Evaluating the Concentration of a Candida albicans...

Infectious Diseases in Obstetrics and Gynecology I: 134-136 (1993)(C) 1993 VViley-Liss, Inc.

Evaluating the Concentration of a Candidaalbicans Suspension

Acficio Rodrigues, C. Pina Vaz, A. Freitas da Fonseca, andJ. Martinez-de-Oliveira

Departments of Microbiology (A.R., C.P.V., A.F.d.F.) and Gynecology (J.M.-d.-O.), Porto Facultyof Medicine, University of Porto, Porto, Portugal

ABSTRACT

Objective: The objective of this study was to develop a reproducible method of establishing theconcentration 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 werehighly 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, ofthe concentration ofyeast cells insolution. This will facilitate experimentation involving precise inocula or requirement for specificconcentrations ofyeast cells for various experiments. (C) 1993 Wiley-Liss, Inc.

KEg WORDS

Candida albicans, spectrophotometry, nephelometry, hemocytometer, Neubauer’s chamber

aginal candidosis, first described in 1849, isone of the most common lower genital tract

infections. 1-3 Since Candida albicans can be foundin up to 40% of healthy women, its diagnosis andtreatment can be problematic.

Vaginal culture continues to be the most sensi-tive method for the detection of vaginal yeast infec-tion. Culture of Candida species is definitely supe-rior to direct microscopic detection and latexagglutination methods. 4,s Isolation of a yeast (withgrowth) in culture from a vaginal specimen is con-sidered to be definitive proof of its presence and the"gold standard" in clinical microbiology and re-search. However, the true sensitivity of culture hasnot yet been determined. Few studies have beenpublished concerning the sensitivity of culture tech-

niques in establishing the presence of yeast fromclinical specimens. Furthermore, no studies havebeen published with regard to the quantitationof yeasts from clinical specimens or in media toestablish precise concentrations of yeast cells forstudy.

The classical method of determining the concen-tration of a yeast cell suspension is counting in a

Neubauer’s chamber or similar method (Burke’schamber, hemocytometer, etc.). However, its useis time-consuming and not practical for evaluatinga large number of suspensions.

The aim of" the present study was to compare thecounts obtained with a Neubauer’s chamber to thevalues obtained by turbidimetry (spectrophotome-try) and by nephelometry.

Address correspondence/reprint requests to Dr. Acicio Rodrigues, Department of Microbiology, Porto School of Medicine,Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal.

Received April 2, 1993Basic Science Article Accepted August 25, 1993

CANDIDA ALBICANS SUSPENSION RODRIGUES ET AL.

TABLE I. Results of the three different methods ofassessing the concentration of yeast suspensions

Absorbance/ Volt/Cells/ml/chamber spectrophotometry nephelometry

1,008 0. 116 15.5540 0.058 8.9387 0.037 5.78284 0.029 4.74252 0.022 3.76234 0.018 3.2208 0.014 3162 0.012 2.52120 0.01 2.1770 0.004 1.24

MATERIALS AND METHODSClinical isolates of C. albicans were grown on Sa-bouraud’s agar and identified using auxanography(API32C, BioMrieux, France). A dense yeast sus-

pension was prepared in 0.9% NaC1 solution atroom temperature from a 24-hour-old culturegrown on Sabouraud’s agar medium at 37C. Thesuspension of yeast cells was homogenized to breakup aggregates of yeast cells into individual cells insuspension. Then, serial dilutions were performed11 times (each one with a 10-fold dilution from thepreceding one). One-milliliter aliquots were re-moved from each dilution and analyzed spectro-photometrically (Spectronic 2001) at 540 nm. Asimilar sample was analyzed by nephelometry(Bhering laser automatic nephelometer); 0.001 mlof the suspension was placed in a Neubauer’s cham-ber and counted under 400 magnification. Quan-

titative determinations were made in duplicate ina double-blind fashion by two independent ob-servers.

