Glycoprotein methods protocols - biotechnology
Mucin Degrading Bacteria in Biofilms 43943936Growth of Mucin Degrading Bacteria in BiofilmsGeorge T. Macfarlane and Sandra Macfarlane1. IntroductionMucins are important sources of carbohydrate for bacteria growing in the humanlarge intestine. As well as being produced by goblet cells in the colonic mucosa, sali-vary, gastric, biliary, bronchial, and small intestinal mucins also enter the colon ineffluent from the small bowel. Particulate matter, such as partly digested plant cellmaterials, are entrapped in this viscoelastic gel, which must be broken down to facili-tate access of intestinal microorganisms to the food residues. It is estimated thatbetween 2 to 3 g of mucin enter the large bowel each day from the upper digestive tract(1), however, the rate of colonic mucus formation is unknown. Complex polymers,such as mucin must be degraded by a wide range of hydrolytic enzymes to smalleroligomers and their component sugars and amino acids before they can be assimilatedby intestinal bacteria.Pure and mixed culture studies have established that in many intestinal bacteria,synthesis of these enzymes, particularly β-galactosidase, N-acetyl β-glucosaminidase,and neuraminidase (2–4), is catabolite regulated, and is therefore dependent on localconcentrations of mucin and other carbohydrates. Although some colonic microorgan-isms can produce several different glycosidases, which allows them to completelydigest heterogeneous polymers (5–8), the majority of experimental data points to thefact that the breakdown of mucin and other complex organic molecules is a coopera-tive activity.In the large bowel, bacteria occur in a multiplicity of different microhabitats andmetabolic niches, on the mucosa, in the mucous layer, and in the colonic lumen, wherethey exist in microcolonies, as free-living organisms, or on the surfaces of particulatematerials (9,10).Wherever there are surfaces, bacteria form biofilms. They are usually complexmicrobial assemblages that develop in response to the chemical composition of thesubstratum and other environmental constraints. The available evidence shows that inthe colon, these microbiotas are heterogeneous entities that form rapidly on the sur-From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 440 Macfarlane and Macfarlanefaces of partly digested foodstuffs in the intestinal lumen, and in the mucous layercovering the mucosa (9). Sessile microorganisms in biofilms often behave very differ-ently from their nonadherent counterparts, and, in particular, the nature and efficiencyof their metabolism may be changed (10–12). Close spatial relationships between bac-terial cells in biofilms are ecologically important in that they minimize potential growthlimiting effects on crossfeeding populations (13).Although the large gut is often likened to a continuous culture system, this is anoversimplification. Consideration of colonic motility and the way in which intestinalmaterial is processed suggests that only the cecum and ascending colon exhibit char-acteristics of a continuous culture. The inaccessibility of the large bowel for experi-ments on the digestion of mucin by colonic microorganisms inevitably means that themajority of studies are made in vitro. A variety of models are available that enablepure and mixed populations of intestinal bacteria to be grown under anaerobic condi-tions, ranging from small screw-capped or serum bottles to more complex batch andcontinuous fermentation systems (chemostats).The effectiveness of in vitro model systems varies depending on the problem to beinvestigated, and each method has advantages and disadvantages. For example, fer-mentation experiments made using serum bottles are inexpensive, allow screening of anumber of substrates and/or fecal samples from different individuals, and require smallamounts of substrate and test sample (14). Depending on bacterial cell numbers, theexperiments can be designed to be of short duration, thereby minimising potentialdistortions in the data resulting from the selection of nonrepresentative populations ofmicroorganisms. Longer-term experiments effectively become enrichment cultures,selecting