In Situ Determination of Bacterial Growth by Multiple Headspace

Anal. Chem. 2008, 80, 7820–7825

In Situ Determination of Bacterial Growth by Multiple Headspace Extraction Gas Chromatography Xin-Sheng Chai,*,† Chunxu Dong,†,‡ and Yulin Deng†,§ Institute of Paper Science and Technology and School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Alanta, Georgia 30332, and State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Dalian University of Technology, Dalian, China 116012 This paper demonstrated an in situ headspace gas chromatography (GC) technique for monitoring bacterial growth using a commercial GC system with a multiple headspace extraction (sampling) mode. The technique was based on measuring the carbon dioxide mass in the headspace of a closed sample vial during the bacteria growth. A mathematic equation was derived in order to calculate the integrated amount of carbon dioxide produced by bacterial growth during the incubation. The method can be used to monitor the bacterial growth rate in a given cultural medium. The present method is very simple, sensitive, and safe. The prevention of the growth of pathogenic bacteria is a major concern for many industries. Considerable efforts have been focused on not only elucidating the effects of environmental factors such as temperature, pH, chemical environment, and substrates to bacteria growth but also looking for the effective materials or additives that could inhibit the bacteria growth or kill the bacteria.1 Clearly, fast, sensitive, and reliable methods for determination of bacteria growth play an important role in this research. Many traditional methods, either direct or indirect, are available for determining bacterial growth rate and cell number. Generally speaking, direct methods such as dry cell mass gravitometry and microscopic count cannot distinguish living from dead cells. Indirect methods, typically the viable cell (colony) counts and turbidity measurement, have been widely used in bacterial growth study.2 However, these methods are usually less sensitive, thus a long incubation time (such as overnight) is required in order to produce a sufficient amount of bacterial cells that are able to be measured or detected by these methods. For bacterial growth rate determination, a periodically manual sampling from the incubation medium is usually required. This might disturb the incubation environment and affect the bacterial growth especially for anaerobic microorganisms. In many cases, the conditions used in bacterial growth measurement have to be * To whom correspondence should be addressed. E-mail: xin-sheng.chai@ ipst.gatech.edu. † Institute of Paper Science and Technology, Georgia Institute of Technology. ‡ Dalian University of Technology. § School of Chemical and Biomolecular Engineering, Georgia Institute of Technology. (1) Cagri, A.; Ustunol, Z.; Ryser, E. T. J. Food Prot. 2004, 67, 833. (2) Hard, S. P. Human Microbiology; CRC Press: New York, 2002; p 56.

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Analytical Chemistry, Vol. 80, No. 20, October 15, 2008

different from the actual conditions. As a result, the data could not truly reflect those in the bacterial growth system. Headspace gas chromatography (HSGC) is an effective technique for determining the volatile species in the sample with a complicated matrix and has been widely used in studies of microorganisms,3 such as for the evaluation of microbial contamination of foods,4-10 in analyses of fungi,11,12 in studies on bacteria of the genus Clostridium,13 and identification of anaerobic bacteria.14 In these studies, carbon dioxide, one of the major volatile microbial metabolites, above a cultural medium during bacterial growth was used as a marker and was measured by headspace GC. With headspace technique, a direct sample injection to the GC system can be avoided, which eliminates the risk of the injector and column contamination caused by the nonvolatile species in the samples. High sensitivity in the GC detectors, typically thermal conductivity detector (TCD), makes it possible to detect a low level of carbon dioxide produced by bacterial cells at an early incubation time, so that a rapid determination of bacterial level and growth rate can be achieved. In a review article,15 Gardini et al. have provided a detailed description of the experimental approaches for not only checking the bacterial contamination level but also monitoring the activity of bacteria after incubation by measuring the carbon dioxide in the vapor phase of the closed sample vials using headspace GC. In their bacterial growth studies, a batch of vials containing a series of incubation samples was placed in a temperature controlled environment, and the carbon dioxide in the headspace of these sample vials was measured by GC at the different incubation times. (3) Hachenberg, H.; Schmidt, A. P. Gas Chromatography Headspace Analysis; Wiley: New York, 1984; p 1. (4) Bassette, R.; Claydon, T. J. J. Dairy Sci. 1965, 48, 775. (5) Bawdon, R. E.; Bassette, R. J. Dairy Sci. 1966, 49, 624. (6) Guarino, P. A.; Kramer, A. J. Food. Sci. 1969, 34, 31. (7) Pierami, R. M.; Stevenson, K. E. J. Dairy Sci. 1976, 59, 1010. (8) Guerzoni, M. E.; Piva, M.; Gardmi, F. Lebensm.-Wiss. Technol. 1985, 17, 128. (9) Guerzoni, M. E.; Gardmi, F.; Cavazza, A.; Piva, M. Int. J. Food Microbiol. 1987, 4, 473. (10) Basem, A.; Gardini, F.; Paparella, A.; Guerzoni, M. E. Lett. Appl. Microbiol. 1992, 14, 255. (11) Norrman, J. Arch. Microbiol. 1969, 68, 133. (12) Gardmi, F.; Castellari, L.; Guerzoni, M. E. Int. J. Food Sci. 1990, 2, 103. (13) Drasar, B. S.; Goddard, P.; Heaton, S.; Peach, S.; West, B. J. Med. Microbiol. 1976, 9, 63. (14) Larsson, L.; Mardh, P.-A.; Odham, G. J. Clin. Microbiol. 1978, 1, 23. (15) Gardinia, F.; Lanciottib, R.; Sinigagliab, M.; Guerzonia, M. E. J. Microbiol. Methods 1997, 29, 103. 10.1021/ac801537x CCC: $40.75  2008 American Chemical Society Published on Web 09/25/2008

