Journal of Microbiological

Journal of Microbiological Methods 30 (1997) 125±132 Methods

An inexpensive chemostat apparatus for the study of microbial bio®lms Marvin Whiteley1 , Erin Brown, Robert J.C. McLean* Department of Biology, Southwest Texas State University, 601 University Drive, San Marcos, Tx 78666, USA Received 11 February 1997; received in revised form 13 June 1997; accepted 13 June 1997

Abstract

Continuous culture is a powerful technique for studying microbial bio®lms because it allows for the control of growth rate through nutrient limitation. These conditions offer a realistic view of how microorganisms interact in natural ecosystems. The vast majority of bio®lm research is performed with batch cultures, due to the high cost of commercially produced chemostats. We describe a chemostat that could be assembled on a limited budget and could be used in a variety of continuous culture experiments, including bio®lm assays. Our design consists of an Erlenmeyer ¯ask with custom-blown ports for aeration and waste removal/direct sampling, and a third port that allows microorganisms in the reaction ¯ask to be circulated through a modi®ed Robbins device and returned via the mouth of the ¯ask. This device enables the formation of highly reproducible bio®lm populations, of for example, Aeromonas hydrophila, at various growth rates. As such, it is well-suited for the study of the physiology and genetics of bio®lm bacteria.  1997 Elsevier Science B.V.

Keywords: Bio®lm; Chemostat; Continuous culture; Modi®ed Robbins device

1. Introduction direct observation has shown that surface-attached cells are profoundly different from their free-¯oating Microbiology often depends on the laboratory counterparts. For example, adhesion has been shown evaluation of planktonic microorganisms in test to activate genes involved in capsule production [2] tubes. Although these studies have provided signi®- and provide increased resistance to antibiotics [3±5] cant information on the morphology and physiology as well as resistance to other antimicrobials [6±8]. of microorganisms, many of these observations are Aggregates of attached cells, termed a bio®lm, thus not applicable in natural ecosystems. The reason for provide a new avenue of research on the natural this is that most microorganisms in nutrient-suf®cient aspects of microorganism response and development. environments are attached to surfaces [1]. While this A bio®lm may be de®ned as a biologically active would not be important if sessile and planktonic cells population of microorganisms that is attached to a were morphologically and physiologically identical, surface and enclosed by an extracellular matrix. Numerous aspects of bio®lm biology have been shown to be related to the growth rate of the *Corresponding author. Tel.: ϩ1 512 2453365; fax; ϩ1 512 2458713; e-mail: [email protected] planktonic microorganisms before their attachment 1Present address: Department of Microbiology, College of Medi- [9]. As the growth rate of planktonic microorganisms cine, University of Iowa, Iowa City, Ia 52242, USA. is changed, due to varying degrees of nutrient

0167-7012/97/$17.00  1997 Elsevier Science B.V. All rights reserved. PII S0167-7012(97)00054-7 126 M. Whiteley et al. / Journal of Microbiological Methods 30 (1997) 125 ±132 availability, aspects of their physiology, including antimicrobial susceptibility [10], production of virul- ence factors [11] and metabolic activity [12], change. To study the effects of growth rate, continuous culture techniques involving a chemostat can be utilized. Chemostats have long been utilized by microbiologists to provide continuous cultures of bacteria at various growth rates. Within the chemos- tat, there is an excess of nutrients, except for one that is growth-limiting. Under steady-state conditions, the loss of cells from the chemostat is the dilution rate and is equal to the growth rate of the bacterial population. The chemostat allows for the evaluation of the relationship between growth rate and factors such as antimicrobial susceptibility and propensity to form bio®lms. Batch cultures, as opposed to chemostats, are closed systems that are continually changing due to metabolic activities of the bacterial population. The initial growth rate of the population of microorga- nisms in a batch culture is high, but it decreases as Fig. 1. Chemostat apparatus for growing Aeromonas hydrophila. the nutrient concentration within the culture is (A) Air inlet, (B) waste outlet and (C) MRD outlet. depleted. With the growth rate in a constant state of ¯ux, accurate evaluations of bacterial responses at speci®c growth rates is impossible. Even with the aforementioned disadvantages, batch cultures are drilled into a rubber stopper that ®t tightly into the routinely used when evaluating bio®lm structure and top of the modi®ed Erlenmeyer ¯ask (chemostat). A susceptibility to antimicrobial agents, because many 1-ml syringe with the plunger removed was placed researchers do not possess the resources to utilize into one of the holes of the rubber stopper, a Pasteur chemostats. Thus, the design of an economically pipet was placed into another and a 20-cm piece of viable chemostat would facilitate the study of growth glass tubing bent in a 90-degree angle and containing rate affects on bio®lm physiology. This study pro- a cotton plug was placed into the third hole (Fig. 2). vides an evaluation of the reproducibility of a The three inserts ®t snugly within the holes and chemostat designed from normal laboratory glass- provided tight seals. A small hole was drilled into ware and utilized in combination with a modi®ed the plunger of the 1 ml syringe and 3 cm of a 50-cm Robbins device (MRD) [5] to produce bio®lms of piece of 1.8ϫ0.5 mm capillary tubing (Millipore Aeromonas hydrophila. 19-7477-01) was drilled through the plunger. The plunger was inserted into the syringe, as indicated in Fig. 2. This capillary tubing serves to deliver fresh 2. Materials and methods media into the chemostat and the syringe serves to protect this media source from aerosol contamina- 2.1. Development of the chemostat tion. A 40-cm length of 5/16 in. (0.79 cm)ϫ3/16 in. A standard 1000 ml Erlenmeyer ¯ask was modi- (0.48 cm) tubing (Fisherbrand) was attached to the ®ed to include an air inlet, a waste outlet and an waste outlet and closed with a clamp. A 40-cm outlet to which a MRD could be attached (Fig. 1). length of 5/16 in. (0.79 cm)ϫ3/16 in. (0.48 cm) Modi®cations were performed by a custom glass silicon tubing (Millipore XX80 0024) was then blower for $25 (US). Three 0.5 cm holes were attached to the Robbins device outlet and closed with M. Whiteley et al. / Journal of Microbiological Methods 30 (1997) 125 ±132 127