bacteria that are most efficient at utilizing the test substrates.However, these fermentations are uncontrolled, and yield little information on bac-terial metabolism, the organisms involved, or how the processes are regulated. Thelimited data provided by such experiments relate simply to the input of substrates andthe output of products. Other problems may also be encountered; for example, if high-substrate concentrations are used, strong nonphysiological buffers are needed to con-trol pH, which may have unpredictable effects on bacterial metabolism. If culture pHis not regulated, the environment in the vessel will change rapidly such that the fer-mentation conditions become physiologically irrelevant.Another factor to be considered with batch fermentations is that they are closedsystems, in which bacterial metabolic activities and environmental conditions in thecultures are constantly changing. Thus, at the beginning of bacterial growth, substrateconcentrations are high, and become depleted as the cells grow, whereas bacterialfermentation products and other autoinhibitory metabolites progressively accumulatein the culture.Many of these problems are avoided in continuous cultures, since they are opensystems that work efficiently at high bacterial population densities. Cell growth isstrictly controlled by the concentrations of limiting nutrients in the feed medium. Forthis reason, the organisms grow suboptimally at specific growth rates (µ) set by theexperimenter, through alterations in dilution rate (D), which is regulated by varyingthe rate at which culture medium is fed to the fermentation vessel. Mucin Degrading Bacteria in Biofilms 441The principal advantage of the chemostat in physiologic and ecologic studies onmicroorganisms is that it enables long-term detailed investigations to be made under amultitude of externally imposed steady-state conditions that are not possible withclosed batch-type cultures. A two-stage continuous culture model can be used forstudying the formation of mucin-degrading biofilms under nutrient-rich and nutrient-depleted conditions. The system comprises paired glass fermentation vessels fittedwith modified lids (Fig. 1), and several extra sampling ports fitted to the vessel sidesto which removable mucin baits or mucin gel cassette holders (Fig. 2) are attached.The fermenters are connected in series, with fresh culture medium being fed to ves-sel 1 (V1), and spent culture from this vessel being pumped into vessel 2 (V2). Thisfacilitates the study of mucin colonization under relatively carbohydrate-rich V1 andextremely carbon-limited (V2) environmental conditions, comparable to the proximaland distal colons.Three main protocols are outlined in this chapter for studying (1) mucous-degrad-ing bacterial consortia occurring in biofilms on the rectal mucosa, (2) mucinolyticspecies growing in artificial mucin biofilms in continuous culture models of the colonin the laboratory, and (3) mucinolytic microorganisms colonizing the surfaces of foodparticles in fecal material.Methods outlined for the isolation of bacteria from the rectal mucosa are essentiallydestructive, and primarily provide details of the types and numbers of different speciesthat take part in this process. They do not contribute information concerning the mul-ticellular organization of biofilm communities. However, the chemostat modeling pro-tocols afford useful comparative data on the enzymology and physiology of thebreakdown of mucin by adherent (mucin baits) and planktonic bacterial communities,under varying environmental conditions, while the use of mucin-coated glass slidesattached to cassettes facilitates microscopic examination of biofilm development.2. Materials1. Samples for scanning electron microscopy (SEM) are placed in 3% (v/v) glutaraldehydein 1 M PIPES buffer, pH 7.0. Then fix the samples with 4% (w/v) aqueous OsO4, dehy-drated stepwise in ethanol, which involves three changes (10 min) in each of 50, 75, 95,and, finally, 100% ethanol. Then dry the samples on a Poleron E 5000 critical-point drier,place on stubs, and gold-coat to a depth of 30 nm.2. Glass tubes, mucin gel cassettes, and fermentation vessels for baiting