NACE TM0194-04 Field Monitoring Ofbacterial Growth in Oil

NACE Standard TM0194-2004Item No. 21224 THE CQRROSION SQCIETY Standard Test Method Field Monitoring of Bacterial Growth in Oil and Gas Systems This NACE International (NACE) standard represents a consensus of those individual members who have reviewed this document, its scope, and provisions. Its acceptance does not in any respect preclude anyone, whether he has adopted the standard or not, from manufacturing, marketing, purchasing, or using products, processes, or procedures not in conformance with this standard. Nothing contained in this NACE standard is to be construed as granting any right, by implication or otherwise, to manufacture, sell, or use in connection with any method, apparatus, or product covered by Letters Patent, or as indemnifying or protecting anyone against liability for infringement of Letters Patent. This standard represents minimum requirements and should in no way be interpreted as a restriction on the use of better procedures or materials. Neither is this standard intended to apply in all cases relating to the subject. Unpredictable circumstances may negate the usefulness of this standard in specific instances. NACE assumes no responsibility for the interpretation or use of this standard by other parties and accepts responsibility for only those official NACE interpretations issued by NACE in accordance with its governing procedures and policies which preclude the issuance of interpretations by individual volunteers. Users of this NACE standard are responsible for reviewing appropriate health, safety, environmental, and regulatory documents and for determining their applicability in relation to this standard prior to its use. This NACE standard may not necessarily address all potential health and --``,`,``,`,````,,`,,``,,`-`-`,,`,,`,`,,`--- safety problems or environmental hazards associated with the use of materials, equipment, and/or operations detailed or referred to within this standard. Users of this NACE standard are also responsible for establishing appropriate health, safety, and environmental protection practices, in consultation with appropriate regulatory authorities if necessary, to achieve compliance with any existing applicable regulatory requirements prior to the use of this standard. CAUTIONARY NOTICE: NACE standards are subject to periodic review, and may be revised or withdrawn at any time without prior notice. NACE requires that action be taken to reaffirm, revise, or withdraw this standard no later than five years from the date of initial publication. The user is cautioned to obtain the latest edition. Purchasers of NACE standards may receive current information on all standards and other NACE publications by contacting the NACE Membership Services Department, 1440 South Creek Drive, Houston, Texas 77084-4906 (telephone + I [281] 228-6200). Revised 2004-1 1-15 Approved 1994 NACE International 1440 South Creek Dr. Houston, Texas 77084-4906 + I 281/228-6200 ISBN 1-57590-197-7 O 2004, NACE International Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale, 10/24/2005 13:11:09 MDT TMOI 94-2004 Foreword This standard describes field test methods that are useful for estimating bacterial populations, including sessile bacterial populations, commonly found in oilfield systems. The described test methods are those that can be done on site and that require a minimum of laboratory equipment or supplies. The described test methods are not the only methods that can be used, but they are methods that have been proved to be useful in oilfield situations. This standard is intended to be used by technical field and service personnel, including those who do not necessarily have extensive or specific training in microbiology. However, because microbiology is a specialized field, some pertinent and specific technical information and explanation are provided to the user. Finally, the implications of the results obtained by these test methods are beyond the scope of this standard. The interpretation of the results is site- and system-specific and may require more expertise than can be provided by this standard. This standard is loosely based on a document produced by the former Corrosion Engineering Association (CEA). CEA operated in the United Kingdom under the auspices of NACE and the Institute of Corrosion (Icorr).") This NACE International standard was originally prepared in 1994 by NACE Task Group T-IC-21 under the direction of Unit Committee T - I C on Corrosion Monitoring in Petroleum Production. It was revised in 2004 by Task Group 214 on Bacterial Growth in Oilfield Systems-Field Monitoring: Review of NACE Standard TM0194, which is administered by Specific Technology Group (STG) 31 on Oil and Gas Production-Corrosion and Scale Inhibition and by STG 60 on Corrosion Mechanisms. It is issued by NACE under the auspices of STG 31. (I) Institute of Corrosion (icor), P.O. Box 253, Leighton, Buzzard Beds, LU7 7WB, England In NACE standards, the terms shall, must, should, and may are used in accordance with the definitions of these terms in the NACE Publications Style Manual, 4th ed., Paragraph 7.4.1.9. Shall and must are used to state mandatory requirements. The term should is used to state something good and is recommended but is not mandatory. The term may is used to state something considered optional. NACE International I --``,`,``,`,````,,`,,``,,`-`-`,,`,,`,`,,`--- Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale, 10/24/2005 13:11:09 MDT ....... NACE International II --``........................................... .................. 10/24/2005 13:11:09 MDT .......... ... . 16 Table D2: Duplicate Serial Dilution ..`--- Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale..... ... 15 Appendix E: Alternative SRB Growth Medium Formulation....................... 10 Appendix B: Rapid Methods for Assessing pulations .................... .............................................`-`-`................................................`...... Sampling Procedure onic Bacteria ........`.............. Culture Techniques ............. 17 . Assessment of Sessile Bacteria.........................................`................. Appendix C: Membrane Filtration-Aided Ba Appendix D: Bacterial Culturing by Serial Dilution ....................````.``.... . 1 3.... .......................... ............ ........................ ............................................................................. Table D I : Single Serial Dilution ..... 17 Table 1: Results Interpretation Table .``...........`........ TM0194-2004 Standard Test Method Field Monitoring of Bacterial Growth in Oilfield Systems Contents 1........................................... Appendix A: Glossary............................ 3 4...... ............`....... General.. Evaluation of Chemicals for Control of P 5....... 1 2...........`........................................ 17 Table D3: Five Replicate Serial Dilution................. etc.2 Some systems may exhibit large profiles is a goal of the work.`-`-`. In other cases.1. population variations over a short time. --``.``.1 .1. However.3 This standard deals only with bacteria and does commercially available “test kits” for detecting various not consider other organisms that may be found in types of microorganisms.1. While these outlined here.1..3 Sampling Frequency hamper accurate assessment of bacteria numbers..5 Media formulations for enumerating common bacterial contamination cannot be given. shut-ins.`. 1. associated with bacterial growth in oilfield systems is given in NACE Publication TPC #3. the user could use this (algae). To establish the natural variation in bacteria numbers.1. these methods standard to evaluate the suitability of these test kits for are not applicable to marine organisms such as any particular situation. greater NACE International 1 Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. TMOI 94-2004 Section 1: General 1. However. to five days is often used.2 Field operators should be solicited for valuable samples (bulked or otherwise) should normally be information.7 Methods for evaluating sutface attached (sessile) bacteria are addressed.``. phytoplankton this standard.1 Sampling frequency depends on how the in reported bacterial populations. or system. Communication with sample points that are representative of the operators can also ensure that baseline sampling system.10 Further information on the corrosion problems applications. these are not discussed in oilfield fluids.1. I . Attached bacterial populations and gas systems. (bulked).1.. 2. technologies are not specifically recommended.