A fast, practical and reproducible procedure for the standardization of the cell density of an Aspergillus suspension

Recent outbreaks of infection caused by Aspergillus, particularly by Aspergillus fumigatus among neutropenic and critical care patients (Fridkin & Jarvis, 1996), led the scientific community to perform comprehensive research on this topic, and particularly on antifungal susceptibility testing (Espinel-Ingroff et al., 1995, 2001; Espinel-Ingroff, 2001a, b). In an attempt to enhance inter- and intra-laboratorial reproducibility, the National Committee for Clinical Laboratory Standards published the Document M38-A (NCCLS, 2002), which standardizes susceptibility testing for several fungal species, including members of the genus Aspergillus.

Inoculum concentration may significantly affect the determination of MIC and minimal fungicidal concentration values for filamentous fungi (Gehrt et al., 1995; Manavathu et al., 1999), as well as germination studies of Aspergillus sp. (Manavathu et al., 1999; Araujo & Rodrigues, 2004). Accordingly, both the NCCLS and the European Committee for Antifungal Susceptibility Testing (EUCAST-AFST) recommend that inoculum standardization should be based on determination of cell density by spectrometry (Espinel-Ingroff & Kerkering, 1991; NCCLS, 2002; Rodríguez-Tudela et al., 2001). Nevertheless, colour, shape and size of spores are morphological characteristics that can affect spectrometric readings of optical density (Petrikkou et al., 2001).

Taking into account the variance of spectrometric readings taken from different species of Aspergillus, Petrikkou et al. (2001) suggested that spectrometry might be used for inoculum standardization, provided that each species was standardized separately, resulting in a time-consuming methodology that makes cell-counting with a haemocytometer a better alternative.

The objective of the present work was to develop and standardize a fast and reliable procedure, based on photometric readings, for the standardization of an Aspergillus inoculum, as an alternative to the classical, haemocytometer-based counting method and devoid of the drawback of observer dependency.

Organisms and growth conditions.
Clinical isolates of A. fumigatus (39 strains), Aspergillus flavus (20 strains) and Aspergillus niger (15 strains), belonging to the collection of the Department of Microbiology of Porto Faculty of Medicine, were used. Organisms were cultivated in Sabouraud agar slants (Difco) at room temperature (20 °C) for 5 or 11 days. Spores were harvested by flooding the agar surface with PBS (Sigma) and then filtered and suspended in PBS with 0.01 % Tween 80 (Difco), in serial concentrations. Spore suspensions were stored at 4 °C for up to 5 days.

Cell-counting.
Spore concentration of different spore suspensions was evaluated by using a Neubauer's chamber (haemocytometer), according to the classical procedure.

Optical readings.
Spore suspensions were submitted to spectrometric readings with a Shimadzu UV-160A spectrometer and to photometric readings with a Densimat photometer (bioMérieux). The suspensions were vortexed before reading. Spectrometric readings were taken at 550 and 620 nm; photometric readings were taken at 550 nm (as established by the manufacturer). All determinations were perfomed in triplicate and each value was entered individually for data analysis.

Statistical analysis.
The program SPSS 11.5 was used for data elaboration and analysis. The Wilcoxon signed-rank test (Bradford Hill, 1991) and Student's t-test for paired samples were used for statistical analysis. Data were compared at a significance level of 0.05.

No significant difference was noted when comparing spectrometric readings at 550 and 620 nm, for all tested strains of A. fumigatus (Fig. 1). Similar results were found with the other two Aspergillus species (data not shown). A correlation between spectrometric readings and cell-counting with a haemocytometer was established for each of the three tested species of Aspergillus (Fig. 2). Within each species, no significant differences were noticed.

(12K): Fig. 1. Correspondence between spectrometric readings at 550 and 620 nm, for all A. fumigatus strains.

(21K): Fig. 2. Correspondence between spectrometric readings at 550 nm and spore-counting: (a) A. fumigatus (39 strains); (b) A. flavus (20 strains); (c) A. niger (15 strains).

A correlation between spectrometric readings (Shimadzu) and the MacFarland density scale, evaluated by the Densimat, is shown in Fig. 3.

(19K): Fig. 3. Correspondence between spectrometric readings at 550 nm and Densimat readings: (a) A. fumigatus (39 strains); (b) A. flavus (20 strains); (c) A. niger (15 strains).

A correlation between the MacFarland density scale and cell-counting was established for each of the different species of Aspergillus tested (Fig. 4). By using the correlation found with each of the three species tested, it was possible to define the upper and lower limits of evaluation of the concentration of a spore suspension with the Densimat, as shown in Table 1.

(19K): Fig. 4. Correspondence between Densimat readings and spore-counting: (a) A. fumigatus (39 strains); (b) A. flavus (20 strains); (c) A. niger (15 strains).

Table 1. Limits of evaluation of the cell concentration (cells ml1) of an Aspergillus spore suspension, by photometric readings with a Densimat photometer

No significant difference was found between suspensions of spores that were 5 or 11 days old with either spectrometric or photometric readings (data not shown).

It is a general consensus that antifungal susceptibility testing of moulds represents an area of clinical interest. With resistance demonstrated in A. fumigatus (Denning et al., 1997; Mosquera & Denning, 2002), reproducible and standardized susceptibility methods are urgently needed in order to obtain meaningful data, particularly from a clinical perspective. Inoculum standardization represents one of the main pitfalls in antifungal susceptibility testing.

Previous reports emphasized that spectrometry could be used for evaluation of the cell density of an Aspergillus suspension in cases where each species had been standardized separately (Petrikkou et al., 2001), a fact that was confirmed by our results. Good correlation coefficients were found between Densimat and spectrometric readings, particularly in the cases of A. fumigatus and A. flavus.

Futhermore, by defining a correlation between cell-counting and the MacFarland scale by using the Densimat for all tested species of Aspergillus, a significant improvement was achieved from a technical perspective. Spectrometry involves the use of a very expensive and sturdy apparatus, which is unavailable in most clinical laboratories. Preparation of a large number of spore suspensions, e.g. for susceptibility testing, as will surely be needed in the near future (according to the most recent epidemiological findings of a steady increase of invasive fungal infections), makes cell-counting also a much too time-consuming and observer-dependent procedure an impractical method for clinical laboratories.

Densimat can replace, with considerable advantage, both spectrometric determination and cell-counting in the preparation of a spore suspension with a precise inoculum size, to be used in antifungal susceptibility testing, as well as in other in vitro and in vivo studies. Nevertheless, uncommon species or atypical strains of Aspergillus, particularly with uncharacteristic pigmentation, may require specific standardization. It is important to emphasize that the limits of resolution of Densimat include the range of cell concentration values that are needed for microbial susceptibility testing, as well as for other laboratorial studies (Gehrt et al., 1995; Bouchara et al., 1997; Wasylnka et al., 2001; taudohar et al., 2002; Wasylnka & Moore, 2002). By reducing inter- and intra-laboratorial variation, this methodology could play an important role in inoculum standardization.

The portability, easiness-of-use and reproducibility of Densimat, together with its correlation with cell-counting, make it a valuable tool for use in clinical mycology laboratories.