studies are manu-factured by Soham Glass, Ely, Cambs, UK.3. All formulated bacteriologic culture media and growth supplements are supplied byOxoid. Use as per manufacturer’s instructions. Unless stated otherwise, all chemicals areobtained from Sigma Aldrich Ltd. (Poole, UK). Agars for isolating specific bacterialgroups in mucin-degrading consortia are as follows:a. Nutrient agar (total facultative anaerobes).b. MaConkey agar no. 2 (lactose-fermenting and nonlactose-fermenting enterobacteria,enterococci).c. Azide blood agar base (facultative anaerobic cocci, some Gram-positive anaerobiccocci).d. Wilkins-Chalgren agar (total anaerobe counts). 442 Macfarlane and Macfarlanee. Wilkins-Chalgren agar plus nonsporing supplements (nonsporing anaerobes). Thesupplements contain hemin, menadione, sodium pyruvate, and nalidixic acid.f. Wilkins-Chalgren plus Gram-negative supplements (Gram-negative anaerobes). Theselective agents in this culture medium are hemin, menadione, sodium succinate,nalidixic acid, and vancomycin.g. MRS agar (lactobacilli).h. Perfringens agar and supplements (Clostridium perfringens and certain otherclostridia). The selective supplements (A and B) contain sulfadiazine, oleandomycinphosphate, and polymyxin B. All antibiotic additions are added at 50°C after auto-claving for 121°C at 15 min.Fig. 1. Two-stage continuous culture model used to study mucin-degrading biofilms undercarbon-excess (vessel 1, left) and carbon-limited (vessel 2, right) conditions. Mucin Degrading Bacteria in Biofilms 443i. Fusobacterium agar (fusobacteria). This comprises: 37.0 g/L Brucella agar base, 5.0g/L Na2HPO4, 1.0 g/L NaH2PO4, 1.0 g/L MgSO4·7H2O, 0.005 g/L hemin, at pH 7.6.This is autoclaved, cooled to 50°C and the following antibiotics are then added afterfilter sterilisation in 5 mL distilled water: 20 mg/L neomycin, 10 mg/L vancomycin,6.0 mg/L josamycin (ICN Biomedicals, Aurora, Ohio).j. Beerens Agar for selective isolation of bifidobacteria is made as follows: 42.5 g/LColumbia agar, 5.0 g/L glucose, 0.5 g/L cysteine HCl, 1.5 g/L purified bacteriologicagar. Five milliliters of propionic acid is added to these constituents, after they havebeen boiled and cooled to 70°C. The pH of the medium is then adjusted to 5.0 beforepouring the agar into Petri plates.Fig. 2. Mucin gel cassette used for investigating bacterial colonization of mucous surfacesin continuous culture experiments. The glass frame contains several slots into which are fittedremovable mucin-coated glass plates or microscope cover slips. 444 Macfarlane and Macfarlanek. Bacteroides mineral salts medium for selective isolation of members of the B. fragilisgroup consists of: 1.5 g/L KH2PO4, 1.0 g/L K2HPO4, 9.0 g/L NaCl, 1.2 g/L cysteineHCl, 1.2 g/L NaHCO3, 0.1 g/L CaCl2·2H2O, 0.15 g/L MgCl2·6H2O, 0.05 g/LMnCl2·4H2O, 0.05 g/L CoCl2·6H2O, 0.001 g/L FeSO4·7H2O, 0.005 g/L hemin, 0.005g/L vitamin B12, 1.0 g/L NH4SO4, 5.0 g/L glucose, 20 g/L purified bacteriologic agar.After autoclaving and cooling to 50°C, 5 mL of a filter sterilized antibiotic solution isadded, containing: 3.0 mg/L vancomycin, 10.0 mg/L nalidixic acid.4. Freezer vials containing Wilkins-Chalgren broth supplemented with 10% glycerol and2% porcine gastric mucin (Sigma Type III, partially purified), with pH adjusted to 6.5.5. Neuraminidase substrate (1 mg/mL N-acetylneuraminlactose). The neuraminidase stan-dard is 1 mg/mL N-acetylneuraminic acid (NANA). Make both in 0.1 M acetate buffer(pH 5.5).a. Solution A: 0.2 M sodium periodate (meta) in 9 M phosphoric acid.b. Solution B: 10% sodium arsenite in 0.5 M sodium sulfate/0.2 M H2SO4.c. Solution C: 0.6% thiobarbituric acid in 0.5 M sodium sulfate.Store solutions A and B at room temperature, and make solution C fresh daily.6. Make glycosidase assays using the following p-nitrophenyl substrates: N-acetyl α-D-galactosaminide, α-L-fucopyranoside, N-acetyl β-D-glucosaminide, and β-D-galacto-pyranoside, all prepared as 15 mM solutions in 0.01 M Tris buffer, pH 6.5. The stopsolution is a mixture of 0.5 M Na2CO3and 0.5 M NaHCO3.A standard curve using vary-ing dilutions of p-nitrophenol is used to calculate enzyme activities.7. PYG broth: 20 g/L glucose, 10.0 g/L Yeast