Although the method assumed the samples in different vials prepared under the same conditions except the time, the method gave a larger data scattering in the determination of the timedependent bacterial growth profiles due to the minor differences in sample collection and container contamination between the incubation samples. For bacterial cell number detection, they used the modified Gompertz equation5 (a model for describing bacterial growth) to calculate the minimum detection time (MDT), which corresponds to 3% of carbon dioxide in the headspace that was assumed to reach at GC detecting limit. However, besides the modeling complexity with the Gompertz equation, it also showed a very significant data scattering in the correlations between MDT and plate cell counts. This can cause larger errors in both the microbial contamination level detection and growth model development.16 In a previous work,17 we have demonstrated a multiple headspace extraction (MHE) technique for monitoring the methanol formation in black liquor (alkaline pulping spent liquor) during its storage using a commercial headspace GC system. It was based on a periodic sampling and measuring of the vapor phase of a given headspace sample vial (regarded as a minireactor) at a desired temperature without disturbing the liquid phase, so that the changes of the vapor species during the reaction can be in situ followed. Compared with the total mass of methanol in the system, the amount of vapor methanol removed from the sample vial at each headspace sampling can be neglected, which is due to an extremely low Henry’s Law constant of methanol in the aqueous system.18 With an appropriate calibration, the kinetic formation of methanol during the reaction can be obtained by MHE HSGC measurement. The major advantages for this in situ MHE HSGC method are that the commercial headspace autosampler system not only provides a precise temperature and shaking controlled environment during the experiment (this might be very important to environment sensitive bacteria) but also performs a highly repeatable headspace measurement. The in situ measurement based on a single sample system eliminates the uncertainty found in the batch sample preparations, which is often the case in dealing with the liquid samples containing the suspended substances. Instead of measuring methanol in situ as reported in our previous work,17 carbon dioxide in the vapor phase of the headspace GS system was used as a marker in this report for studying the bacterial growth in a solution. If the MHE HSGC was applied to determine the carbon dioxide changes in the vapor phase during the bacterial growth, a direct relationship between the concentration of carbon dioxide in the vapor phase and the liquid phase after each sampling should be developed first because a portion of carbon dioxide in the sample vial (incubator) is removed at each headspace sampling from the system and transferred to GC for measurement. Therefore, it is the key to find the relationship between the amounts of carbon dioxide produced during the bacterial growth and those measured at each headspace sampling time in the MHE HSGC method. Thus, the (16) Zwietering, M. H.; Jongenberger, I.; Rombouts, F. M.; van’t Riet, K. Appl. Environ. Microbiol. 1990, 56, 1875. (17) Chai, X.-S.; Luo, Q.; Zhu, J. Y. J. Chromatogr., A 2002, 946, 177. (18) Gupta, A. K.; Teja, A.; Chai, X.-S.; Zhu, J. Y. Fluid Phase Equilib. 2000, 170, 183.