RESULTSThe results obtained by each of the three proce-dures are shown in Table 1. The results obtainedfrom Neubauer’s chamber were expressed as themean of the two determinations. These results werecompared to the values obtained from both thespectrophotometric (Fig. 1) and the nephelometricdeterminations (Fig. 2). The results obtained uti-lizing spectrophotometry and nephelometry wereconverted to quantitative values utilizing the for-mulas presented in Table 2. These formulas allow a

conversion to the number of cells per milliliter ofmedium. This method provides a simple and quickmethod to determine the number of yeast cells permilliliter in a suspension.

DISCUSSIONInocula of known concentrations as well as suspen-sions of precise concentrations require a method ofmeasurement that is fast, accurate, and reliable.Determination of the number of cells per milliliterusing a counting chamber is commonly consideredto be the "gold standard" or reference method.However, it is a tedious and inconvenient methodwhen preparing a large number of suspensions.The use of the visual McFarland scale or Brownetubes does not seem to be accurate. 6 Turbidimetryis based upon a measurement of the reduction of theintensity of a light passing through a suspension bya photometer or spectrophotometer. 7 Nephelome-

1200

=, 1000

800

600

= 400E.=: 200

i1

0 0,02 0,04 0,06 0,08 0,1 0,12

Spectrofotometry (Ab$)

Comparison of the results of spectrophotometry with counts in a counting chamber.

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 135

CANDIDA ALBICANS SUSPENSION RODRIGUES ET AL.

1200

800

F... 1000

600

E 400

0 200

0 5 10 15 20

Nephelometry (Volt.)

Fig. 2. Comparison of the results of nephelometry with counts in a counting chamber.

TABLE 2. Formulas for conversion

Chamber (x 103/ml) 64.3 + 8,206 x spectrophotometry(absorbance)

Chamber (x 103/ml) -0.2 + 64 nephelometry (volt)

try measures the intensity of a reflected light beam,by the particles in the suspension, at a 90 angle tothe incident beam. 7

The present study shows a direct and reliablecorrelation between the three methods employed.Any one of these methods can be used indepen-dently to evaluate the concentration of yeast cells insuspension or to prepare a suspension of a definiteconcentration. The use of the spectrophotometer orthe nephelometer facilitates these calculations as wellas makes it simpler and faster than utilizing a cellcounting chamber. The yeast cell suspensions usedin the present study were easily converted fromreadings obtained with the spectrophotometer or

nephelometer into a quantitative number of yeastcells per milliliter of medium. These two methodswere found to be as accurate as counting individual

yeast cells with a counting chamber such as Neu-bauer’s chamber.

REFERENCES1. Wilkinson JS: Some remarks upon the development of

epiphytes with the description of new vegetable formationfound in connection with the human uterus. Lancet 2:448,1849.

2. Hillier SL: Laboratory diagnoses of yeast vaginitis. InHorowitz BJ, Mrdh PA (eds): Vaginitis and Vaginosis.New York: Wiley-Liss, pp 121-124, 1991.

3. Odds FC: Candidosis of the genitalia. In Odds FC (ed):Candida and Candidosis. London: Balliere Tindall, pp124-135, 1988.

4. Odds FC: Isolation, identification and other laboratoryaspects of Candida. In Odds FC (ed): Candida and Candi-dosis. London: Balliere Tindall, pp 60-67, 1988.

5. Martinez-de-Oliveira J, Fonseca AF: Diagnosing vaginalcandidosis by a slide latex agglutination test. In GuaschinoS (ed): Infectious Diseases in Obstetrics and Gynecology.Bologna: Monduzi Editore, pp 171-172, 1990.

6. Raphael SS, Spencer F, Culling CFA: Microbiology--Applied microbiology: Some basic concepts, techniques,and methods. In Raphael SS (ed): Lynch’s Medical Labo-ratory Technology. Vol 1. Toronto: W.B. Saunders, pp532-573, 1976.

7. Hyde TA, Mellor LD, Raphael SS: Chemistry: Analyticalsystems and applications. In Raphael SS (ed): Lynch’s Med-ical Laboratory Technology. Vol 1. Toronto: W.B. Saun-ders, pp 73-126, 1976.

136 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY

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