extract, 5.0 g/L Tryptone Soya broth, 5.0 g/LPeptone water, 0.5 g/L cysteine HCl, 0.005 g/L hemin (see item 8). Add 40 mL of PYGsalt solution, 0.2 mL of vitamin K1solution (see item 8), and 10 mL of Tween-80 to950 mL of distilled water.a. To make PYG salt solution, add 0.2 g of CaCl2·2H20 and 0.2 g of MgSO4 to 300 mLof distilled H2O and dissolve by mixing. Then add a further 500 mL of H2O, togetherwith 1.0 g K2HPO4, 1.0 g of KH2PO4, 10.0 g of NaHCO3, and 2.0 g of NaCl. Finally,make up the volume to 1 L with distilled water.8. MIDI PYG broth is made as follows: 5.0 g/L peptone water, 5.0 g/L Pepticase (QuestInternational, Norwich, New York), 10.0 g/L Yeast extract, 0.5 g/L cysteine HCl, 10.0 g/Lglucose. In addition, the following solutions are added: 40.0 mL salts solution, 10 mLhemin solution, 0.2 mL vitamin K1. Add the hemin solution, vitamin K1, and cysteineafter the medium is boiled, but before it is dispensed into metal capped glass Universalbottles at 100°C and autoclaved. The salt solution is made in the same way as for normalPYG medium, but the NaCl concentration is increased to 50 g. The haemin solution ismade as follows: Dissolve 50 mg of hemin in 1 mL of 1 M NaOH; make to 100 mL withdistilled water, then autoclave at 121°C for 15 min. Store at 4°C. Vitamin K1: Dissolve0.15 mg in 30 mL 95% of ethanol. Store at 4°C in a brown bottle. Discard after 1 mo. Foridentification of Gram-positive organisms, add 2.5 mL of 1:10 Tween-80 in distilled waterat the same time as cysteine to the medium.9. To make Balch trace elements solution (15), add the following constituents to 600 mL ofdistilled water: 3.0 g MgSO4·7H2O, 0.45 g MnCl2·4H2O, 1.0 g NaCl, 0.10 g FeSO4·7H2O,0.18 g CoSO4·7H2O, 0.10 g CaCl2·2H2O, 0.18 g ZnSO4·7H2O, 0.01 g CuSO4·5H2O, 0.018g Al(SO4)2·12H2O, 0.01 g H3BO4, 0.01 g NaMoO4·2H2O, 0.19 g Na 2SeO4, 0.092 gNiCl2·6H2O. Adjust the solution to pH 7.0 with 1M KOH, then make up to 1 L. Store at4°C until use. Mucin Degrading Bacteria in Biofilms 4453. Methods3.1. Enumeration and Identificationof Mucinolytic Bacteria in Rectal Biopsies1. Rectal biopsy material is obtained from hospital out-patients. Tissue samples should beimmediately placed in preweighed sterile Bijoux bottles containing 4 mL of a suitableanaerobic transport medium, such as Wilkins-Chalgren broth (see Note 1).2. Weights and sizes of the samples are measured before placing them in an anaerobic cabi-net (atmosphere 10% H2, 10% CO2, 80% N2) at 37°C. Speed is important during this step(see Note 2).3. Mascerate the biopsy material using a sterile glass tissue homogeniser. One mL of thissample is serially diluted (10-fold dilutions to 10–5) in test-tubes containing 9 mL half-strength sterile anaerobic Peptone water (see Note 1).4. Plate out 50 µL of the original sample and 100 µL of all dilutions to 10–5in triplicate, ontoa range of selective and nonselective culture media, using sterile tips and glass spreaders(see Subheading 2., item 3). Plates for aerobic incubation are removed from the anaero-bic cabinet and incubated at 37°C.5. Aerobic plates are incubated for 2 d, and anaerobic plates for up to 5 d, with periodicexamination, before counting of colonies.6. The bacteria are then characterized on the basis of their Gram staining characteristics,cellular morphology, fermentation products (16), and cellular fatty acid methyl ester(FAME) profiles (see Note 3).7. Fermentation products (short chain fatty acids, lactate, succinate) are analysed by growingthe organisms as pure cultures in PYG broth (see Subheading 2., item 7) for 24 h, thencentrifuging (13,000g, 10 min) to obtain a clear supernatant for GC or HPLC analysis.8. Bacterial cellular fatty acids are extracted from overnight cultures of the organisms in MIDIPYG broth (see Subheading 2., item 8). After centrifugation to obtain a cell pellet, FAMEsare produced by saponification, methylation, and finally, solvent extraction. FAMEs are thenseparated using a 5898 A Microbial Identification System. (Microbial ID, Newark, DE).9. FAMEs are automatically integrated and numerical analysis done using standard MISLibrary Generation Software which identifies the organisms.10. Colonies for further study are grown on