formation kinetics of carbon dioxide during the bacterial growth can be established. In this paper, we reported a novel method for in situ monitoring of bacterial growth using a commercial headspace GC system. The main focuses were to (1) develop a mathematical equation for calculating the accumulated amount of carbon dioxide produced during the bacterial growth in MHE HSGC measurement and (2) address the importance for selecting the experimental parameter in the MHE method. In the application, a bacterium, Escherichia coli, was used to demonstrate the method for the determination of bacterial growth. EXPERIMENTAL SECTION Materials. All materials used in the experiment were from commercial sources. E. coli JM109 strain was purchased from Promega Co. (Madison, WI). Luria broth (LB, containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L sodium chloride) was purchased from Sigma-Aldrich, Inc., St. Louis, MO. Apparatus and Operations. All measurements were carried out using an HP-7694 automatic headspace sampler and model HP-6890 capillary gas chromatograph (Hewlett-Packard, CA) using a thermal conductivity detector (TCD). GC conditions were as follows: capillary column with an i.d. ) 0.53 mm and a length of 30 m (model GS-Q, J&W Scientific, Folsom, CA) at 30 °C; carrier gas helium flow rate of 3.1 mL/min. The temperature for TCD operation is 220 °C. Headspace sampler operating conditions were as follows: oven temperature of 37 °C; vial pressurized by helium and pressurization time of 0.2 min; sample-loop fill time of 0.2 min; loop equilibration time of 0.05 min; and loop fill time of 0.2 min.19 HP 8453 UV-visible spectrophotometer (Hewlett-Packard, Germany) was also used to measure the optical density (OD) of the sample for the determination of the growth curve of the bacteria. A 3 mL volume of the liquid sample was withdrawn from the incubator and placed in a 1 cm cuvette for optical density measurement at a wavelength of 600 nm.20 Sample Incubation and Measurement Procedures. A volume of 200 µL of E. coli culture with approximately 107 colony forming units per milliliter (CFU/mL) was inoculated in a 20 mL autoclaved LB medium. After strong shaking to make them well distributed, a 2 mL volume of the culture was added into a 22 mL headspace sample vial. The vial was immediately sealed by a rubber septum and then placed in the headspace sampler at a constant temperature 37 °C under a gentle agitation condition to allow the bacteria growth. The headspace of the vial was periodically sampled followed by GC measurement. The headspace sampling cycle time is 25 min. RESULTS AND DISCUSSIONS Methodology Development. Since the amount of carbon dioxide produced was found to be proportional to the number of bacterial cells,15 carbon dioxide can be used as a marker to indirectly determine bacteria growth. In a closed container, the carbon dioxide produced by the bacteria in a cultural medium has a partition equilibration between the liquor and vapor (headspace) phases and agrees with Henry’s Law, i.e., (19) Chai, X.-S.; Luo, Q.; Zhu, J. Y. J. Chromatogr., A 2001, 909, 249. (20) Gautam, S.; Sharma, A.; Thomas, P. Int. J. Food Sci. Nutr. 1998, 49, 11.


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Hc )

CO2(g) CG ) CO2(l) CL

(1)

All symbols are defined in Table 1. The total mass of carbon dioxide in this system can be expressed as m ) CLVL + CGVG

(2)

Combining eq 1 with eq 2, the total mass of carbon dioxide can be further written as

(

m ) CG

)

VL + VG ) pCG Hc

(3)

Thus, the amount of carbon dioxide produced in the system can be obtained by determining the carbon dioxide in the vapor phase using HSGC. In many commercial headspace autosampler systems, the sampling is achieved by injecting a certain amount of an inert gas to create a headspace pressure followed by releasing the pressure through a sampling loop. The headspace sample in the loop is then measured by GC. During each headspace sampling, a portion of the species in the headspace is removed from the closed system (sample vial). In a multiple headspace extraction (MHE) approach, the headspace sample is periodically removed at a desired time-interval. If there is no volatile species generated during the process, the concentration of the species in the headspace decreases at each extraction. According to the previous work,21,22 the relationship between the amount of species in the headspace and its extraction number can be described as log(CGn) ) log(CG0) - bn

