi ed 3-stage method could achieve a high level of reproducibility. The statistics show that the methodology, as a whole, is more than 80% suitable. In the meantime, with the counting routines of the participating laboratories having been adjusted, it was possible to achieve a coeffi cient of determination R 2 of 0.9.
It is important to have a high recovery rate when the adhesive tape samples have a low surface distribution, such as the remediation control of inventory or archaeological samples [7]. The recovery rate for cell numbers below 500 spores per cm 2 is 70% in the LINE process. In the 3-STAGE method, the result is slightly lower at 65%.
The results will be comparable and highly accurate, if the same procedure is adopted for counting, regardless of the equipment used. Further, using a microscope of 100x magnitude or 600x is a matter of minor importance only.
The most important factor is the reference basis of cm 2 . This is best achieved through looking at the linear extension of the sample. The measurement width depends on the device and determines, therefore, the calculation factor.
The 3-LINE method should be used for greater accuracy.
Here, however, the user has the option of defi ning the termination criteria, if the result in the interpretation is unlikely to ensure any further accuracy. The 3-STEP method off ers a fast and accurate analysis, especially for the determination of damages and the classifi cation of the biomass according to the German Mould Guideline UBA [6].
The investigation has shown that that high precision   Table 2 presents the recommended target value of mould contamination after a precision cleaning of indoor surfaces.

Removal control
As seen, the diff erentiation between porous surfaces like render or concrete and smooth surfaces like glass or metal yields two values regarding to the eff ect of the cleaning.
There no need to diff erentiate between spores and hyphae, which are counting as one.

Case histories
The surface assessment in case histories diff ers to    which should be tested. This is described in [8,9] but not in the interest of this work. In practice the analyst needs to decide, what cell count is expected to choose the right method (more or less counting areas). The analyst can also start with the enhanced 3-LINE method and break up using the Cancelation criteria. If the Analyst needs information about the genius of the species this method will be not the right one at once. Therefore, the cultivation methods are the better choice. In lab practice of the authors booth methods are used as an inhouse standard.

DECLARATIONS
Ethics approval and consent to participate. We confi rm that any aspect of the work covered in this manuscript has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript.
No confl ict of interest exists. We wish to confi rm that there are no known confl icts of interest associated with this publication and there has been no signifi cant fi nancial support for this work that could have infl uenced its outcome.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.