A RAPID METHOD FOR ESTIMATING VIABILITY INDESICCATED CELLS OF THE BIOCONTROL AGENT

TSUKAMURELLA PAUROMETABOLA C-924

ARMANDO HERNÁNDEZ1,3, ALAIN MOREIRA1, EULOGIO PIMENTEL1,JORGE MARTÍNEZ2, JESÚS MENA1 and NEYLEN DEL TORO2

1Center for Genetic Engineering and BiotechnologyPO Box 387, Camagüey 70100, Cuba

2Faculty of BiologyUniversity of Havana

Havana, Cuba

Accepted for Publication February 27, 2008

ABSTRACT

A new method for the rapid estimation of viability in anhydrobiotic cellsof the biocontrol agent Tsukamurella paurometabola C-924 was developed.The method is based on the absorbance due to hydrogen sulfide production. Alinear model was obtained by plotting decimal logarithm of the correctedabsorbance at 670 nm (log[Abscorrected]) versus the decimal logarithm of cul-turable cells (log[Xv]) (r2 = 0.9428). After comparing the results obtained bythe proposed method to the ones obtained by the plate counting technique, nosignificant difference was observed in the number of culturable cells. The newtechnique, which is faster than plate counting, permits the estimation ofbiologic activity in anhydrobiotic cells of the strain C-924 in only 2 h.

PRACTICAL APPLICATIONS

No previous reports concerning the method presented in this paper werefound in the reviewed literature; in our particular case, this method allows arapid evaluation of the viability and physiologic state of Tsukamurella pau-rometabola C-924. The method presented here permits the estimation ofviability in desiccated cells of bacteria that produce hydrogen sulfide fromcysteine as a sulfur source. This methodology could be applied in researchrelated to the isolation of anhydrobiotes hydrogen sulfide producers. In addi-tion, industrial application could be found, for example, the assessment of the

3Corresponding author. TEL: 53-322-61295; FAX: 53-322-61587; EMAIL: armando.hernandez@cigb.edu.cu

Journal of Rapid Methods & Automation in Microbiology 16 (2008) 222–229. All Rights Reserved.© 2008, The Author(s)

Journal compilation © 2008, Wiley Periodicals, Inc.222

viability of bacteria hydrogen sulfide producers formulated in wettablepowders used in bioremediation or bioprecipitation of heavy metals. Further-more, the biologic activity of these microorganisms could be estimated (i.e.,desulfurase activity).

INTRODUCTION

Nematodes are among the most important parasites causing economiclosses in the agricultural sector (Mena et al. 2003). During many years, plantparasitic nematodes have been controlled using chemical pesticides; neverthe-less, the negative impact of such chemicals on ecosystems is a real problemnowadays. Hence, the biologic control of nematodes is a valuable alternativeto the use of chemical nematicides (Mena et al. 2003).

Tsukamurella paurometabola C-924 is a gram-positive bacterium whosenematocidic action has been reported (Mena et al. 1996, 2003). This bacterialstrain was isolated from the soils of Cuba as a good candidate for the biologiccontrol of phytonematodes such as Meloidogyne incognita, Radopholus similisand Pratylenchus coffeae (Mena et al. 2003), and its mechanism of action hasbeen explained by the combined effect of desulfurase and chitinase activitieson eggs and larvae of Haemonchus sp. (Mena et al. 2003).

In order to preserve the cells of this biocontrol agent, freeze-drying andspray-drying technologies have been successfully applied (Hernández et al.2006, 2007). The viability of formulated powders has been determined by theplate counting technique; nevertheless, plate incubation at 37C takes 48 h,which delays the analysis of the results.

In addition, the ability of this microorganism to produce hydrogen sulfidefrom cysteine as a sulfur source has been previously reported (Mena et al.2003). Taking into account this biochemical characteristic of the strain C-924,the aim of our work was the development of a method for a rapid estimationof viability. In this paper, we described a rapid method for the estimation ofviability in the desiccated cells of T. paurometabola C-924, based on thecorrelation between culturable cells and the absorbance due to hydrogensulfide production. The method described here could be applied in the rapidestimation of viable cells of other bacterial strains that are hydrogen sulfideproducers.