CGn ) CGn-110-b

b CG0 t n ∆CGn j f ∆An ∆mn ∑0n ∆mn mt An A0 K g τ k R a

concentration of carbon dioxide in vapor phase concentration of carbon dioxide in liquid phase Henry′s Law constant total mass of carbon dioxide in the system ratio constant volume of headspace volume of liquid sample concentration of carbon dioxide in vapor phase at nth headspace sampling slope of eq 4 intercept of eq 4 (at t ) 0) incubation time headspace extraction number amount of carbon dioxide produced between (n 1)th and nth headspace sampling headspace extraction cycle time response factor in headspace GC measurement net GC signal increase (related to carbon dioxide produced) between the (n - 1)th and nth headspace measurement mass of carbon dioxide produced between (n - 1)th and nth headspace sampling integrated mass of carbon dioxide produced during bacteria growth total mass of the carbon dioxide produced at the reaction time t GC peak area at the nth headspace measurement GC peak area for the ambient above a blank sample overall calibration coefficient number of bacteria generation generation time factor slope of eq 14 intercept of eq 14

where the relationship between incubation time and headspace extraction number is t ) j(n - 1)

CGn ) CGn-110-b + ∆CGn

(6)

The accumulated mass of carbon dioxide at the incubation time t is the sum of carbon dioxide generated between every two headspace measurements, i.e., n

∑ ∆m

n

(7)

1

(21) Kolb, B.; Ettre, L. S. Static Headspace-Gas Chromatography-Theory and Practice; Wiley-VCH: New York, 1997; p174. (22) Chai, X.-S.; Zhu, J. Y. Anal. Chem. 1998, 70, 3481.


(8)

According to eq 3 and the relationship between the GC signal and the carbon dioxide concentration in the headspace, CG = fA, eqs 7 and 6 can be further written as n

(5)

If the system involved bacteria growth, carbon dioxide is produced during the time period between two adjacent headspace extractions. Thus, the concentration of carbon dioxide in the headspace can be expressed as

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CG CL H m p VG VL CGn

(4)

For any two adjacent headspace measurements (i.e., n and n -1), eq 4 can be written as

mt ) ∆m1 + ∆m2 + ... + ∆mn )

Table 1. Symbols and Definitions

mt )

∑ 1

n

pf∆An ) K

∑ ∆A

n

(9)

1

and ∆An ) An - An-110-b

(10)

respectively. Base MHE GC measurement on carbon dioxide in a given sample vial during the bacteria growth, a set of GC signals, A1, A2, A3,... An at 1, 2, 3,... nth headspace sampling can be recorded. Thus, the mass of carbon dioxide during bacteria growth can be calculated according to eqs 10 and 9. In a batch bacterial cultural growth cycle, the bacteria population is significantly increased in the logarithmic growth phase and can be expressed as the following2 t

Nt ) N02g or Nt ) N02 τ

(11)

Thus, there is a linear relationship between the logarithm of the number of bacteria and the incubation time, i.e., log Nt ) log N0 + Rt

(12)

Since the amount of carbon dioxide produced is proportional to the number of bacteria generated during incubation,15 i.e., mtk ) (Nt - N0) or mtk ) Nt (when Nt . N0) (13) Substituting eq 13 into eq 12, we can obtain log mt ) log(N0 ⁄ k) + Rt ) a + Rt

(14)

By the mass (mt) of carbon dioxide produced vs the incubation time (t) being plotted and conducting a linear fit, the slope and intercept of the curve (eq 14) can be obtained. The slope (R) represents the bacteria growth rate. The numbers of the bacteria initially inoculated in the given cultural medium can be calculated according to the intercept, i.e., a

N0 ) k10

(15)

Selection of the Major Parameters. Inoculated Sample Size. Although GC thermal conductivity detector (TCD) is fairly sensitive in detecting carbon dioxide, it is difficult for the system in the presence of a very small number of bacterial cells, in which the amount of carbon dioxide produced is below the TCD detection limit. As reported,15 the amount of carbon dioxide can be detectable by headspace GC when the bacterial cells reach a level of 106-107 per mL. For a rapid testing, a larger sampling size introduced to the cultural medium is helpful to improve the sensitivity in the headspace GC measurement. Otherwise, a longer incubation time is required in order to produce a sufficient amount of carbon dioxide to reach the GC detection limit. MHE Measurement Cycle Time. A significant amount of metabolic products formed during bacterial growth in incubation could inhibit the further growth of the bacterial cells. Traditionally, releasing the carbon dioxide from the system by a regularly ventilation is a common approach for reducing the effect.23 In the present HSGC system, the headspace sampling is based on pressurizing an external gas (helium) to replace a part of old headspace before each GC measurement. It also provides an opportunity to vent a part of carbon dioxide from the system. If the amount of bacteria cells in the system is large, a significant amount of carbon dioxide could be produced during the incubation, which will affect the bacterial growth and lead to a nonlinear relationship between the amount of carbon dioxide produced and incubation time. Thus, a frequent headspace sampling, i.e., a short MHE cycle time, should be chosen during the bacteria growth experiment to minimize the carbon dioxide inhibition effect. Clearly, the longer MHE cycle time will make the experiment less efficient. In this work, the MHE cycle time was 20 min. Gas for Headspace Replacement. The atmosphere composition above the culture medium is also important for bacterial growth. Oxygen in air is essential for aerobic bacteria, however, it could (23) Coyne, F. P. Proc. R. Soc. London, Ser. B 1933, 113, 196.