agar plates and removed with sterile swabs into2-mL freezer vials which are then stored at –80°C (see Subheading 2., item 4).3.2. Mucin-Degrading Enzymes in Mucosal Bacteria1. Grow individual isolates at 37°C in Wilkins-Chalgren Broth, supplemented with 5 g/Lpartially purified porcine gastric mucin, in anaerobic Universal bottles (prepare by boil-ing and dispensing the media into the bottles at 100°C, and then autoclaving).2. After the cultures have grown, keep a portion of the whole culture, and harvest some ofthe bacteria by centrifugation (13,000g, 30 min). Retain the cell-free supernatants and thewhole-cell cultures for comparative determinations of cell-bound and extracellular mu-cin-degrading enzymes.3. Calculate culture dry weights (see Note 4) by spinning down 1 mL of the culture in amicrocentrifuge at 13,000g for 5 min. Discard the supernatant and add a further 1 mL,repeating the process until a total of 5 mL of culture have been collected. Finally, washthe bacterial pellets with distilled water. Place the microcentrifuge tubes containing thebacteria in a drying oven at 90°C for 3 d, or until dry. Determine the culture weights byweighing the sample and calculating the dry weight per milliliter of original culture. 446 Macfarlane and Macfarlane4. Neuraminidase assay: Test solution (0.05 mL) and boiled controls are incubated with0.1 mL N-acetylneuraminlactose for 1–2 h at 37·C. Stop the reaction by boiling at 100·Cfor 2 min. Add solution A (0.1 mL), mix, and allow to stand for 20 min at room tempera-ture. Next add solution B (0.4 mL) and mix until the yellow colour disappears. Then addsolution C (1.0 mL), and heat the mixture in a boiling water bath for 15 min, beforecooling in cold water for 5 min. After centrifuging at 13,000g for 5 min to remove theprecipitate, read the absorbance of the supernatant at 549 nm. Prepare a standard curve byusing known amounts of NANA and developing these with the test after the boiling stage(see Subheading 2., item 5).5. In glycosidase assays, incubate 0.5 mL of test solution at 37°C with 0.25 mL of substrate,until a yellow color begins to appear, or for 1 h. Terminate the reaction by adding 0.75mL of stop solution and then centrifuge at 13,000g for 5 min before reading the absor-bance at 420 nm (see Subheading 2., item 6).3.3. In Vitro Modeling System Using Mucin Baitsand Mucin Gel Cassettes1. Use glass fermentation vessels (560 mL working volume) with modified lids, containingseveral extra sampling ports in these experiments (see Fig. 1).2. Use fresh feces to prepare 20% (w/v) inocula in 100 mM anaerobic sodium phosphate buffer(pH 6.0), by macerating the stool in a stomacher for 5 min and then sequentially filteringthrough 500- and a 250-µm metal sieves to remove particulate material. Add 200 mL of thisinocula to 200 mL of double-strength culture medium (see step 4) in the fermenter.3. Constantly stir the fermentation vessels and set the dilution rates at 0.1/h (see Note 5)operating pH at 6.0, and temperatures at 37°C. Maintain anaerobic conditions by spargingcultures with O2-free N2 at a low gas flow rate (2.4 L/h).4. A suitable culture medium (see Note 6) comprises: 2.0 g/L starch (soluble), 0.5 g/L pec-tin (citrus), 0.5 g/L inulin, 0.5 g/L xylan (oatspelt) 0.5 g/L arabinogalactan (larchwood),0.5 g/L guar gum, 2.0 g/L mucin, 3.0 g/L Tryptone, 3.0 g/L Peptone water, 4.5 g/L Yeastextract, 0.015 g/L hemin, 4.5 g/L NaCl, 2.5 g/L KCl, 0.45 g/L MgCl2·6H2O, 0.2 g/LCaCl2·6H2O, 0.4 g/L KH2PO4, 0.8 g/L cysteine, 0.4 g/L Bile salts No.3, 20 mL Balchtrace elements (see Subheading 2., item 9); 0.5 mL Tween-80.5. When the chemostats reach steady state, at least 10 turnovers in culture volume, as indi-cated by analysis of short chain fatty acid (SCFA) profiles (see Note 7), extracellular andcell-associated samples of the lumenal populations, for comparative purposes, are takenfor both chemical and enzymic measurements, and for viable counts of bacteria, using arange of selective and nonselective agars (see Subheading 2., item 3). The range of dilu-tions of samples for plating should be increased to 10–3–10–8, to take into account thegreater numbers of bacteria in these samples.6. Place sterile