Evaluating the Concentration of a Candida albicans Suspension

Objective: The objective of this study was to develop a reproducible method of establishing the concentration of yeast cells per milliliter of solution. Methods: Three methods of determining the number of yeast cells in solution were compared: Neubauer's counting chamber, spectrophotometry, and nephelometry. Results: All three methods were comparable and reproducible. The following formulas were highly effective in determining the number of yeast cells in solution: chamber (× 103/ ml) 64.3 + 8,206 × spectrophotometry (absorbance); and chamber (× 103 /ml) –0.2 + 64 × nephelometry (volt). Conclusions: Utilization of spectrophotometry or nephelometry and the appropriate formula allow for the precise determination, which is easily reproducible, of the concentration of yeast cells in solution. This will facilitate experimentation involving precise inocula or requirement for specific concentrations of yeast cells for various experiments.

aginal candidosis, first described in 1849, is one of the most common lower genital tract infections. [1][2][3] Since Candida albicans can be found in up to 40% of healthy women, its diagnosis and treatment can be problematic.
Vaginal culture continues to be the most sensitive method for the detection of vaginal yeast infection. Culture of Candida species is definitely superior to direct microscopic detection and latex agglutination methods. 4,s Isolation of a yeast (with growth) in culture from a vaginal specimen is considered to be definitive proof of its presence and the "gold standard" in clinical microbiology and research. However, the true sensitivity of culture has not yet been determined. Few studies have been published concerning the sensitivity of culture tech-niques in establishing the presence of yeast from clinical specimens. Furthermore, no studies have been published with regard to the quantitation of yeasts from clinical specimens or in media to establish precise concentrations of yeast cells for study.
The classical method of determining the concentration of a yeast cell suspension is counting in a Neubauer's chamber or similar method (Burke's chamber, hemocytometer, etc.). However, its use is time-consuming and not practical for evaluating a large number of suspensions.
The aim of" the present study was to compare the counts obtained with a Neubauer's chamber to the values obtained by turbidimetry (spectrophotometry) and by nephelometry.

RESULTS
The results obtained by each of the three procedures are shown in Table 1. The results obtained from Neubauer's chamber were expressed as the mean of the two determinations. These results were compared to the values obtained from both the spectrophotometric (Fig. 1) and the nephelometric determinations (Fig. 2). The results obtained utilizing spectrophotometry and nephelometry were converted to quantitative values utilizing the formulas presented in Table 2. These formulas allow a conversion to the number of cells per milliliter of medium. This method provides a simple and quick method to determine the number of yeast cells per milliliter in a suspension.

DISCUSSION
Inocula of known concentrations as well as suspensions of precise concentrations require a method of measurement that is fast, accurate, and reliable.
Determination of the number of cells per milliliter using a counting chamber is commonly considered to be the "gold standard" or reference method. However, it is a tedious and inconvenient method when preparing a large number of suspensions. The use of the visual McFarland scale or Browne tubes does not seem to be accurate. 6 Turbidimetry is based upon a measurement of the reduction of the intensity of a light passing through a suspension by a photometer or spectrophotometer. 7   The present study shows a direct and reliable correlation between the three methods employed.
Any one of these methods can be used independently to evaluate the concentration of yeast cells in suspension or to prepare a suspension of a definite concentration. The use of the spectrophotometer or the nephelometer facilitates these calculations as well as makes it simpler and faster than utilizing a cell counting chamber. The yeast cell suspensions used in the present study were easily converted from readings obtained with the spectrophotometer or nephelometer into a quantitative number of yeast cells per milliliter of medium. These two methods were found to be as accurate as counting individual yeast cells with a counting chamber such as Neubauer's chamber.