`.2 A glossary of terms used in this standard is technology that can be useful. I Natural bacterial population fluctuation and uneven bacterial distribution within water systems may 2.’ methods that are used. However. 2.1.8 Emerging technologies for the rapid proved useful. critical past biological monitoring (background) baseline.). The importance of 1.1 This standard describes field test methods for these bacteria in oilfield problems is usually not estimating bacterial populations commonly found in oil adequately considered. several samples field system operates and should encompass the should be taken on each occasion and combined various stages of its operation. “action” concentrations for 1. determine the applicability of these new methods to the sitekystem. selection of proper sample sites can best be made in cooperation with operators. These operators can often provide. this procedure may mask fluctuations in population profiles. oilfield bacteria are given.1 Scope 1. Similarly. I .1.. In addition. if determining such 2. may be harmless in another.. twice-daily sampling over three excursions (pigging. or fungi. such as archaebacteria. bacterial population determinations are one more diagnostic tool useful in assessing oilfield problems. protozoa. it is not the intent of this standard to prevent the use of any 1.1 Baseline Sampling In addition.`.`.1.’ Section 2: Sampling Procedures for Planktonic Bacteria 2. 10/24/2005 13:11:09 MDT . the user must provided is Appendix A. If baseline studies described here show a large variation 2.`.9 The simple presence of bacteria in a system 1.`--- 1. there are a number of 1. This work should also establish the data taken from the system.6 This standard describes dose-response (time- kill) testing for evaluating biocides used in oilfield 1.1.````. zooplankton (copepods).3.`. problems in one situation.. In addition. Rather. or taken randomly over several days to establish a obtain. However. biocide treatments. It is not the intent of this standard to exclude additional techniques that can be 1. they are not the only microbial ecology.4 Because effective sampling is essential to any does not necessarily indicate that they are causing a successful analysis. caution should be exercised determination of bacterial populations and bacterial with any technique that is at variance from those activity are addressed (See Appendix B). emphasis is given to sampling problem. bacterial populations causing methods that are suitable for use in oilfield conditions.3. I . Although these techniques have are often the most important component of a system’s been successful in the oil field. Therefore. As an example of what sample frequency occurs during normal operations and not during might be required. 2.`.. a sample thief. system water.5 If the detection of very low bacterial populations --``.. 2.g. a glass container may be used to estimate the water volume used in the should be used. samples should normally be taken immediately Prior consultation with operators may identify prior to treatment and at random intervals over alternatives to avoid some sampling problems.3 The sample container should be rinsed with the 2. 10/24/2005 13:11:09 MDT . it should be noted that errors culturing procedure (for those workers who feel more in bacterial population estimates still could result. This practice helps minimize population be used. occur before the water is used.g. with 2. but any new containers usually tight emulsions may not contain enough water for suffice.. preferably on site. Sterile containers are water cuts (¡.4 During sampling of systems containing both oil time while being held in any sample container. and then closed with a screw cap (preferably with an airtight 2. if 2. For example. is required. The cap should only be removed just prior to sampling and replaced immediately afterwards. In the latter case.3. 2. Sample and water. bacteria. enough water for a particular test.2 To minimize changes. several days after each treatment. 2. time delay occurring between sampling and analysis should be held constant for all testing. or polypropylene.. and location of the sample. phase separation should be permitted to containers should be made of sterile glass.3.. even under 2.3.`.3 Samples may be taken from either flowing (e. additional at or close to the suspected location of the problem).2 Sample temperature and pH.5 To fully understand the ecology of a samples should be obtained by cracking a valve and system.2 Sampling Bottles sampling bomb. these variables can only be established with extensive background monitoring. are of dubious value.3. the samples that were testing. A good consultation on sample point location should take place procedure would be to match the sampling during the design and construction phase of the facility. time.``. The recorded water cut more than one hour is unavoidable.. schedule used with the baseline sampling for the system.3. Samples with low polyethylene. 2.1. or a pumped line. the entire system should be surveyed allowing the fluids to flow for several minutes (to rather than only areas where elevated bacterial thoroughly flush out dead-space fluids) before populations are expected or where obvious collecting the sample. nature of bacterial activity in the system.````. low percentage of water) or those with obviously preferred.4 Any production chemicals present.3 Dissolved oxygen and hydrogen sulfide (H2S) Touching the internal sutfaces of the container neck content.4.4.e. taking samples: 2. it on only planktonic bacterial testing for problem solving is important to account for any system variables may lead to serious misunderstanding of the extent or that are related to seasonal changes.1.4 During biocide treatments. then completely filled to flush out air. In some instances (such as with bacterial problems are occurring.`.`. If a delay of emulsion for bacterial isolation. and cap should be avoided. Relying 2..2 The available sampling points may not be suitable for identifying a microbiological problem (¡. For handling thermophilic with the membrane filtration technique. Usually.`-`-`. collection.1 It should be assumed that bacterial populations undergo both qualitative and quantitative changes with 2..3 Possible Sampling Problems concentration noted.2.`.e.. Usually.2. tank bottoms or when sampling from open waters).3. liner).3. e. caution should be exercised to prevent contamination during sample 2.1 Date.3 If the evaluation spans several months.4. storage tank) systems.`.4 The following information should be recorded when refrigeration.4. special precautions may need to be observed. all samples should be held for four hours special means to increase the bacteria numbers must before testing.2.. The accurate bacterial population estimates will result). Ideally. less than one viable cell per mL). 2. Sterile sample containers must be used refrigerated (4°C [4O"F]).. a specially designed sampling apparatus.e.1. pipeline) or static (e. See Appendix C for Samples to be held more than four hours should be more detail. the sample should be bulking.3. Samples held for longer than 48 hours. Special procedures are required for sampling sessile bacteria (See Section 5).1 These sampling procedures pertain only to be required. a 2.g. planktonic bacteria. 2 NACE International Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale.``.. membrane filtration technique. 2. TM0194-2004 sample frequencies over longer time periods may 2. It is usually satisfactory to directly use an analyzed without delay. If an additional sample is necessary to obtain collected in nonsterile containers should be so noted. however. One common method for doing this is the variability caused by the sample handling procedure.`--- some samples are normally analyzed four hours after is required (¡. .``.`.. 10/24/2005 13:11:09 MDT .0 g needed.1..0 with NaOH.4. Distilled water 1..0 g culturing procedures.5 Observations on color (particularly suspended 2. osmotic balance.25 g magnitude are required.S). ¡. because Sodium chloride 2.og of some bacteria and enhance the growth of Peptone 10.3 mL media provides sufficient accuracy. The pH should be adjusted to 7.0 g 3.. Classical methods using agar-solidified media Yeast extract 5.`..2. For those Agar 0.2. In addition.1.0 with NaOH. etc. the methods detailed here are usually The broth should be distributed to tubes or vials sufficient.) prevent the growth Beef extract 1. However.1 Thioglycolate Broth: 3. Section 3: Culture Techniques 3. underestimate the number of bacteria in a sample.`--- 3.