mucin gels in glass tubes (17 × 12 mm, 2 mL/vol), or mucin gel cassettes inthe fermenters, in each of the five side sample ports. Prepare the baits by placing the glasstubes in a covered glass beaker, autoclave them, and when cool, pour over 2% (w/v)porcine gastric mucin with the addition of 0.2% (w/v) purified bacteriologic agar, afterautoclaving and cooling. Then place the gels in an anaerobic cabinet to set. Make the gelcassettes by autoclaving a solution containing 0.8% (w/v) purified bacteriologic agar and2% (w/v) mucin in distilled water, then aseptically coating sterile glass microscope cov-erslips, or custom made glass plates, with this solution at 60°C, before fitting them withsterile forceps to the cassette holder.7. Remove gels periodically over 48 h for analysis. Wash the surfaces gently with 100 mManaerobic sodium phosphate buffer, pH 6.0 to remove loosely adherent planktonic micro- Mucin Degrading Bacteria in Biofilms 447organisms. Resuspend the gel material in 10 mL anaerobic glycosidase buffer at pH 6.5for enzymic analysis, and bacterial enumeration (methods as for biopsy samples). Use themucin-coated glass coverslips directly for microscopic analysis. Take the samples andfreeze at –20°C for carbohydrate analysis (see below).8. Keep samples of the planktonic populations and fermenter media for measurements ofmucin carbohydrate uptake. Rates are calculated as follows: qs= D (So–S)/x, where D =dilution rate, So= substrate entering fermenter, S = residual substrate in fermenter, and x= community dry weight (qs = substrate utilized/[min·mg dry weight bacteria]).9. Mucin oligosaccharides are determined by hydrolysing samples in 2 M H2SO4 for 2 h at100°C. A standard sugar mix containing 1 mg/mL of fucose, galactosamine, glucosamine,galactose, glucose and mannose is also hydrolyzed in 2 M of H2SO4. To 100 mL ofhydrolysate, or standard sugar mix (in 2 M of H2SO4), add 5 mL of internal standardsolution (0.02 mg/mL deoxygalactose in high purity water), mix, and then run on a DionexDX 500/ED 40 analytical system.10. Neutral and amino sugars are separated by high-pressure anion exchange chromatogra-phy with pulsed amperometric detection (HPAEC-PAD) on a Dionex CarboPac PA 10(4 × 250 mm) column equipped with a Dionex PA 10 guard column (4 × 50 mm) and aDionex ED 40 detector using the Dionex DX500 system (see Note 8). High-puritydeionized water (18 MΩ cm) should be employed in these tests, after being filteredthrough 2-mm filters. Sodium hydroxide (50%, low in carbonate) is purchased from BDH,Poole, Dorest, UK. Solution 1 is 0.2 M NaOH, and solution 2 isdistilled water. Duringpreparation of these solutions the water is sparged with helium for 15 min before andduring the addition of NaOH. Carry out monosaccharide detection using a gold cell andpreset carbohydrate waveforms. Achieve isocratic separation of neutral and amino sugarsat 1.0 mL/min with 30 mM NaOH. After 20 min, the column is purged with 100 mMNaOH for 10 min, then re-equilibrated with the starting conditions for 10 min before thenext sample is injected. Use a PC 10 Pneumatic controller to introduce 0.3 M NaOH at aflow rate of 0.5 mL/min to the column effluent, before the PAD cell, which minimizesbaseline drift and increases the analytical signal. Use a Dionex Eluant De-gas Module tosaturate the eluants with helium gas to minimize CO2absorption. Transfer the samples topolyvials with 20-mm filters and inject with a Dionex AS40 Automatic sampler via aDionex high pressure valve. Use a Dionex Peaknet Software data handling system to plotand integrate results.11. Determine NANA by hydrolyzing samples in 0.05 M of H2SO4for 1 h at 80°C. Thenvisualize released NANA colorimetrically as in the neuraminidase assay (see Subhead-ing 3.2., item 4), after the boiling stage.3.4. Short-Term Fermentation Studieson Biofilm and Lumenal Populations in Chemostats1. Take culture from both culture vessels, together with material from mucin baits or gelcassetes. At this time, also remove the biofilms that form on the vessel walls. After wash-ing and resuspension in 0.1 M sodium phosphate buffer, pH 6.0, the samples are centri-fuged at 13,000g for 20 min. Resuspend each of the resulting pellets in 20 mL anaerobic0.4 M phosphate buffer, pH 6.0. Add 10 mL of each suspension to 40 mL of chemostatmedium in 70 mL Wheaton serum, bottles under N2, at 37°C. Take samples hourly for 6 h(see Note 9), centrifuge at 13,000g for 10 min then freeze the supernantants at –20°C forsubsequent measurement of SCFA and other organic acids. Also freeze samples for analy-sis of residual mucin. Make dry weight determinations on the samples as in Subheading3.2., item 3 for calculations of specific rates of substrate uptake and utilization. 