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The entomopathogenic fungus Neozygites parvispora (Entomophthorales: Zygomycetes) was recently cultivated for the first time in a medium containing insect hemolymph, fetal bovine serum (FBS), and Grace's insect cell culture medium (5). This fungus grows in liquid culture as irregularly shaped, discrete hyphal bodies. Since hemolymph is difficult to obtain and available in only limited amounts, cultivation is limited to small volumes. The identification of growth factors and the simplification of this complex medium required the determination of cell densities in many samples. We were therefore looking for an accurate and rapid method for the determination of cell densities in small culture volumes. Methods commonly used for this purpose are hemocytometer counting, determination of protein content, wet or dry weight measurement, and determination of the optical density (OD). While hemocytometer counting and protein determination have the disadvantage of being time-consuming and tedious, the measurement of wet or even dry weight is not practicable for very small culture volumes. The measurement of the OD works well if cell shapes are regular, as for example in yeasts, but in our case it is problematic because of the irregular cell shapes and dimensions of N. parvispora.

An alternative method that was originally developed as a rapid assay for growth and survival of mammalian lymphoma cells is based on the transformation and colorimetric quantification of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (8). The respiratory chain (11) and other electron transport systems (7) reduce MTT and other tetrazolium salts and thereby form non-water-soluble violet formazan crystals within the cell (1). The amount of these crystals can be determined spectrophotometrically and serves as an estimate for the number of mitochondria and hence the number of living cells in the sample (2). These features can be taken advantage of in cytotoxicity or cell proliferation assays, which are widely used in immunology, toxicology, and cellular biology (10).

The MTT assay was performed as first described by Mosmann (8) with the modifications suggested by Denizot and Lang (2). Additionally, some adjustments for the use of fungi had to be made. An MTT stock solution (5 mg of MTT/ml of distilled water) was filter sterilized and kept for no more than 2 weeks at 4°C. To start the coloring reaction, stock solution was added to growing cultures (final concentration, 0.5 mg/ml). The mixture was incubated for 16 h on a shaker (160 rpm at 20°C). Cells were pelleted by centrifugation in Eppendorf tubes (15,000 × g, 5 min), the medium was removed, 500 µl of 1-propanol was added to the cells, and the tubes were vortexed. Lysed cells and debris were pelleted (15,000 × g, 5 min), and 100 µl of the supernatant was transferred into a 96-well plate. The OD was measured with a spectrophotometer (SPECTRAmax PLUS, Molecular Devices) at 560 nm, with 690 nm as a reference read-out. A blank with propanol alone was measured and subtracted from all values. Measurement of a dilution series showed that the linear range extended up to an OD of 2. For all experiments, an isolate of N. parvispora was grown on a shaker (160 rpm at 20°C) in 48-well microtiter plates with 500 µl of medium per well. The inoculum consisted of 2.5 × 10⁴ cells per well. The standard liquid medium consisted of Grace's insect cell culture medium, hemolymph, and FBS as described by Grundschober et al. (5). In a first experiment, the fungus was grown for 1 week to a density of 7 × 10⁶ cells/ml of culture volume (cells in stationary phase) and was then incubated with MTT for different time periods. The results showed that formazan production was saturated after 16 h, and therefore this was chosen as the incubation period for all further experiments.

The method was validated and compared with conventional hemocytometer counting. A culture of N. parvispora was grown and diluted with fresh medium to yield cell densities between 8.45 × 10⁶ and 8.25 × 10⁴ cells/ml of culture. The cell densities in the different dilutions were determined as the mean value of four hemocytometer counts (about 300 cells counted except for the lowest cell densities). From each dilution, five samples were measured with the MTT method. Comparison of the results obtained with the two methods showed a linear relationship between the OD measurements obtained by the MTT method and by hemocytometer counting (Fig. 1). Additionally, the variability of the values determined with the MTT method was smaller.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Comparison of the results from the MTT method with the hemocytometer counts. A dense culture of N. parvispora was diluted to cell densities between 8.45 × 106 and 8.25 × 104 cells/ml of culture volume. The samples were then incubated with MTT, and the absorbance (OD) was measured after 16 h. The results of both methods show a linear relationship (R2 = 0.996; n = 8; P < 0.0001). Each data point represents the mean and standard error calculated from five MTT measurements and four hemocytometer counts.