`.2 Bacteria Testing: Media and Determinations 3.0 g impractical for routine field use. users should be aware of the limitations of the culture technique: Several different liquid bacterial culture media are widely used for enumerating heterotrophic bacteria in 3. and operator time required for this method also makes it impractical for routine field work ’ NACE International 3 Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. the large amount of bench space.e.0 g others.1.1 Any culture medium grows only those oilfield waters. glassware.4 Only liquid culture methods are described herein.`.0 with NaOH.5 Some bacteria cannot be grown on culture media at all. such as for finished water quality testing. 3.000 mL may hamper the growth of strict anaerobes. Examples are: bacteria able to use the nutrients provided.`.6 Other relevant information pertaining to the metallic sulfide or black water).1.1.2 A test for hydrocarbon-oxidizing organisms Distilled water 1.2.000 mL should be used in the rare instance when such organisms are important to a particular situation.000 mL most probable number (MPN) method7 can be used.0 g Peptone 5.3 Conditions induced by sampling and NaCI 5.0 g 3.1. redox potential.2 Culture medium conditions (pH. odor sample.1.1 Bacterial culturing in artificial growth media is 3.1.`-`-`.2 Anaerobic and Facultative Anaerobic Bacteria sulfur-oxidizing bacteria4a5and iron bacteria6 are not Testing: described here.1.`.1. (particularly H.1 Heterotrophic Bacteria (Aerobic and Facultative accepted as the standard technique for the estimation Anaerobic Bacteria) Testing: of bacteria numbers.1.1.4. such as exposure to oxygen.2 Standard Bacteriological Nutrient Broth: 3. TMOI 94-2004 2.2. The pH should be adjusted to 7. The pH should be adjusted to 7.1. Beef extract 3. However. These test methods are described elsewhere Otherwise. 3. the Distilled water 1.0 g can be found e l ~ e w h e r e . and the presence of slime and deposits. turbidity. 3.1.75 g occasions when estimates of greater precision are Dextrose 5.3 Procedures for the detection or enumeration of 3. and autoclaved for 15 min at 121°C (250°F).2. incubator space. culture media methods may and autoclaved for 15 min at 121°C (250°F).5 g only population estimates to the nearest order of L-cystine 0.4 Only a small percentage of the viable bacteria in a sample can be recovered by any The broth should be distributed to tubes or vials single medium. --``.````..2.1 Phenol Red Dextrose Broth: 3.``.1 General 3.1. duplicate culturing in liquid Thioglycolic acid 0. ~ Such methods are Casitone 15.1.018 g Dextrose 5. Phenol Red 0. 3. then use another acid-producing bacteria.2. medium preparation date and stored at 4°C (40°F) Complete dilution-to-extinction determinations unless stated otherwise. see Appendix sterile syringe.4.3). are not usually necessary. Adding to the confusion is the fact that bacterial media inherently underestimate bacterial 3.2. the ranges of bacterial This may require a I O 9 dilution factor (or populations shown in Table 1 are actually too g reater).4. For more details.`. With the duplicate testing by a complete dilution-to-extinction procedure.`.. and the more periodically determine the bacterial population precise the estimate.) 22-gauge needles fluids at temperatures of 80°C (176°F) or higher.2. it must be noted that using this table is factor depends on the expected bacterial simplistic.5 Incubation approximate the temperature of the sample to avoid “shock effects on the microbes in the 3. Then withdraw 1 mL of this well-mixed broth and 3. 3..2. the values practiced): Vigorously agitate the initially reported in Table 1 are considered acceptable for inoculated vial as before. with 9 desired. formulated to approximate that of the field water being Cleanse the syringe again by rinsing three tested. Repeat this procedure in the same manner until an appropriate dilution 3.1. If all the serial dilution series. Stopper the vials with butyl or natural latex rubber stoppers.2. yellow accompanying the turbidity is a positive for Vigorously agitate this vial.3 Salt composition and concentration should be inject it into the third vial of the dilution series. several milliliters of broth three times.`-`-`.2. discard the must be within f5”C (f9”F) of the recorded syringe. 10-mL nominal capacity. and autoclaved for 15 thoroughly by drawing up and expelling min at 121°C (250°F). the bacterial contamination. More detail can be found in serial dilution method is a subject for statistical Appendix D. Inject this 1 mL of red dextrose media.1.4 Fill serum vials.4. 10/24/2005 13:11:09 MDT . then the previous (¡. invert the vial. using it to transfer 1 mL to the next dilution vial.3 Estimate bacteria numbers using Table 1.2 Alternative Procedure (widely populations. The bacterial estimate reported is the one and inject it into the second vial of the dilution shown in the fourth column. then report the results as 4 NACE International Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. Using another oilfield situations.4. but may be done in special cases.” The media temperature should 3. As with the classical (to any salinity requirement) from biological supply procedure outlined in Paragraph 3. Then complete the serial dilution with one temperature of the water when sampled. If this is not felt to be the case or conditions (See Paragraph 3. Inoculate the first dilution vial with a sterile critical to growing bacteria removed from the field disposable syringe containing 1 mL of sample system.. vial. and rinse the syringe distributed to tubes or vials. However. are convenient. All work must be done incubation temperature must be recorded.2 Vials that become turbid in between 1 and sterile syringe. This of the following procedures. using another 3.`. With phenol inoculated broth. factor is reached. the incubation temperature collected as described in Section 2. All media should be marked with the normally a 106 dilution factor is sufficient.4. NOTE: In cases of severe analysis.. mL of media.3.2. prescribed in this standard.2. classical) dilution method 3.2. Protect and seal the rubber NOTE: The occasional spurious result is more stopper with a disposable metallic cap.````. the user may wish to tighter the statistical distribution.1 Classical Procedure: Vigorously agitate the inoculated vial and.5. The appropriate dilution However. dilution vial.5.. Some workers prefer an occasional spurious result is usually to bottle and cap these media under reduced-oxygen acceptable.`--- in duplicate.2.``. withdraw 1 mL from this vial D.2 Arrange media vials into a “dilution series.`.5. Steam sterilize likely when using this method.2. 3. narrow.e. a color change from red to inoculated broth into the second dilution vial. --``.1 The proper incubation temperature is sample.1 These media can be obtained ready made should be used. spurious results are common. NOTE: Disposable 3-mL plastic Because oilfield bacteria can grow in produced syringes with 25-mm (1 . formulations in Paragraph 3. Keeping that syringe needle in this vials used are positive. However. houses. the filled and sealed vials in accordance with the media because of inherent inaccuracies of culturing. Repeat this procedure for each dilution 3.2.``.`..2. special incubation procedures may be required when high-temperature fluids are encountered. Estimating bacterial populations by the population.O-in.`. by convention. Therefore. TM0194-2004 The medium should be heated to boiling.2.2. These vials may be sterile syringe to transfer 1 mL to the third discarded after 14 days’ incubation. withdraw 1 mL of the 14 days shall be scored as positive. The salinity should be approximated within times (in the inverted third dilution vial) before 10%. The more replicate samples done.. 0 2.1 Sodium Lactate SRB M e d i ~ m ( ~ ) ~ ~ ~ ~ 3. 10/24/2005 13:11:09 MDT .000.PO. the medium is referred to in this standard as Sodium Lactate SRB Medium. Many different media 3.````. such as pH.000 100 to 999 1. Without such information.000. one of Sodium lactate solution the following may be added to this (60 to 70%) 4.`. Fe(S04)2(NH4)2’6H20 0.0 g possible.``.2. and whenever MgS04.6 Also.5 g 3. redox potential.3.2.3.. Therefore.`--- (Paragraph 3.1 g etched iron nail to provide adequate iron MgS04.0 mL medium: (1) 0.3 Preparation of SRB Media in a liquid medium should be used to estimate SRB to the nearest order of magnitude. They should be stored at 4°C (40°F). ()’ Formerly referred to as AP1(3)RP 388 Medium.000 to 9. 3.O g --``.000 100.0 0.000 mL As with heterotrophic bacterial culturing.1 g FeS04.000 3. general heterotrophic bacterial population evaluations Sodium lactate 2.000 5 1: I 00.3.000 mL and then examined periodically for deterioration.2.999 1. total dissolved solids.og additional redox reduction. (anhydrous) 0.2 Postgate Medium BIo Determination KH..og oxygen content.2 Media Distilled water 1. 1220 L St. it may be difficult to estimate Thioglycolic acid 0.