448 Macfarlane and Macfarlane3.5. Desorption of Mucinolytic Bacteria from Food Materials in Feces1. Fresh fecal samples are homogenized in anaerobic 0.1 mol/L sodium phosphate buffer(pH 6.5) to give 10% (w/v) slurries. Pass fecal slurries sequentially through 500- and250-mm diameter sieves. Retain filtrates containing nonadherent bacteria under anaero-bic conditions for enzymic analysis, fermentation studies, and bacterial counts.2. Material retained on the filters is washed twice with 500 mL of the anaerobic buffer toremove loosely adherent organisms. Washed food particles are subsequently incubated at37°C under anaerobic conditions (O2-free N2atmosphere) in phosphate buffer in the pres-ence of a surfactant such as 0.001% (w/v) cetyltrimethylammonium bromide (CTAB)(BDH) for 30 min, with mixing. Samples are then refiltered to remove food materials.Retain filtrates containing adherent bacterial populations and residual food materialsunder anaerobic conditions (see Note 10).3. Place samples of particulate material, washed particulate material and CTAB treated par-ticles in 3% (v/v) glutaraldehyde in PIPES buffer (0.1 M, pH 7.4) at 4°C for SEM (seeSubheading 2., item 1).4. Perform enzymic analysis on bacteria extracted directly from feces and organismsremoved from particulate materials with CTAB resuspended in 0.1 M sodium phosphatebuffer (pH 6.5), as described in step 2.5. Serially dilute unattached fecal bacteria and organisms desorbed from particulate mate-rial with 0.001% CTAB on a variety of selective and nonselective agars for enumeration(see Subheading 2., item 3).3.6. Mucin Fermentation Experimentswith Biofilm and Nonadherent Fecal Bacteria1. Incubate biofilm and nonadherent faecal bacteria from fecal material at 37°C under O2-free N2in 0.1 M sodium phosphate buffer (pH 6.5), in sealed 70-mL serum bottles(Wheaton) with mucin. Take samples (2 mL) periodically over a period of 6 h (see Sub-heading 3.4.) and freeze for analysis of fermentation products and residual mucin carbo-hydrate. Determine culture dry weights (see Subheading 3.2., item 3) to calculate specificrates of substrate utilization and fermentation product formation.4. Notes1. The benefits of using rectal biopsies to study mucosal bacterial populations are that, formost of the time, the rectum is empty and the mucosa is clean, and uncontaminated withlumenal material, and samples are relatively easy to obtain since the patients/volunteersdo not need to be cleaned, or otherwise specially prepared, as would be required whenremoving tissue from the proximal or distal bowel during colonoscopy. Wilkins-Chalgrenbroth is sterilized by autoclaving (121oC, 15 min). The bottles are prereduced by beingplaced in an anaerobic chamber or gas jar (Don Whitley Scientific, Shipley, Yorks) withloose lids, and allowed to cool. This is also used to prepare anaerobic peptone water forthe dilution series.2. Weights and physical dimensions of the biopsy samples are needed to estimate bacterialcell densities, either as per unit area or as per unit tissue weight. Rapid handling of samplesis essential to prevent growth of facultative anaerobes, and inactivation of strict anaer-obes during transport.3. Bacterial CFAs are highly stable and reproducible taxonomic markers. This allows phe-notypic analysis of pure and mixed populations of intestinal microorganisms to be under-taken by extracting their CFAs and comparing patterns of the methyl esters by GC, using [...]