For further evaluation of the MTT method, N. parvispora was grown for 7 days in media with various hemolymph concentrations and different concentrations of bovine serum albumin (BSA) as a substitute for FBS. In this manner, different cell densities were obtained and the MTT method could be tested under growth-limiting conditions. The cell densities of four independent replicates were determined with a hemocytometer (four counts per replicate, 500 to 1,000 cells counted except for the lowest cell densities) and compared with the MTT measurement in these four replicates. The two methods again showed a linear relationship, and in comparison with hemocytometer counts the MTT method was less variable (Fig. 2).

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Comparison between MTT assay results and hemocytometer counts for N. parvispora grown with various medium compositions in order to get different cell densities: Grace's insect cell culture medium and FBS with increasing hemolymph concentrations (, 0%, a; 5%, c; 10%, g); Grace's insect cell culture medium and 10% hemolymph with different concentrations of BSA as a replacement for FBS (; 0 mg/ml, b; 0.625 mg/ml, d; 1.25 mg/ml, e; 2.5 mg/ml, f; 3.75 mg/ml, i; 5 mg/ml, h; 10 mg/ml, j). The two methods still exhibit a linear relationship (R2 = 0.968; n = 10; P < 0.0001). Each data point represents the mean and standard error of four independent replicates.

Finally, the same method was tested with another entomopathogenic fungus, Entomophthora thripidum (Zygomycetes: Entomophthorales). This fungus does not require hemolymph for cultivation and grows as protoplasts that form small aggregates in liquid culture (3). To determine the cell numbers, cultures were first pipetted gently to disrupt the pellets and then the protoplasts were counted as described above, in a hemocytometer (four counts per sample, 200 cells counted except for the lower cell densities). After a serial dilution with fresh medium, the cell densities for E. thripidum varied between 4.75 × 10⁵ and 2.5 × 10³ cells/ml of culture volume. For each cell density, six samples were measured with the MTT method and the results were compared with the hemocytometer counts. As for N. parvispora, the two measurements correlated linearly and the values from the MTT method were less variable.

The results of this study demonstrated that the transformation of the tetrazolium salt to formazan and its quantification could serve as a measure for cell densities of the two fungi N. parvispora and E. thripidum. In comparison with the procedure published by Mosmann (8), the incubation period for the MTT method had to be prolonged. This was expected because these fungi grow at relatively low temperatures (20°C) compared with the temperature (37°C) at which vertebrate cells grow. Furthermore, the cell wall might act as an additional barrier for the uptake of MTT. In most other reports on the MTT method the OD measurement reached a maximum after 4 to 8 h and remained constant thereafter (4). However, incubation periods up to 24 h did not have a negative influence and have been used (6). Another change to the original protocol was the removal of the medium prior to the measurement. This resulted in improvement of the accuracy and reliability (2). Because all measurements were made in propanol, different complex media did not directly affect the ODs. This was an advantage over a similar method using the tetrazolium salt XTT (sodium 3'-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis[4-methoxy-6-nitro]benzene-sulfonic acid hydrate), which forms a water-soluble formazan (9). The centrifugation and exclusion of the lysed cells from the solution before the determination of the OD was an additional step that further improved the accuracy and reliability of the results.

The advantages of the MTT procedure are accuracy and reliability and the saving of time (2, 8). In our case the new method reduced the time needed for the assays by at least 80%. We also observed that the variability of the results from the OD measurement was smaller than that from the hemocytometer counts. This was not surprising since the MTT method considers all cells in a sample rather than only a small subsample. Therefore, the MTT assay resulted in more accurate and reliable estimates of cell densities than hemocytometer counts.

In conclusion, the results of this study confirmed the MTT assay to be a fast, simple, cheap, and accurate method (8) for the determination of cell densities of the entomopathogenic fungi tested. In particular, the MTT method proved to be useful to estimate cell densities in small culture volumes and was more accurate and reliable than hemocytometer counting. The cultivation in small culture volumes and the sensitive evaluation with the MTT assay allow the screening and testing of many different substances, fractions, and nutrients indispensable to the development of defined media for the cultivation of such biotrophic fungi.