`.000 10.CI 1.999 100.3. Two widely used media formulations for gentle heating and adjust the pH to 7.3.`. to avoid referring to a publicationthat is no longer in print. DC 20005.3 SRB estimation are given below: k0.. Washington. The RP 38 standard that previouslyserved as a referencefor this medium was not reauthorized for publication by API..1 SRB testing should be conducted in association “.3.000 to 999.3 with NaOH solution. (2) an acid- Ascorbic acid 0.0 0.2 g 3. NACE International 5 Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale.og with other analyses.`.3.000 4 1:10.000 6 1:1. and/or (3) 2.`-`-`.5 g sodium K. 0. serial dilution 3.``.05 mL thioglycolic acid for Yeast extract 1...1 g the contributions of SRB to the problems found.7H.7H.HPO.3 All vials should be marked with NaCI 10. sulfide and sulfate content. (3) American Petroleum Institute (API).2) should be conducted simultaneously.000 1.3. TABLE 1 RESULTS INTERPRETATION TABLE Number of Positive Actual Dilution of Growth (+) Indicates Bacteria Reported Bacteria Vials Sample per mL per mL 1 1:lO 1 to 9 10 2 1:lOO 10 to 99 1O0 3 1:1.2.2.`.3 Sulfate-Reducing Bacteria (SRB) Testing: Media and 3.`. Ascorbic acid 0.8 g Yeast extract 1.2 Because of the difficulty in growing some field strains of SRB.0 g the date that the medium was prepared Distilled water 1.2 g concentrations.7H.3.1 Dissolve the ingredients with may be used.2.000 to 99. TMOI 94-2004 “equal to or greater than” (2) the highest dilution used in the testing. Cas04 1.5 g 3.01 g acetate.3.3.999 10. NW. 3. etc.4 Salt composition and 3.3. The salinity should be gas atmosphere (e. These compounds can allow 3.4 There are SRB that use carbon sources until 28 days. fill serum concentrations should be formulated to vials (nominal 10-mL capacity) with 9 mL of hot approximate that of the field water being bacterial growth medium while maintaining an inert tested. the H. subcultures cases.5 Incubation non-SRB to grow in these media and be reported as SRB rather than more 3.). These nonlactate-utilizing SRB may the blackening is caused by the presence of be present in some oilfield systems and may not sulfide in the water sample. nitrogen or argon).) should be avoided. Some source waters 3.. especially when sulfur sources methods are available (one commercially) for other than sulfate are present. reagent grade.3.1 with 15 glL agar added. waters may contain high levels of CO. SRB culturing in traditional media can shall be made into fresh medium to serve as a seriously underestimate the total SRB population check.3.2 If iron nails are used.3).. water rinsing to medium is black on initial preparation).S may be removed container of acetone for storage.7 Some workers report that the sampled. In addition.2. bisulfite.3.3.``. 10/24/2005 13:11:09 MDT .``..2.e. Appendix E lists an alternative SRB growth Other bacteria (e. (NOTE: It is acceptable to make these present.5 The medium may be made up covers. The nails make them inappropriate for use in should be prepared by degreasing in acetone. sterilize the filled vials at in source water..) microbiologically influenced corrosion. TM0194-2004 3. Because oilfield bacteria can grow in a sample of field water improves SRB produced fluids at temperatures of 80°C (176°F) or recovery from the water. 3..`. Incubation must be within k 5 T (9°F) of the recorded temperature of the water when 3. other The time that it takes each vial to blacken shall be media options should be screened to determine noted because this can be used as an indication of the most appropriate one for a particular system. However. medium preparation (because the soaking in 2 N HCI for 0. estimating SRB populations (see Appendix B). that may lead to pH instability.e. dilutions according to Paragraph 3.`.5 hours.3. sulfite. The required when high-temperature fluids are SRB agar is a commercial product similar encountered.3.5. activity) of the growing culture. propionate.3. This method higher.5. Vials shall not be scored as negative 3.3.1 Collect water samples according to the 3. vials with stoppers made of butyl or natural latex rubber and cap them with disposable metallic 3.. other source substituted for the iron nail.2.g.3. may be preparation.1 To limit oxygen contamination. remove all acid. Make serial compounds other than sulfate (¡. situations in which high SRB populations are backup subcultures must be made after 28 days to expected (as indicated by sulfide production.2.2. Likewise.2.`.`. special incubation procedures may be should be considered qualitative.. 3. The incubation temperature must be addition of 20 to 30% melted SRB agar to recorded. and then transferring into a In such cases.`.3. Iron wire or by boiling the water prior to medium reduced iron powder. If lactate-based media invariably and subcultures after only 7 days to reduce the unexpectedly yield low SRB populations in turnaround time for obtaining results. hours are discounted (¡.g.`.2. specifically acetate.3. ¡..3. Seal the approximated within 10%. confirm these results in case they are negative. substituting filtered 100 kPa (15 psig) steam pressure for 15 minutes.. If these vials are the grow in media containing only lactate. After sealing. In these only ones blackening after 28 days.2 Vials that turn black shall be scored as positive.`--- Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. 6 NACE International --``. the “strength” (¡.5. 3. they should be may contain sulfide concentrations that added prior to filling and stoppering.````. several rapid in some cases.1 Proper incubation temperature is critical appropriately as “sulfide-producing for growing the bacteria present in the field bacteria.3.3.6 Adding sulfu r-containing technique described in Section 2. thiosulfate.” system.3 Estimate bacteria numbers using Table 1 3. Vials that turn black within two other than lactate. Buffering 3.`-`-`.. not scored) because and butyrate.4.4 Inoculation may be needed.3. etc. to the medium described in Paragraph 3.3 Medium Bottling Procedure (also See Paragraph 3.3.5.2.e..e.3.3.4. source water for distilled water in the medium formula. Shewanella pufrefaciensl I ) medium that has given improved SRB recovery in can produce sulfide (and cause media blackening) some situations..3.2. Use this dose rate and 4.2. as controls for the field water. system may be used.`.``. both those in the first or second serial dilutions with the containing the biocide dilutions and the control higher biocide concentrations used because bottles.2.4.2.m L samples from each dilution of each biocide being tested (and the 4.7 Examine the test systems for evidence of immediately after sample collection. The total including the presence of normal amounts of biocide stock solution added should not exceed production chemicals.`. number of viable bacteria initially present in the test bottles. Use this tabulation to determine the minimum effective biocide dose rate.1 To assess a potential biocide application. done in duplicate. the tests should be field system. 4.2.2..9 Notes sterile bottles (30 to 200 mL).2. performed in nitrogen. standard 4.3 Add distilled water-based biocide stock solutions (10. up to a 1% inoculum effectiveness must be confirmed once the of a fully grown culture originating from the field chemical is added to the actual field system.. system holding times) to match the likely contact times for the biocide within the field system.3 The tests described here are only previously for general heterotrophic bacteria (see for planktonic organisms. A goal is to match populations.6 Following growth medium incubation. Begin testing 4.. procedure for biocide testing.2.2. 10/24/2005 13:11:09 MDT .2.2. Alternatively. At the 4.````.2.2 Biocide Time-Kill Testing for Planktonic Bacteria end of these times. (once the bottle is filled with field water). Therefore. Make testing biocide/water incompatibilities. withdraw I .`. It is unrealistic to describe a single.2.5 Choose biocide exposure times (test selecting biocides. --``.2.2. Use the methods described 4. for does not preclude compatibility problems in the anaerobic systems (typical).`--- TMOI 94-2004 Section 4: Evaluation of Chemicals for Control of Planktonic Bacteria 4. the fluids and components in the system.8 Field experience shows that time-kill 4.1 When a chemical inhibitor (biocide) is desired to application) to make this determination. Septum control microbial activity in a system.4 Fill the above test bottles.2.2.2. These biocide tests must be each biocide dose rate and each exposure time.2 The organisms used to challenge the test testing can only serve as a guide for the field biocides should be the population normally found application of the biocide. it is necessary to seals should be used to limit oxygen ingress into select an effective chemical agent that is compatible with the test systems. The ability of Paragraph 3. with the test fluid (containing bacteria)..m L concentrations from the test fluid to the samples from the control bottles to determine the growth medium.000 mg/L recommended) to small 4.