Fig. 3. Closed loop structure attaching a Pasteur pipet to the Robbins device outlet for sampling microbial bio®lms. (A) Chemostat, (B) MRD outlet, (C) peristaltic pump, (D) MRD or 60 Fig. 2. Components of the top of the chemostat. (A) Air outlet, (B) cm glass tubing and (E) Pasteur pipet in chemostat top. Arrows Pasteur pipet, (C) capillary tubing, (D) rubber stopper and (E) 1 indicate the direction of ¯ow. ml syringe.

и и a clamp. Silicon tubing was attached to the Robbins 0.06 mg; ZnSO427H O, 0.06 mg; CuSO 425H O, device outlet because it was necessary for the 0.006 mg; NaBO324 , 0.006 mg; Na MoO , 0.006 mg peristaltic pump that would be used at this location. and NaCl, 0.6 mg. This media was determined to be A 75-cm length of 5/16 in. (0.79 cm)ϫ3/16 in. carbon-limiting for Aeromonas hydrophila. All re- (0.48 cm) tubing (Fisherbrand) was attached to the agents utilized were of analytical grade or equiva- Pasteur pipet in the rubber stopper. The Robbins lent. A 5-cm stir bar was placed into the chemostat device outlet tubing was connected to the Pasteur to provide adequate mixing. pipet tubing by a 60-cm piece of glass tubing (I.D.ϭ Preparation of the aforementioned minimal salt 0.5 cm), which served to form a closed loop between media was performed through the use of stock the Robbins device outlet and the Pasteur pipet in the solutions. The stock solutions served to prevent rubber stopper (Fig. 3). A 75-cm length of 5/6 in. precipitation and the formation of unwanted products (0.79 cm)ϫ3/16 in. (0.48 cm) tubing (Fisherbrand), that often occurs during heat sterilization. A 12.5% with an attached 0.22 ␮m ®lter (Gelman), was (w/v) stock solution of glucose was prepared to attached to the air outlet of the chemostat. avoid the well-known formation of fructose and other sugars that occur during heat sterilization [13]. ϫ 2.2. Media preparation A 100 stock solution of CaCl2 as well as a stock solution containing the following compounds: и и и A 700-ml volume of sterile minimal salt media CoCl226H O, FeSo 427H O, NaCl, ZnSO 427H O, и и containing glucose as the sole carbon source was CuSO425H O, NaBO 3 , Na 2 MoO 42H O, MnSO 4 and prepared in the chemostat apparatus described in nitrilotriacetic acid was prepared and autoclaved at Section 2.1 with the rubber stopper tightly attached. 121ЊC for 15 min. Appropriate amounts of the stock The media contained, per liter of deionized water: solutions were added to the chemostat, which con- и glucose, 0.025 g; CaCl222H O, 0.017 g; NH 4 Cl, 0.24 tained an autoclaved solution of deionized water, g; MgSO424 , 0.0258; KH PO , 1.0 g; nitrilotriacetic KH 244 PO and NH Cl at pH 7.2. The total volume и и acid, 0.9 mg; CoCl226H O, 0.06 mg; FeSO 427H O, after addition of the stock solutions was 700 ml. 128 M. Whiteley et al. / Journal of Microbiological Methods 30 (1997) 125 ±132

