I Acta Biotechnol. 9 (1989) 1, 89-93 I

Flow Cytometric Determination of Yeast Sterol Content

MULLER, S., SCHMIDT, A.

Academy of Sciences of the G.D.R. Institute of Biotechnology, PermoserstraDe 15, Leipzig, 7050 G.D.R.

Summary

A method for the flow cytometric determination of the unesterified t/?-hydroxysteroI content in yeast populations by the fluorescence shifted macrolide nystatin is presented. Preliminary in- vestigations of changing sterol content in aerobic and anaerobic batch cultivation revealed the technological usefullness of this information.

The sterol content of yeast cells, mostly ergosterol, are important for brewing [l] and wine making [2] as survival factor and for the rehydration of baker’s yeast [3]. This is understandable because the molecular structure of cell membranes is altered by its sterol content. Details of this were published by PARKS et al. [4] and SHMITZKY et al. [5]. The aim of our work was to develop a method for the flow cytometric determination of unesterified 3~-hydroxysterols in single yeast cells, because usually determined popula- tion sterol mean values are a restrictive information in control strategies [6]. MULLER et al. [7] published a flow cytometric determination of cholesterol in murine cells by means of the macrolide filipin. Our selfmade flow cytometer [S], fitted out with an argon- ion laser, as most others, requires the fluorescence shifting of the macrolide. Thus we take the amino group containing nystatin, a macrolide with a similar reaction specifi- city €9-111. The problems to be solved resulted from the fluorescence shifting of nysta- tin by fluorescein isothiocyanat and some peculiarities of the yeast cell. The Sacchromyces cerevisiue strain H 155 was batch cultured on a READER medium containing 2% glucose at pH 5.4 and a t 30°C aerobically and anaerobically. The nystatin, original of drug store quality, was purified according to [12]. The efficiency was controlled by comparing to a nystatin specimen from PIERREL, Milan, Italy. 2 mg nystatin dissolved in 200 pJ dimethyiformamide were treated with 50 pJ FITC solution (1 mg FITC/ml 0.5 rn carbonate/bicarbonate buffer of pH 9.5) to shift the excitation range. The resulting conjugate was purified and isolated by gel chromatography on Sephadex G 15 in 0.5 m carbonate/bicarbonate buffer of pR 9.5. The ergosterol and cholesterol used were from FERAK, West Berlin, Sephadex G 15 waspurchased from PHARMACIA, Sweden, and FITC isomer I from SERVA, F.R.G. The yeast cells were counted by the coulter counter Laborscale from MEDICOR, Buda- pest. The sterol content of the yeast population was determined by the antimony trichloride method published by IAMB et al. [13].

90 Acta Biotechnol. 9 (1989) 1

In preliminary studies the conditions for nystatin-ergosterol complex formation and for nystatin-cholesterol complex formation with pure substances in buffer were investigated and checked by the alteration of the emission spectra. For complexing the yeast cell sterols the harvested and separated yeast cells were fixed by 70% ethanol and washed twice with 0.5 m carbonate/bicarbonate buffer of pH 9.5 and after that passed through a 45 pm nylon mesh to withdraw aggregates. Every yeast specimen was set at 5 x lo6 cells by a coiilter counter technique and resuspended in 1 ml conjugate buffer solution to form the nystatin complex with the yeast cell sterol. The cells were then separated

Temperature [OC]

Fig. 1. Comples formation with yeast cell sterols in 0.5 m carbonate/biocarbonate buffer of pH 9.5 after 16 hours at different temperatures. The conjugate concentra- tion was 25 pmol

at 3.000 g for 5 min and washed twice with 1 m10.5 m carbonate/bicarbonate buffer of pH 9.5. After centrifugation and resuspension the nystatin-sterol complex was stable for at least 5 hours. The cell suspension in buffer was analysed by our flow cytometer after excitation a t 488 nm. The emission signals were detected at 520 nm and classified. The integrated histogram values are proportionally connected with the corresponding sterol mean values indicating the obvious accuracy of the cytometry. To elaborate optimal complexing conditions the influence of the following parameters were investigated : temperature (Fig. l), pH-value (Fig. 2), reaction time (Fig. 3) and conjugate concentration (Fig. 4). For all parameters the most favorable condition for stable staining corresponds to the relative fluorescence at highest frequency of the partic- ular histogram. Figures 5 and 6 illustrate the alteration of the sterol histograms in dependence on oxygen supply of the yeast culture and the time of culturing. The first results clearly indicate that the method is very suitable for obtaining valuable fermentation information. More detailed investigations are going on.

W e wish to thank Mrs. I. KONARSKI for her skilful assistance throughout this work.

MULLER, S., SCHMIDT, A., Flow Cytometric Determination 91

3 ~ , 1 1 1 , 1 , s 5 2 4 6 8 10 12 14

PH

Fig. 2. Yeast cells were labelled for 16 hours a t room temperature with a conjugate concentration of 25 pmol. The pH-value varied from 3 to 12

I ' I I I !/- I 2 4 6 8 16 I8 20 22

Time [h] '

Fig. 3. Time course of complex formation with yeast cell sterols at R conjugate con. centration of 25 pnol at room temperature and pH 9.5

92 A c h Biotechnol. 9 (1989) 1

I I I I I I

Fig. 4. Yeast cells were labelled for 16 hours at room temperature and pH 9.5 with different conjugate concentrations

Channel number Fig. 6. Histograms of sterol contents of aerobic batch cultivation after 4 hours (thick line) and 24 hours (thin line)

MULLER, S., SCHMIDT, A., Flow Cytometric Determination 93

Channel number

Fig. 6. Histograms of sterol contents of anaerobic batch cnltivation after 4 hours (thick line) and 72 hours (thin line)

Received March 7, 1988

References

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