inhibit the growth of some anaerobic bacteria. Therefore, it is important to select an appropriate atmosphere during bacteria incubation. As mentioned above, a part of the headspace is withdrawn from the system and replaced by a fresh external gas at each MHE sampling. Thus, by selection of either air, or an inert gas or a makeup gas, as a pressurizing gas in the MHE sampling, a desired atmosphere of the culture medium can be guaranteed. pH Effect. In the present method, carbon dioxide is used as a marker for monitoring bacterial growth. When the carbon dioxide is dissolved in water, there are several equilibriums among CO2, H2CO3, HCO3-, and CO32- in the solution, which is highly pH related. At equilibrium, only a small fraction (∼0.2-1%) of the dissolved CO2 is actually converted to H2CO3 at room temperature.24 Thus, the total mass of carbon dioxide produced during the bacterial growth, mt, could be expressed as mt ) mCO2,L + mHCO3-,L + mCO32-,L + mCO2,V

(16)

where m is the mass of the chemical species and L and V represent the liquid and vapor phases, respectively. In eq 16, only the concentration (so the mass) of CO2 in the vapor phase could be directly measured by HSGC. It is well-known that the equilibriums among CO2, H2CO3, HCO3-, and CO32strongly depend on the pH of the solution. Although the equilibriums among these chemical species are complex, they are well studied in literature. Drimal et al. 25 indicated that, at pH < 7.2, the total amount of carbonates (HCO3- and CO32-) in the solution is insignificant, and the partition of CO2 in the vapor and liquid phases follows Henry’s law as expressed by eq 1. In this study, the initial pH of the solution was adjusted to 6.5. Therefore, the total mass of CO2 produced in the bacterial growth in the system could be simply obtained by measuring the CO2 in the vapor phase and calculated using eq 3. Method Calibrations In the present method, the slope (b) in eq 10 and the overall calibration coefficient (K) in eq 9 are two key parameters for calculating the amount of carbon dioxide produced during the process. The values of these parameters can be determined by MHE HSGC measurement based on a broth (1.5 mL) spiked a known amount of carbonate (a standard sodium carbonate solution) in a sealed headspace sample vial. After injecting 0.5 mL of 2 M hydrochloric acid, carbonate is complete converted to carbon dioxide. The vial is then placed into the headspace sampler for an automatic MHE HSGC measurement. The GC signals (A1, A2, A3,... An) of carbon dioxide at 1, 2, 3,... nth headspace sampling in the MHE HSGC measurement are recorded. The value of the slope (b) in eq 9 is determined by plotting the logarithm of the GC signals of carbon dioxide vs headspace extraction number and conducting a linear curve fit. The overall calibration coefficient (K) in eq 9 is calculated based on the peak area at the first MHE HSGC measurement using eq 17

K)

mcarbonate A1 - A0

(17)

(24) Carroll, J. J.; Mather, A. E. J. Solution Chem. 1992, 21, 607. (25) Drimal, P.; Hmcirik, J.; Hoffmann, J. J. Polym. Environ. 2006, 14, 309.


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Figure 1. (a) Signal of CO2 measured by MHE HSGC and (b) accumulated CO2 generation during Escherichia coli growth.