2.3. Bacterium identi®cation and chemostat 2.4. Chemostat stabilization and Robbins device inoculation attachment

Aeromonas hydrophila was isolated from the The chemostat was allowed to equilibrate for six Edward's Aquifer (San Marcos, TX, USA) using days at 25ЊC. Conditions within the chemostat were R2A media (Difco 1826-17-1) and was identi®ed monitored daily by measuring the optical density at using Analytical Pro®le Index (API) 20E strips. The 600 nm and the pH of aseptically removed 5 ml bacterium was stored at Ϫ80ЊC, and this frozen stock aliquots of chemostat culture. Six days was found to culture was used to inoculate the chemostat. A be suf®cient for population stabilization to occur at constant ¯ow of compressed atmospheric air, sup- all dilution rates (Fig. 4). After the sixth day, the 60 plied by a standard wall outlet at a rate of 2000 cm piece of glass tubing was removed and replaced ml/min, as measured by water displacement, was by a sterilized MRD containing circular silicon plugs bubbled into the ¯ask. Before entering the chemostat, of 7 mm diameter (Tyler Research, Edmonton AB, the air was bubbled into a 500-ml water trap, to Canada). The MRD was sterilized with 100 ppm increase the humidity and remove oils and/or par- sodium hypochlorite for 15 min and rinsed in sterile ticulates. This air was then sterilized through a 0.22 distilled water. Procedures for the use of the MRD ␮m ®lter before entering the chemostat through the are provided in Nickel et al. [5]. Flow through the air inlet of the chemostat (Fig. 1). The chemostat MRD was established at 100 ml/min. culture was mixed using the 5 cm magnetic stir bar at approximately 700 rpm, with a Thermolyne 2.5. Batch culture bio®lm formation Cimarec 2 hot/stir plate (Baxter Scienti®c SP46925). The bacteria were allowed to grow in the chemostat Aeromonas hydrophila was grown overnight in as a batch system for 24 h at 25ЊC. the minimal salt media outlined in Section 2.2. A After 24 h, the clamp was removed from the 70-ml volume of the overnight culture was placed Robbins device outlet tubing, and the chemostat into 630 ml of sterile minimal salt media. This culture was pumped through the closed loop connect- culture was then pumped through a sterilized MRD ing the Robbins device outlet (Fig. 1) and the at 100 ml/min to allow bio®lm formation under Pasteur pipet in the top of the chemostat (Fig. 2) at batch conditions. 100 ml/min using a peristaltic pump (Millipore XX80 20E LO) positioned as shown in Fig. 3. The 2.6. Sampling and data analysis clamp was removed from the waste outlet and the decant was allowed to ¯ow down into a sterile 1000 After 24 and 48 h, ten randomly selected silicon ml Erlenmeyer ¯ask. The decant was removed discs were removed from the MRD [5]. Each disc throughout the experiments by gravity as the volume was then washed with 5 ml of sterile phosphate of the chemostat reached the level of the waste buffered saline (PBS), to remove planktonic bacteria, outlet. It is important to remove the clamp from the and placed into 2 ml of sterile PBS. The discs were Robbins device outlet tubing and begin pumping sonicated in a bath sonicator (Sonicor Instruments, through this closed loop before opening the waste SC-200TH) for 5 min and then vortex-mixed for 5 outlet, because this allows the volume of the entire min, to remove the adherent organisms. This pro- set-up to remain constant throughout the experiment. cedure maximized the number of organisms retained At this time, a second peristaltic pump (Pharmacia from the bio®lm discs. Serial dilutions were per- 19-4611-02) was used to add sterile media to the formed onto R2A agar and colony forming units chemostat through the capillary tubing and syringe, (CFUs) were noted. Log10 values were calculated for to yield dilution rates of 0.0083, 0.033 and 0. 133 the CFUs obtained. The set-up and sampling of the Ϫ h1 . The media was contained in a sterile reservoir bio®lms was performed in duplicate for each of the and stirred with the 5 cm stir bar, to maintain an three dilution rates and for the batch culture (Fig. homogenous solution. 5a,b). One-way analysis of variance (ANOVA) fol- M. Whiteley et al. / Journal of Microbiological Methods 30 (1997) 125 ±132 129

Fig. 4. Mean optical density measurements for the three dilution rates throughout a 21-day period (nϭ3). Optical density did not differ signi®cantly each day. Error bars are not shown to retain ®gure clarity. lowed by pairwise comparisons of the eight trials ous environment, with little visible attachment to the within each time period (24 and 48 h) were per- walls of the chemostat. formed to determine variation in the log10 number of The syringe adequately protected the incoming adherent organisms. Pairwise comparisons of trial media and no contamination of the incoming media means within each time period were performed using line was observed for 21 days of chemostat utiliza- a Tukey multiple comparison test with equal sample tion. The pH within the chemostat for all of the sizes at ␣ϭ0.05. dilution rates was maintained at between 6.8 and 7.0.