MATERIALS AND METHODS

Preparation of Cells for Assays

Desiccated cells of T. paurometabola C-924 (Cell Bank of Center forGenetic Engineering and Biotechnology, Havana, Cuba; this strain was depos-

223ESTIMATING VIABILITY IN T. PAUROMETABOLA

ited in Centraalbureau voor Schimmelculcultures, Baarn, Netherlands) wereobtained by spray-drying as previously described (Hernández et al. 2007).

Samples of powders containing anhydrobiotic cells (0.1 g in triplicate)were rehydrated and washed with 10 mL of buffer A (pH 8.6), composed ofNaCl (0.14 mol/L), Na2HPO4 (0.01 mol/L), KH2PO4 (1.8 mmol/L) andMgSO4 ¥ 7H2O (61 mmol/L). The washed cells were centrifuged at 3,000 rpmfor 15 min, and the pellets were resuspended in 10 mL of reaction buffer B(pH 8.6), composed of NaCl (0.14 mol/L), Na2HPO4 (0.01 mol/L), KH2PO4

(1.8 mmol/L), MgSO4 ¥ 7H2O (61 mmol/L) and cysteine (1.5 mmol/L).Several dilutions (six replicates per dilution) were made in 1.5-mL microfugetubes up to the final volume of 1 mL using buffer B. For each dilution, threetubes were capped with parafilm and incubated at 37C for 1 h with gentleagitation in a roller; the other three replicates were used to determine viabilityby the plate counting technique.

Standard Curve for Spectrophotometric Analysis of H2S

A standard curve of sulfide was prepared in 1.5-mL microfuge tubes usingbuffer B to dilute sodium sulfide as the sulfide standard according to Wanget al. (2000). Total sulfur concentration was kept constant considering that thefree sulfide detected is produced by the cysteine transformation according to

Cysteine pyruvate H S NH→ + +2 3

Sulfide concentrations were set (1–40 mmol/L), and the total volume ofstandards was 1 mL. To each microfuge tube, 100 mL of N,N-dimethyl-p-phenylene-diamine oxalate (0.02 mol/L) in HCl (7.2 mol/L) and 100 mL ofFeCl3 (0.3 mol/L) in HCl (1.2 mol/L) were added and mixed homogenously.Color was allowed to develop at room temperature for 15 min. The absorbanceat 670 nm was measured, and the absorbance values were plotted againstsulfide concentration.

Spectrophotometric Analysis of Sulfide Produced by Rehydrated Cells

The reaction of cysteine desulfuration from rehydrated cells previouslyincubated at 37C (preparation of cells for assays section) was stopped byadding 100 mL of N,N-dimethyl-p-phenylene-diamine oxalate (0.02 mol/L) inHCl (7.2 mol/L) and 100 mL of FeCl3 (0.3 mol/L) in HCl (1.2 mol/L) mixinghomogenously. Color was allowed to develop for 15 min at room temperature.These samples were centrifuged at 12,000 rpm for 5 min.

Simultaneously, the blanks for sulfide background correction (in tripli-cate) were prepared as described in the preparation of cells for assays section,

224 A. HERNÁNDEZ ET AL.

but these samples (control cells) were not incubated at 37C. Once the controlcells were suspended in the buffer containing cysteine, color was developedimmediately as described above; then, the samples were centrifuged at12,000 rpm for 5 min. The absorbance of supernatant (670 nm) was measuredfor blanks and samples. The corrected absorbance (absorbance due to thehydrogen sulfide produced after the incubation at 37C) was calculated bysubtracting the blank absorbance from the total absorbance.

From the corrected absorbance, amounts of sulfide produced in 1 h weredetermined by interpolation in the sulfide standard curve (standard curve forspectrophotometric analysis of H2S section).