The slope (b) of 0.148 and the coefficient (K) of 0.00354 µmol (0.155 µg) were obtained from the calibration at our given experimental conditions. By conducting a MHE HSGC experiment for bacterial growth incubation with a known amount of cell numbers (N0 ) 2.4 × 106), a linear relationship between the logarithm of the amount of carbon dioxide and the incubation time can be calculated from eq 14, i.e., lg mt ) lg(N0 ⁄ k) + Rt ) -0.432((0.031) + 0.465((0.018)t (18) Thus, the value of factor k () 6.5 × 106) was calculated. Applications. Determination of Bacteria Growth Rate. Figure 1a shows the carbon dioxide signals that were real-time measured by the MHE HSGC method during E. coli growth, in which a part of carbon dioxide (φCGn) was withdrawn from the system at each headspace sampling/measurement. The GC signal of carbon dioxide was increased during the MHE HSGC measurement if the amount of carbon dioxide produced is greater than that of being removed. However, if no carbon dioxide is produced or if the carbon dioxide produced is less than the amount that is removed from the system, a decrease of GC signals for carbon dioxide during the process should be observed. On the basis of the data in Figure 1a and eqs 10 and 9, we can obtain the time-dependent profile of the accumulated mass of carbon dioxide produced during E. coli growth at the given conditions, as shown in Figure 1b. 7824


Figure 2. Bacterial growth during the incubation: (a) monitored by the present method and (b) compared with the optical density method.

Figure 2a shows the relationship between the logarithms of mass of carbon dioxide vs incubation time for E. coli growth at two different bacterial inoculation levels. It is noticed that the bacterial growth rate was affected after a few hours incubation when helium was used as a pressurizing gas. It was further proved by a separate experiment that the lack of oxygen inhibits the growth, as observed in a previous work.26 However, such effect is not significant in the early incubation stage of E. coli bacteria growth, in which the level of oxygen in the system is sufficient for the bacteria growth. The present experiment was conducted at 37 °C, and the bacteria were inoculated in the cultural medium (LB) overnight in a cold room before the testing. Results show that E. coli growth rates are 0.472(±0.007) and 0.476(±0.013) h-1, respectively, indicating that there is no statistical difference for the E. coli between these experiments. In Figure 2b, it shows the comparison in the determination of E. coli growth rate between the present method and the optical density method, in which the slopes are 0.389 and 0.371 h-1, respectively, in the linear bacterial growth period. It can be seen that an increase of CO2 in the system was detected by HSGC at a much earlier stage than the optical density of the solution measured by spectroscopy, which is attributed to the high sensitivity of TCD in the GC system. Therefore, the present technique, i.e., HSGC with the MHE mode, has the advantages over the traditional methods not only for a fully automatic but (26) Goldberg, J. J.; Bramley, A. J.; Sjogren, R. E.; Pankey, J. W. J. Dairy Sci. 1994, 77, 3338.

It should be pointed out that a longer incubation time is required in order to get a sufficient amount of carbon dioxide in the system to reach the GC detection limit if the level of bacterial cells in the examined sample is very low. By selection of a longer equilibration time for the first MHE HSGC measurement, the frequency of headspace replacement is reduced. Thus, the oxygen level in the sample vial will not drop too much during the bacterial growth when an inert headspace pressurized gas is used. In the case that the bacterial growth is very sensitive to the oxygen level, compressed air should be selected as the headspace pressurized gas.

Figure 3. Comparison between the plate counting and present methods (slope ) 0.9941).

also more efficient measurement in the rate determination during E. coli growth. Determination of Bacteria Cell Numbers. A set of samples were prepared by inoculating different amounts of bacterial cells in the LB medium based on an E. coli culture with approximately 107 colony forming units per milliliter (CFU/mL). The exact cells numbers in the concentrated E. coli culture was measured by the plate counting method using a diluted culture (∼104 CFU/mL). Thus the bacterial numbers in these samples were obtained. The above samples (2 mL for each test) were placed in the headspace sample vials and measured by MHE HSGC. In Figure 3, it shows an excellent correlation between the data measured by the plate counting method and the present MHE HSGC method. Therefore, the present method is justifiable to be applied to examine the bacterial levels.

CONCLUSIONS We have proposed a MHE HSGC method using a commercial system for the determination of bacteria growth rate and the numbers that were initially inoculated in a cultural medium. The method was based on GC measuring the carbon dioxide produced in the headspace of a closed sample vial during the incubation. The present method is very simple, sensitive, and safe, which can easily perform an automatic measurement for bacteria growth at various desired incubation conditions. ACKNOWLEDGMENT The China Scholarship Council, State Education Department, is gratefully acknowledged for helping support Chunxu Dong as a visiting researcher at the Institute of Paper Science and Technology (IPST), Georgia Institute of Technology, Atlanta, GA. Received for review July 22, 2008. Accepted August 21, 2008. AC801537X


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In Situ Determination of Bacterial Growth by Multiple Headspace

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