3.2. Variability within bio®lms

3. Results When comparing the log10 values of the number of microorganisms contained in the bio®lms at the 3.1. Variability within the chemostat three dilution rates, one-way ANOVA analysis re- vealed signi®cant population variation at 24 h ϭ Ͻ ϭ The variability from day to day in the number of (F7,729.48, p 0.0005) and at 48 h (F 7,72 17.26, microorganisms within the chemostat stabilized with- pϽ0.0005). Comparisons of means showed that in 48 h of the introduction of fresh media, as shown batch culture trials differed signi®cantly from by measurements of the optical density at 600 nm. chemostat trials at 24 and 48 h. These comparisons The ODs at the three dilution rates were identical also showed that the bio®lms that developed at Ϫ (Fig. 4). The volume of the chemostat was checked 0.0083 h1 were signi®cantly denser than those Ϫ at various intervals and was seen to ¯uctuate only grown at 0.033 or 0.133 h1 . No differences were slightly from day to day, between 595 and 607 ml, as observed between trials at identical dilution rates and measured with 250 ml graduated cylinders. The times. Greater variability was observed within the combination of stirring with bubbling of incoming batch cultures, compared to chemostat-born bio®lms, air in the chemostat served to maintain an homogen- as expressed in standard errors of the mean (Fig. 130 M. Whiteley et al. / Journal of Microbiological Methods 30 (1997) 125 ±132

Ϯ Fig. 5. Mean values ( SE) of the number of Aeromonas hydrophila (log10 ) present in bio®lms after 24 h (a) and 48 h (b) for batch culture and chemostat dilution rates of 0.0083, 0.033 and 0. 133 hϪ1 for nϭ10. Error bars for a dilution rate of 0.0083 hϪ1 are too small to be seen.

5a,b). Similar results were obtained with other obvious advantage of continuous data collection organisms and mixed cultures [14]. We observed that compared to batch culture. Although there was slight the number of microorganisms attached to the discs variation in the volume of the chemostat (595±607 was independent of the location of the disc within ml), this variation was of little signi®cance because it the MRD, as others have found in related applica- caused only slight deviations in the dilution rate of tions involving the MRD [15]. the chemostat. The variation in the volume can be attributed to the gravity over¯ow utilized. To be more precise, a pump could be attached to the 4. Discussion over¯ow tubing and set to decant the ideal amount of the culture from the chemostat. However, by choos- Planktonic population variability from day to day ing dilution rates that are adequately varied, a small within the chemostat was very low for the 21-day variation in volume is not a parameter that would monitoring period (Fig. 4), indicating that the popu- signi®cantly alter results. lations within the chemostat were stable. Thus, The syringe adequately protected the incoming multiple sampling within each chemostat set-up is media from contamination by aerosols from the possible throughout this stable period, providing the chemostat for up to 21 days. The use of capillary M. Whiteley et al. / Journal of Microbiological Methods 30 (1997) 125 ±132 131 tubing was instrumental in the protection of the fresh velocities on bio®lm formation, a parameter that has media because it provided for high ¯ow velocity been determined to be important [16]. Problems with while maintaining low ¯ow volume and so prevented the system lie in limitations concerning the automatic contamination of the sterile media reservoir. No control of parameters in the chemostat, such as pH problem in dispensing the ¯ow rates utilized was and measurement of dissolved oxygen, however, the noted with the 1 ml syringe. At higher ¯ow rates, versatility of this inexpensive system provides inves- however, the volume of the syringe may need to be tigators with an alternative to an expensive continu- increased by introducing a larger syringe into the ous culture system. rubber stopper. Maintaining ¯ow through the tubing and glass rod attached to the MRD outlet during equilibration of the chemostat allowed for bio®lm formation to occur Acknowledgements on the tubing and to equilibrate as the chemostat itself equilibrated. Therefore, when the glass tubing This project was supported by a faculty research was removed and the MRD attached, the volume of enhancement grant and the Biology Department at the system did not change (60 cm glass tube had Southwest Texas State University. We thank Clay approximately the same volume as the MRD), and Fuqua, Mary Barnes and David Villareal for helpful the MRD provided the only location for new coloni- suggestions. zation. If the closed loop attached to the MRD outlet was not allowed to equilibrate, the volume of the system would be altered at each sampling time and colonization could occur throughout the tubing. This References latter problem may cause a reduction in the number of organisms in the chemostat, depending on the [1] J.W. Costerton, G.G. Geesey, K.-J. Cheng, How bacteria length of tubing utilized. stick, Sci. Am. 238 (1978) 86±95. [2] D.G. Davies, A.M. Chakrabarty, G.G. 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