Determination of Viability by the Plate Counting Technique

Viability was determined by the plate counting technique as previouslydescribed (Hernández et al. 2007). In triplicate, 0.1 g of powder was sus-pended by vortexing in 10 mL of NaCl (0.15 mol/L), and serial dilutions weremade to plate in Luria-Bertani agar plates. The plates were incubated at 37Cfor 48 h and cfu/mL was calculated.

Mathematical Correlation Between Viability and Absorbance at 670 nm

In order to estimate viability from the absorbance measurements, the datawere processed and the standard curve was graphed (spectrophotometricanalysis of sulfide produced by rehydrated cells section). The corrected absor-bance transformed to decimal logarithm (log[Abscorrected]) was plotted versusthe decimal logarithm of viability (log[Xv]). The data were fitted using Graph-Pad PRISM 4.00 (GraphPad Software, Inc., La Jolla, CA). The resultingmathematical model was used to estimate the viability of samples frompowders produced by spray-drying.

Comparison of Viability Using the Plate Counting Technique andthe Proposed Method

Samples from powders obtained by spray-drying in three differentbatches were processed as described above (preparation of cells for assayssection), and spectrophotometric determination of sulfide was performed asdescribed in the spectrophotometric analysis of sulfide produced by the rehy-drated cells section. From these measurements, viability was estimated byinterpolation in the curve previously obtained (mathematical correlationbetween viability and absorbance at 670 nm section). Replicates of thesesamples were analyzed by the plate counting method. The mean values ofviability determined by both methods were compared statistically (n = 9)using Statgraphics 5.1 Plus (Statistical Graphics Corp., Rockville, MD).

225ESTIMATING VIABILITY IN T. PAUROMETABOLA

RESULTS AND DISCUSSION

Previous studies (unpublished observations) have shown that the viabilityof T. paurometabola C-924 does not change when cells are incubated for 1 hwith cysteine (1.5 mmol/L) in buffer B. Thus, our assays were made based onthis experimental evidence.

The method presented here is based on the production of hydrogensulfide by the cells of T. paurometabola C-924 (Mena et al. 2003). As it isdescribed here, the absorbance at 670 nm is corrected because the reactionof the color development produces an additional color background becauseof the presence of bacterial cells (Wang et al. 2000). Thus, the addition ofreagents to develop color in the control cells was performed immediatelyafter the control cells were suspended in the buffer containing cysteine; theresulting absorbance was considered nonassociated to the desulfurase activ-ity. Therefore, the absorbance associated to the desulfurase activity was esti-mated by subtracting the blank absorbance from the total absorbance of thesamples.

After analyzing the results shown in Fig. 1, we conclude that it is possibleto estimate viability in desiccated cells of T. paurometabola C-924 consideringthe correlation between log(Abscorrected) and log(Xv). The data were fitted tolinear models, therefore, increasing the cell concentration between 1 ¥ 109 and2 ¥ 1010 cfu/mL; the hydrogen sulfide production increases linearly. The fittingequation for absorbance as a function of viability was

9.0 9.3 9.6 9.9 10.2 10.5-0.5

-0.3

-0.1

0.1

0.3

Absorbance dataHydrogen sulfide data

1.8

2.0

2.2

2.4

2.6

2.8

3.0

Culturable Cells (log Xv)

log

(Ab

s co

rrec

ted)

log

(mm

ol(

H2S

)/h

)

FIG. 1. CORRELATION BETWEEN VIABILITY OF TSUKAMURELLA PAUROMETABOLAC-924 AND ABSORBANCE AT 670 nm

As a measure of viability, the culturable cell concentration was transformed to decimal logarithm. Thecorrelation between viability and desulfurase activity is shown. As a measure of the desulfuraseactivity, the transformed variable log(n[H2S]/h) was considered, where n(H2S)/h is the production rate

of hydrogen sulfide.