``.9. Add distilled water instead must modify the procedure to suit a particular of biocide stock solution to several bottles to serve system.2.2 Basic Test Procedure the biocide unit cost to calculate the most cost- effective biocide.2. For example.1 This testing is most reliable when be the amount calculated to provide one of the the test procedure most closely matches the dose rates expected to be useful in the system normal operating condition of the field system. 4.2. In addition.2. Use no more than 1% Some fine adjustment of biocide dose rates is inoculum to prevent the undue addition of organic almost always required.2.2). the user 1% of the final volume.`.2. However.2. biocide in the test fluid. material to the test systems.2. The stock solution volume added to each bottle (test system) should 4.2.1 Obtain field water samples as previously described (See Section 2).3). for SRB (see Paragraph 3. biocide/system compatibility problems may not become apparent until field trials are performed. or biocides to control sessile bacteria in the both (depending on the objectives of the biocide system cannot be determined by this NACE International 7 Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale.`. Therefore. On-site dose- response (time-kill) testing is often used as a guide for 4.2 False results may be encountered 4.9.or argon-purged bottles.2.. 4. therefore only the basic tabulate the surviving bacterial populations for test design is outlined.`-`-`. Mix of the transfer of significant biocide thoroughly and immediately withdraw I . 4.`. as described in Paragraph 4.. test conditions to those prevailing in the system under scrutiny. the lack conditions as similar as possible to those of apparent incompatibilities in these systems prevailing in the system.2.9. as a minimum. adapt controls) and determine viable bacterial the following basic test procedure. 0. It is necessary to establish that the 5.2 The coupons may be located in suitably Furthermore.4 During any baseline or investigation survey.000 mL and electrically isolated to prevent galvanic effects. the bacteria shall be removed from the produce variable results. or biofilm probes.1 . using a vortex mixer with glass beads or by a sonic bath.1 Coupons bearing biofilms can be used to assess 5. the time taken for the development of a dense biofilm is variable and 20 mL of 2. guidelines should provide a basis for analytical work that If scalpels or swabs are used. TM0194-2004 technique..`-`-`.1.2 Anaerobic PBS Solution 5.'^ is commonly used to process sessile samples. swabbing.``. etc.`--- yields valuable information about sessile bacteria within an associated products must be completely dispersed oilfield system. 5.3 When metal coupons are used. However.2. biocides are much less effective planktonic bacteria.2.1. 10/24/2005 13:11:09 MDT . as well as service specimens. pipe walls at effects).5 While clean coupons inserted in the system may Prepare as above and add: be rapidly colonized by bacteria. The PBS solution companies.. NaCI 8.3. See Section 5 for more detail. must be located such that they are representative of sessile bacterial growth. sterile phosphate-buffered saline 5. procedures can be described... with these bacteria. Good should be added to avoid osmotic shock sources are filter backwashes. The coupons pipelines.2 Sampling Biofilms (or ultra-filtered field water) shall be used to collect the removed bacteria. coupons are 5. “coupons” in this standard.1 PBS Solution often located at the “6 o’clock position in oil and gas piping. The previously discussed them and then applying the techniques described planktonic techniques are of limited value for assaying earlier to count the bacteria (Section 3).1 Any removable field system component can collecting method used is effective and that the assay potentially be used to sample for sessile bacteria. Alternatively.4 g 5.3. In against sessile bacteria than against general. Techniques for sessile bacterial study sessile bacteria.23 g similar in composition to the pipework of the system Distilled water 1.PO.3.`. coupons specially designed for This solution can also be used to suspend microbiological use are available from suppliers of deposits from corrosion failures. coupons.7 g KH.`.H PO..`. provides an environment for maintaining viable bacteria without providing nutrients for growth.````. Consequently. few routine coupon by scraping with a sterile scalpel.4.1% Resazurin indicator (optional) reason. methods allow efficient recovery of the bacteria being These removable components are referred to as analyzed.2. Bottle and autoclave (100 kPa [ I 5 psig]/20 5. some investigators have reported designed side streams or they may be placed within benefits from using anaerobic PBS solutions in actual system flow paths by employing properly processing sessile specimens from gas or oil designed coupons and access fittings. minutes) (for brines.`. 1.4 Assessment of Biocide Efficiency episode. pig run corrosion-monitoring systems. Standard corrosion coupons are a good example.`. A major obstacle in working and/or with sessile bacteria samples is the uneven nature of 20 mL of 5% ascorbic acid sessile growth within the system (patchiness). For this 1 mL of 0.2.2. Corrosion failures should always be tested for sessile bacterial populations.1.1 The above sampling devices can be used to most important biological component of the bacterial monitor biofilm development by periodically removing ecology of an oilfield system.5% cysteine-HCI depends on the system. I Phosphate-buffered saline (PBS) solution the use of removed pipe sections spool^). the following shaking with glass beads. 5. 5. they must be K.3 Monitoring of Sessile Bacteria the efficiency of biocide treatments against sessile NACE International Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. Another alternative is 5. Section 5: Assessment of Sessile Bacteria 5. However.`. multiple sessile samples (or large surface areas) should be removed during each sampling 5.. In each case. unions. the biofilm and --``.1. For example.3. pig runs. greater amounts of NaCI sessile samples should always be collected. or using ultrasonic devices.1 Attached microbes (sessile bacteria) are normally the 5.``. 16. Wagener. H. TX: NACE. 7. Prasad. paper no. Tuovinen. I. References 1. 1023-1036. ?Utilization of Certain 14.G.? Journal of Bacteriology ?Laboratory and Field Tests of Efficiency to Biocides at 41 (1941): pp.? International Symposium of Sphaerotilus-Leptothrix Group. Kuenen.M. G. W. ?Biocide Influenced Corrosion and Biofouling in Oilfield Equipment? Testing Against Corrosion Causing Bacteria Helps Control (Houston. O. R. ?A Radiorespirometric Method ?Mediation of Sulfur Speciation by a Black Sea Facultative for Evaluating Inhibitors of Sulfate-Reducing Bacteria. European Journal of Applied Microbiology and Biotechnology 17 (1983): pp. paper no.````. Truper. Little. 87 (Houston. 19. NACE Publication TPC #3.R. ?Iron Bacteria. H. and after biocide treatment. 5312 (Richardson. P. W. Kostka.A.. Littmann.`. Frank. ~ ~ . 317-329.?4? Journal of Applied Bacteriology 27. B. England: Cambridge University Press. R. B. growth.H. Luther 111.W. Pogemiller. D. J.P. Such tests will time-kill testing. McCance. J.H. sessile bacteria on coupons should be undoubtedly become more widely used in the future. and Industrial Problems. Oil Field Waters. J. J. L. The Procaryotes. T. Pope. Cookingham.P. M.R. Oxidize Thiosulphate.E. 10/24/2005 13:11:09 MDT . 1 8.L. TX: SPE. H. Microscopy.? ed.`-`-`.H. Aldrich. Mansfeld. TMOI 94-2004 bacteria.A.``. 1984).G.S. ?Pros and Cons of ATP Measurement in 5.? CORROSION/89. eds. Morris.. ?An Investigation of Errors in Food and Dairy Microbiology (New York: Academic Press.`. 192 (Houston.R.? Oilfield Chemistry. Harrigan. H.`. 653-673..`. with Reference to a New Method for Dyeing Membrane Filters. J. ?The Influence of Enzyme Systems on MIC.W. D. 12. Syrett. Zintel. Ruseska. Plugging. (Berlin: Springer Verlag.?Soil Science 39 (1935): pp.O. NACE Publication TPC #17.. 1981): pp. Haas. For effectiveness are under development.G. Starr. Bushnell. paper no.A. 1989).? CORROSION/88. Zintel. 253-272. Schlegel TX: NACE.E. 3. 1956-1959. Jones. TX: NACE. J. A.G. ?Isolation of Some Bacteria Which (Houston. 34 4. Nealson. Lashen. Day. 49-51. 1975) (out of print).G. R. K.? International Corrosion in Gas Industrial Facilities. 