... glycosidase assays using the following p-nitrophenyl substrates: N-acetyl - D - galactosaminide, - L -fucopyranoside, N-acetyl - D -glucosaminide, and - D -galacto- pyranoside, all prepared as 15 mM solutions in 0.01 M Tris buffer, pH 6.5. The stop solution is a mixture of 0.5 M Na 2 CO 3 and 0.5 M NaHCO 3 .A standard curve using vary- ing dilutions of p-nitrophenol is used to calculate enzyme... cultures have grown, keep a portion of the whole culture, and harvest some of the bacteria by centrifugation (13,000g, 30 min). Retain the cell-free supernatants and the whole-cell cultures for comparative determinations of cell-bound and extracellular mu- cin-degrading enzymes. 3. Calculate culture dry weights (see Note 4) by spinning down 1 mL of the culture in a microcentrifuge at 13,000g for 5 min.... agar plates and removed with sterile swabs into 2-mL freezer vials which are then stored at –80°C (see Subheading 2., item 4). 3.2. Mucin-Degrading Enzymes in Mucosal Bacteria 1. Grow individual isolates at 37°C in Wilkins-Chalgren Broth, supplemented with 5 g/L partially purified porcine gastric mucin, in anaerobic Universal bottles (prepare by boil- ing and dispensing the media into the bottles at... either a batch or a continuous culture. The reactor is stirred magneti- Fig. 3. Schematic diagram showing a simple single-stage continuous culture system. Mucin Degrading Bacteria in Biofilms 445 3. Methods 3.1. Enumeration and Identification of Mucinolytic Bacteria in Rectal Biopsies 1. Rectal biopsy material is obtained from hospital out-patients. Tissue samples should be immediately placed in preweighed... ecosystems. When the chemostats reach steady-state, sterile mucin gels in baits, or glass coverslips, are fitted to the fermenters and removed periodically for analysis. Partially purified porcine gastric mucin is used in these experi- ments because of its compositional and structural similarity to human mucins. 7. Bacteriologic analysis is time-consuming and labor-intensive, especially when working with... 3.0 mg/L vancomycin, 10.0 mg/L nalidixic acid. 4. Freezer vials containing Wilkins-Chalgren broth supplemented with 10% glycerol and 2% porcine gastric mucin (Sigma Type III, partially purified), with pH adjusted to 6.5. 5. Neuraminidase substrate (1 mg/mL N-acetylneuraminlactose). The neuraminidase stan- dard is 1 mg/mL N-acetylneuraminic acid (NANA). Make both in 0.1 M acetate buffer (pH 5.5). a. Solution... as Wilkins-Chalgren broth (see Note 1). 2. Weights and sizes of the samples are measured before placing them in an anaerobic cabi- net (atmosphere 10% H 2 , 10% CO 2 , 80% N 2 ) at 37°C. Speed is important during this step (see Note 2). 3. Mascerate the biopsy material using a sterile glass tissue homogeniser. One mL of this sample is serially diluted (10-fold dilutions to 10 –5 ) in test-tubes containing... microprocessor, and contains several computer-generated libraries comprising data on over several hun- dred different aerobic and anaerobic species. 4. Culture dry weights or, alternatively, the protein content of the sample (e.g., Lowry method) are used for calculating specific enzyme activities. 5. The basic experimental setup of a semicontinuous or continuous culture type fermenta- tion system, fitted with automatic... stir the cultures using a direct-drive impeller, with spent culture medium and bacterial cells being removed from the reactor using a dedicated pump. Dilution rate (D) is calculated as the working volume of the fermentation vessel divided by the flow rate of growth medium into the system. Specific growth rates (µ) of bacteria in a chemostat are equal to D, under steady-state conditions. The mean residence... in test-tubes containing 9 mL half- strength sterile anaerobic Peptone water (see Note 1). 4. Plate out 50 µL of the original sample and 100 µL of all dilutions to 10 –5 in triplicate, onto a range of selective and nonselective culture media, using sterile tips and glass spreaders (see Subheading 2., item 3). Plates for aerobic incubation are removed from the anaero- bic cabinet and incubated at 37°C. 5. . using the following p-nitrophenyl substrates: N-acetyl α-D-galactosaminide, α-L-fucopyranoside, N-acetyl β-D-glucosaminide, and β-D-galacto-pyranoside, all. heterogeneous entities that form rapidly on the sur-From :Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield