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.: 11 512 2453365; fax; 11 512 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 1.830.5 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)33/16 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)33/16 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. ? ? 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)33/16 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.5 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)33/16 in. (0.48 cm) tubing (Fisherbrand), that often occurs during heat sterilization. A 12.5% with an attached 0.22 mm ®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]. 3 2.2. Media preparation A 100 stock solution of CaCl2 as well as a stock solution containing the following compounds: ? ? ? A 700-ml volume of sterile minimal salt media CoCl226H O, FeSo 427H O, NaCl, ZnSO 427H O, ? ? 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. 1218C for 15 min. Appropriate amounts of the stock The media contained, per liter of deionized water: solutions were added to the chemostat, which con- ? 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 ? ? acid, 0.9 mg; CoCl226H O, 0.06 mg; FeSO 427H O, after addition of the stock solutions was 700 ml.