1 (1964): pp. Coupon-based biofilm samples should be 5. Pope. 151-173. Perry. 2.A. P. Hamilton. Rosser. T. DC: API.? Journal of Applied Bacteriology 39. paper no. ?Corrosion Failures in 1990). J. Corrosion Inhibiting in the Control of Microbiologically Influenced Corrosion. H.? Science 259 (1993): pp.E. (Cambridge. K. Boilers?(Houston. Starkey. Duquette. (Washington. ?Oilfield Bactericide Parameters As --``.H. Particularly Those of the Measured by ATP Analysis.A.? CORROSION/SO. M. 6 (1991): pp. TX: NACE. Balos. E. Box 833836. 197-219. Simon. Boivin. 2nd Accurate Determination of (35S)SulfateReduction Activity. ?Microbiologically 13. Mulder. ?Recommended Practice for Biological (1975): pp. 45 (1983): pp.S. Hydrocarbons by Microorganisms.`. 1996). B.?Oil and Gas Journal 4 (1982): pp.G. The Sulphate-Reducing Bacteria. 128 (Houston.`--- 6. 15. Laboratory Methods in 17. TX: NACE. Postgate.4. exposed to biocides either under static conditions or by but no single recommended procedure can be given at being placed in dynamic flow I o o P s . 1988).? Anaerobe..TX 75083-3836 NACE International 9 Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. Costerton. 801-803. J. Howard. Stolp. Applied and Environmental Microbiology 6. Materials Reviews 36. 1990). K. E. ?Microbiologically ?Mitigation Strategies for Microbiologically Influenced Influenced Corrosion of Metals and Alloys.. API RP 38.F. during. D. 253-264.`. many different test methods to evaluate biocide Surviving bacteria should be assayed as above. D.``.? CORROSION/SO. 1975). F.D. 1990). ~ ~ this time. 9.F. Hardy.? 3rd ed. R. 18.R. 11. E.P. ?Simple Assay for 1O. Richardson. (4) Society of Petroleum Engineers (SPE). TX: NACE.2 In recognition of the importance of biofilm removed before. H. 20. paper no. Analysis of Watetflood Injection Waters. Direct Counts of Aquatic Bacteria by Epifluorescence 1976). G. D.H. See sterilize. Standard corrosion coupons are to number of bacterial cells. an example.. TX: SPE. Zintel. Gawel. 1988).5 billion years ago and exist in extreme environments. Appendix A GLOSSARY 4-6-diamidino-pheynylindole hydrochloride (DAPI): A Autoclave: A chamber that utilizes pressure and heat to DNA-binding molecule that will fluoresce when illuminated sterilize solutions. TM0194-2004 21. TX: NACE).J. NACE Standard TMOI 73 (latest revision).``.`.`--- Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. and derive energy from inorganic molecules or chain. Also termed to the terminal phosphate. Adenosine triphosphate (ATP): A molecule that provides Biocide: A chemical product that is intended to kill or energy to living cells via hydrolysis of the high energy bond render harmless biological organisms. This is usually based on concentration and contact time relative to other biocides Agar: A dried polysaccharide extract of red algae used as a being screened. exopolymer. 24..L. This Bacterial culturing: Techniques used to grow bacteria reagent can be used to stain bacterial cells for enumeration present in a sample inoculum in select growth media in the by fluorescence microscopy. instruments. solidifying agent in microbiological media.`. “Standard Methods for the Examination of Water and of Under-Deposit Microbiologically Influenced Corrosion. When referring to sessile biofilms. 10/24/2005 13:11:09 MDT . allowing killing all microorganisms present.. and damp terrestrial environments. Biomass: The mass per sample volume of microorganisms present. TX: NACE. marine water.`. Horacek. water.`. antimicrobial pesticide. bacteria in the laboratory.````.. paper no. See culture medium. “New Test Kit for Rapid for Determining Quality of Subsutface Injection Water Using Detection of SRB in the Oil Field.`. debris. laboratory.`-`-`. visualization and enumeration of bacterial cells in a sample. 10 NACE International --``. These bacteria evolved over Adults average 1 to 2 mm in size. All algae possess chlorophyll for photosynthesis. (Washington. Bacteria: Prokaryotic microorganisms enclosed by a cell membrane without a fully differentiated nucleus.P. halophilic bacteria. 63rd Annual Technical Conference of the Society of Petroleum Engineers (Richardson. this term may Anaerobic bacteria: Bacteria that grow and reproduce in also include the solids formed by bacterial growth such as the absence of oxygen. T. L.” 17th ed. light. SPE 18199. quantifies the amount of ATP present in a sample and thereby provides an estimate of bacteria present based on Coupon: A removable system component used to sample the assumption that the concentration of ATP is proportional sessile bacteria growth. thermoacidophilic bacteria. and numerous group of metazoans in the water community.. Membrane Filters” (Houston.``. Compare Eubacteria. Cost-effective biocide: A biocide that provides superior kill ATP photometry: A method of bacterial enumeration that of microorganisms based on cost per gallon or pound. Biofilm: A matrix of bacteria. APS-reductase: An enzyme specific to sulfate-reducing Broth: An alternative term for liquid medium used to culture bacteria that is involved in the reduction of sulfate to sulfide.” Wastewater. 1988). Archaebacteria: The kingdom of monerans that consists of Copepods: Aquatic crustaceans comprising the most methanogenic bacteria. Compare acridine orange. “Methods 22. exhibits in killing bacteria.`. See culture medium.” paper no. Acridine orange: A fluorochome that binds to DNA and RNA and fluoresces when excited with UV light. Pope. 1989).. and glassware by with appropriate excitation wavelength of light. They represent an 3. media. and Algae: Unicellular to multi-cellular plants that occur in fresh particulate matter that adheres to a sutface. 249 (Houston. Aerobic bacteria: Bacteria that grow and reproduce in the Biocide efficacy: The degree of petformance a biocide presence of oxygen. “Methods for the Investigation 23. exopolymer. DC: American Public CORROSION/88. are important link between phytoplankton and fish in the food anaerobic. G. Health Association. Specific filters are used to select for proper Microorganisms: Common term used for unicellular emission spectra of the illuminated probe. undiluted concentration of bacteria in the in immunofluorescence methods. or molecules present in the sample. Inoculum: A medium or sample containing microorganisms Emulsion: A mixture in which one liquid. is uniformly distributed (usually as minute globules) in another liquid. dilution. NACE International 11 Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale.``. for fluorescein dye or chomogenic substrate. Membrane filter technique: An enumeration technique for waters with low bacterial concentrations in which a volume Facultative anaerobic bacteria: Bacteria that are able to of sample is passed through a 0. hydrogen and is possibly involved in cathodic depolarization by sulfate-reducing bacteria.2 to 200 pm in has been raised against a specific antigen being size. 10/24/2005 13:11:09 MDT . which allows instance with the serial dilution method.. termed the that is introduced into a culture. antibody specificity to a protein or cell component being analyzed.. in order to obtain quantification of the molecule being investigated. and is coupled to a fluorescein molecule to allow its detection. Most probable number (MPN) method: The essence of this method is the dilution of a sample to such a degree that Fluorescein isothiocyanate:A very common fluorochome inocula will sometimes but not always contain viable that is excited at 420 to 480 nm and fluorescesces at 530 to organisms. sample.`. See serial Hydrogenase: An enzyme that catalyzes the oxidation of --``. ¡. such as a protein or cell component. Microbial ecology: Encompasses a wide range of Fluorescence microscopy: A microscopic method that disciplines and focuses on the interactions of utilizes a specific illuminating wavelength of light to excite a microorganisms with one another and with their fluorescent stain or probe added to a sample for specific environment.. cycle to treat sessile bacteria buildup on the filter media. Immunoassay: A detection method that takes advantage of See time-kill test. dispersed phase. requiring the which no microorganisms are transferred in the final presence of oxygen.e.. called the continuous phase or Iron bacteria: The so-called iron-oxidizing bacteria. investigated.. smuts. structures. the numbers of inocula 540 nm." ¡. microorganisms capable of using hydrocarbons as their extinction refers to continuing the serial dilutions to a point energy source as well as a carbon source for growth.``.`. These cannot be seen without Fluorescent antibody: An antibody or immunoglobulin that magnification and generally range from 0.