226 A. HERNÁNDEZ ET AL.

log . log . .Abs X rcorrected v( ) = × ( ) − =( )0 532 5 165 0 94282 (1)

and the fitting equation for the production of sulfide was

log . log . .n X rH S h v21 20 532 2 847 0 9428[ ]( ) = × ( ) − =( )− (2)

Both equations are useful; in fact, the viability is estimated when theabsorbance is evaluated in Eq. (1). In addition, analyzing the amount of sulfideproduced, it is possible to estimate the desulfurase activity, which finallyindicates the ability of the cells to produce hydrogen sulfide after rehydration.

According to these results, the hydrogen sulfide production follows first-order kinetics in respect to the viable cell concentration, which could bedescribed mathematically through Eq. (3)

dn

dtkX

H Sv

2( )= (3)

where:

n(H2S): amount of extracellular hydrogen sulfide (mmol);t: reaction time (h);k: rate constant (mmol/h/cfu/mL);Xv: cell viability (cfu/mL).

This mathematical behavior could be used in determining the desulfuraseactivity per gram of powder, as an indirect measure of biologic activity of thepowder obtained by spray-drying.

The comparison of both techniques is shown in Fig. 2. No significantdifference was found at 95% confidence level between the mean values of thetransformed viability (log[Xv]) determined by using the spectrophotometricproposed method and the plate counting method (P > 0.05). Thus, this devel-oped technique is suitable for the routine analysis of viability when freshdesiccated cells are analyzed. Moreover, the proposed method gives additionalinformation related to the desulfurase activity of rehydrated cells, which is ameasure of biologic activity (Mena et al. 2003).

Our method is simpler and faster than the classical plate counting tech-nique. Once the regression curve has been plotted with enough experimentalpoints to ensure statistical robustness, the analytical procedure is very simple,and this method becomes a routine analysis technique. The results of estimatedviability are ready in 2 h, which saves time and permits to evaluate primarilythe quality of fresh anhydrobiotic cells obtained, considering the hydrogen

227ESTIMATING VIABILITY IN T. PAUROMETABOLA

sulfide production as one of the main “potency” tests for the powder producedby spray-drying.

Several methods have been employed to assess the viability in desiccatedbacterial cells. Mainly, rapid techniques such as the flow cytometry and thefluorescence microscopy have been used (Parthuisot et al. 2003; Ananta et al.2005), employing fluorescent dyes that are able to discriminate between“viable cells” and “nonviable cells.” Nevertheless, up to date, no previousdescription indicating the application of desulfurase activity reactions in esti-mating the viability of anhydrobiotic bacterial cells have been made.

The method presented here is more economic than the plate countingmethod and could be used by other researchers in a rapid estimation ofviability of desiccated fresh cells, mainly in those bacterial cells that producehydrogen sulfide from cysteine as a sulfur source.

CONCLUSIONS

A new rapid spectrophotometric method for estimating viability in des-iccated cells of the nematocidal agent T. paurometabola C-924 was developed.We show no significant difference in the estimation of viability when thespectrophotometric method and the plate counting technique are applied in theinterval 1 ¥ 109 - 2 ¥ 1010 cfu/mL. The method we developed is advantageousbecause the results are ready in only 2 h. In addition, the use of the proposed

02468

101214

Desulfurase activity per gram of powder

0102030405060

Number of culturable cells by the plate counting

technique

Number of culturable cells by the proposed method

log

(X

vs)

Act

ivit

y (U

g-1

)

FIG. 2. COMPARISON OF THE DEVELOPED METHOD AND THE PLATECOUNTING TECHNIQUE

The transformed specific viability of the powder (log [Xvs]) was the comparison variable. The units ofXvs are cfu per gram of powder (cfu/g). Right y-axis reflects the desulfurase activity per gram of powder.

The unit of desulfurase activity was defined as 1 U = 1 mmol sulfide/h.

228 A. HERNÁNDEZ ET AL.

method permits the estimation of the biologic activity in anhydrobiotic cells ofstrain C-924 by evaluating the desulfurase activity.

ACKNOWLEDGMENT

The authors wish to thank Professor Orestes Padrón for his assistancein the translation of the manuscript.

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229ESTIMATING VIABILITY IN T. PAUROMETABOLA