`--- dilution method. The discrete colonies that through a filter in order to dislodge particles from the filter form on the sutface of the membrane can be easily media.. The passage of nutrients through the filter facilitates the growth of organisms on the Filter backwash: A process of forcing a water stream back upper sutface of the membrane. will imply an estimate of microscopy and can be conjugated with an antibody for use the original. This at which no growth will be encountered. A generally precipitates as iron hydroxide.e. These dispersion medium.`.`-`-`.`. Any organisms in the sample therefore are able to grow and reproduce in both the are concentrated on the sutface of the membrane.. is then placed in nutrient medium. Dose-response test: Biocide screening test to establish concentration and contact time for effective bacterial kill. The antibody is usually conjugated to a Duplicate culturing: Petforming replicate cultures.45-pm filter using a filter carry out both aerobic and anaerobic metabolism and funnel and vacuum system. and mushrooms. This protein dye is often used in fluorescence producing growth at each dilution. dilution.`. detection of cells. dilution-to. In the oil field. reverse emulsion refers to oil dispersed in water (oil-in- water emulsion). more reliable interpretation of the results. Oítentimes biocide may be introduced during this transferred to confirmation media. The "outcome. which dispersed as droplets in oil (water-in-oil emulsion). to a point at metabolic process is generally aerobic. The filter presence and absence of oxygen. Dilution-to-extinction method: During microbial Hydrocarbon oxidizing organisms: Heterotrophic enumeration using the serial dilution method. mildews. rusts. TMOI 94-2004 Culture medium: Formulated solution of organic and Heterotrophic bacteria: Bacteria that are unable to use inorganic nutrients that facilitate bacterial growth in the carbon dioxide as their sole source of carbon and require laboratory. This ensures that a full estimate of the original population in the sample can be determined. Fungi: A group of plants that lacks chlorophyll and includes incubating several tubes or plates (replicates) at each molds. organisms of the plant or animal kingdoms that are structurally related.````. one or more organic compounds. In order to obtain estimates over a broad range of possible concentrations.. typically water is bacteria oxidize ferrous iron (Fe") to ferric iron (Fe3').`. microbiologists use serial dilutions. It segment for the purpose of cleaning or to monitor pipeline contains a septum that can be sealed with a metal ring.``. See Phase separation: Specifically used here to refer to the sampling bomb. Aerobic bacteria grow best in systems with highly positive redox potentials Standard bacteriological nutrient broth: Basic culture (oxidizing environments) while anaerobic bacteria. inner pipe sutface and is launched through a segment of Bacteria that live in biofilms are sessile bacteria. The pig consists of a cylindrical device that forms a seal with the Sessile bacteria: Bacteria that are attached to sutfaces. Sampling thief Another term used for a liquid sampling device used to take samples at discrete depths. Protozoa: Single-celled eukaryotic microorganisms that Sodium lactate SRB medium: Medium for culturing SRB.`--- Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. Pigs can be equipped with brushes or various adaptations to facilitate Shewanella pufrefaciens: Although not true SRB. whereby a representative sample of sessile biofilm growth can be Redox potential: A measure of the relative oxidation. the number of positive cultures heterotrophic bacteria.. The vial assembly can be filled with Pigging: A procedure used for cleaning pipeline scale.`. brine. It is derived from the original Postgate B medium and provides similar nutrients and salts but in a slightly different Radiorespirometry: Sensitive method for bacterial formulation. and solids or to monitor pipeline integrity. See Most Probable Number method.” a sample via transfer to a series of growth media vials using successive 1: I O dilutions in each successive vial. Following Phenol red dextrose broth: Culture medium used to grow an incubation period. feed heterotrophically and exhibit diverse forms of motility. piping. Planktonic bacteria can become sessile bacteria by adhering to a sutface.. culturing sulfate-reducing bacteria characterized by lactate During the shut-in period. vessels. the fluids in that part of the that is used as a carbon source and sulfate to provide the system are stagnant and amenable to increased bacterial terminal electron acceptor required for SRB metabolism.`-`-`. reduction potential of an environment. provides an estimate of the number of bacteria in the original sample. enumeration whereby radioactive nutrients are metabolized by bacteria in a sample and the amount of radiolabeled Spool: A monitoring device for obtaining sessile bacterial gases that are generated are measured to give an estimate samples that often consists of a removable pipe section of the number of viable bacteria in the sample. of hydrogen ions multiplied by the mean ion-activity coefficient. these cleaning or permit inspection of the pipe wall.`. inserted in a side-stream flow loop.. written as: PH = -loglo (aH+) Sampling bomb: A sample device that can be used to take liquid samples at discrete depths in drums. or injection wells. chlorides. the redox potential is less than -100 mV. See Postgate B medium. For statistical validity this test is done with Phytoplankton: A collective term for free-floating aquatic replicates and the population estimate is derived from a plants and plant-like organisms. TM0194-2004 Osmotic balance: In the context used here. integrity. Shut-in: In general. Pig run: The process of launching a pig device in a pipeline Serum vial: A glass vial used for culturing bacteria. Compare zooplankton. The term also refers to closing down a Postgate medium B: Specific medium designed for segment of a system.`. including medium for heterotrophic bacteria containing beef extract SRB. 12 NACE International --``. the pipeline using differential pressure. growth.`. statistical table. The septum can be accessed with a syringe needle for inoculating bacteria.``. deposits. culture media and autoclaved for sterilization. usually reported as total dissolved salts (TDS) or cultured to be able to maintain proper osmoregulation... refers to closing the valves to a well to shut off production. will grow much better in reducing environments where and peptone. acquired. tanks. the proper salt Salinity: The measure of dissolved salts in the system concentration of a medium required for the bacteria being water.`. 10/24/2005 13:11:09 MDT .````. The salinity should be approximated when media for culturing microorganisms from the system water are pH: The negative logarithm of the hydrogen ion activitiy prepared.. and Where aH+ = hydrogen ion activity = the molar concentration sutface water bodies.`. macroscopic separation of oil and water in a sample into the two respective fluids by a allowing time for the fluids to Serial dilution method: Method of enumerating bacteria in “settle. sulfidogenic bacteria exist in biofilms and can act synergistically with SRB to facilitate MIC and hydrogen Planktonic bacteria: Bacteria that are freely floating in sulfide formation. exposing to an method requires a sensitive instrument In view of these enzyme-extracting solution. ATP photometry.`. A fluorescence Users are responsible for determining the appropriateness microscope is used to allow cells to be counted. (2) the results exhibit unacceptable scatter with Hydrogenase testing is best petformed on sessile samples.``. developments in fluorescence stain technology have fluorescent antibody microscopy. above 113°F (45°C).. Strict anaerobe: A microorganism that grows only in the Thermophilic bacteria: Bacteria that grow and reproduce absence of oxygen. a 12-hour exposure is NACE International 13 --``. The total number of bacteria body of this standard rely on growth of bacteria in nutrient in the sample can be determined. fluorescein populations in oilfield systems.`.. 6. These techniques are outlined below. particularly following biocide treatments.17 As with of any of these methods for their needs. dyes. because they stain both the live and the dead cells. and 4..5 to 4 hours. however to evidenced by a color reaction with a dye). used to Thioglycolate broth: A culture medium used to grow evaluate false positives for the presence of SRB in the first anaerobic bacteria in the laboratory. APPENDIX B RAPID METHODS FOR ASSESSING BACTERIAL POPULATIONS The procedures for bacterial analysis outlined in the main Fluorescence Microscopy.`. by filtration).. (3) the method does not differentiate Hydrogenase should be measured by first collecting the between the various types of organisms. Such techniques generally do not allow rapid can be distinguished. involved in energy metabolism. See heterotrophic microorganisms characterized by its autoclave. and live and dead cells media. the presence of hydrogenase may indicate a potential for this corrosion. be expected in 0. These include isothiocyanate (FITC). general hydrochloride (DAPI) are used for total bacteria counts fluorescent microscopy.`--- Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale.`.````. ATP can give an indication of the Hydrogenase Measurement.g. Some intetferences can result from organic and inorganic material suspended in the ATP Photometry. bacteria that derives energy from the oxidation of sulfide or elemental sulfur to sulfate. and measurement of resulted in methods using a combination of dual fluorescent APS-reductase. by fluorescence microscopy. After Time-kill test: A biocide screening test that determines the the 28-day incubation period. Subculture: For the purposes of this standard. with different emission spectra. Zooplankton: Collective term for nonphotosynthetic Sulfur oxidizing organisms: A broad group of aerobic organisms present in plankton. A strong hydrogenase activity can also Disadvantages of ATP photometry include the following: (1) indicate the presence of a microbial biofilm community. metabolism of sulfate to sulfide.`. when ATP is present. Stains such as acridine orange.``. and measurement of hydrogenase. then noting the degree of disadvantages. ATP can be measured analyzes for the hydrogenase enzyme that is produced by using an enzymatic reaction that generates flashes of light bacteria able to use hydrogen as an energy source.. These flashes are detected in a Because it is believed that the use of cathodic hydrogen is photomultiplier. Specific evaluation of bacterial contamination. 10/24/2005 13:11:09 MDT . Because it rapidly disappears on cell death.`-`-`. ATP is present in all living cells and is sample. low bacteria numbers. between live and dead bacteria.. present. in high temperature environments. and chemical additives intetfere with the reaction. ATP photometry is not used for routine hydrogen oxidation in an oxygen-free atmosphere (as monitoring.. two vials of a dilution series that have turned black within two hours due to the presence of hydrogen sulfide. To sterilize a medium or Sulfate-reducing bacteria: A diverse variety of material is to kill all microorganisms that are present. A response can 16 monitor trends. the output being proportional to the amount an important factor in microbiologically influenced corrosion. of ATP. Only total bacteria GENERAL BACTERIA counts can be determined. that can distinguish together with literature references to specific applications. this method requires a delicate instrument and is best suited for the laboratory. It has been used successfully. chloride. The hydrogenase test viable biomass present in a sample. Many techniques stains that fluoresce when irradiated with ultraviolet light are have been used to obtain rapid information about microbial used.” sulfide. and (4) the bacteria in a sample (e. Recent Methods specific for SRB are as follows: radiorespirometry. a I-mL aliquot can be taken efficacy of a biocide against microorganisms cultured from from these vials and re-tested by serial dilution into SRB the system and identifies optimum contact times and media (subculturing) to determine whether SRB are concentrations for effective bacterial kill.diamidino-2-phenylindole measurement of adenosine triphosphate (ATP). Many analytical kits are currently available. TMOI 94-2004 Sterile: Free of living organisms.`. of this method is that. containing the appropriate SRB medium. The test radioactive substances is strictly regulated.lg test fairly general. the disadvantages are similar to those for other detection. only those minutes. and the detection of amount of 35S-labeledsulfate. consequently. While a large number of SRB antibodies can be combined to make the Radiorespirometry. using a liquid must be developed for each microbe to be tested.45 pm. APS-reductase. should also be sterilized between samples. separate antibody “pools” fixed in zinc acetate prior to quantification. The test should be carried out using to the general fluorescent microscopy described above. is used to detect SRB.`--- APPENDIX C MEMBRANE FILTRATION-AIDED BACTERIAL ANALYSES Occasionally.`. which is “any activity of the SRB population can be calculated. the overall advantage of the functional definition of SRB.`. there is always the possibility that new is specific to SRB. does not require bacterial growth to occur (no medium is used) and is independent of sample temperature. NOTE: Permission for using an open flame must 14 NACE International Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. When APS-Reductase Measurement. Like the culture methods described strains that are not detected will be encountered. disposable “kits” that are fully contained and usable either in except that FITC (the fluorescent dye used) is bound to the field or the laboratory. NOTE: This method is most applicable to aerobic or facultative A known volume of water (usually one liter) should be anaerobic heterotrophic bacteria.” bacteria recognized by the antibodies fluoresce under the 21 microscope. The major limitation comparison purposes. however. this technique is limited (for heterotrophic bacteria) or inserted into a bottle to low-turbidity waters. it can be used for any 35S-sulfideproduced by SRB. followed by flaming. because the antibodies are developed against whole SRB cells. Unlike other culture methods for SRB. This method is similar and redox condition. Also. including the most commonly used membrane objects. scintillation counter.. The membrane filte$ technique can be used to test relatively large volumes of sample and generally yields The filter should then be placed on a suitable agar sutface numerical results rapidly. therefore. Forceps should normally be cleaned with 70% types. When the membrane is handled. The entire test takes 15 to 20 antibodies specific to SRB cells. microscope techniques: a high degree of training required... difficulty in dealing with samples containing a lot of debris. it is desirable to test for microbial be obtained from the local operations management. This method. the handling of an estimation of the total number of SRB present. Measurement of However. Fluorescent Antibody Microscopy. Recovery efficiencies for filtered through a presterilized cellulose acetate membrane strict anaerobes and SRB are likely to be low. TM0194-2004 generally used to allow the system to equilibrate for results are obtained within two hours. it produces results in one to two days of total testing time.````.``. The sample should first be incubated with a known trace the need for a laboratory facility.`. then enough presterilized forceps populations (1‘ to 10 cells/mL). alcohol. or the cells in these samples must be concentrated. sterilized forceps should always be NACE Standard TM0173’4 gives details of membrane used and the filter should never be touched to nonsterile filtration. either should be provided. However. it is a highly specialized technique involving the amount of APS-reductase in a sample.``. 10/24/2005 13:11:09 MDT . Other than elsewhere in this standard. they can be quantified in laboratories away from the site. However.. The major advantage is speed. Such sulfides should be other microbes as well. gives expensive laboratory equipment. After incubation.`-`-`. with a pore size no larger than 0.`. bacteria capable of anaerobically reducing sulfate to Radiorespirometry has been applied to quantify SRB in the sulfide. If contamination in waters that contain very low bacterial flaming is not permitted. salinity.. If many samples are to be taken. as currently proposed. the reaction nonviable as well as viable SRB. such as isopropanol or methanol. they are specific only to SRB TESTS the type of SRB used in their manufacture.`. In these situations. as cited..`. NOTE: While this should be terminated with acid to kill the cells and to release method.” presence of an enzyme. Once the 35S-sulfides are fixed. the large volumes of sample must be inoculated into culture parts of the filter holder in direct contact with the membrane media. it requires bacterial growth for that. This immunoassay takes the natural concentration of sulfate is known.” A unique requirement for this process is the field and for testing biocide efficiency in the laboratory. because --``. 2. an RESULTS INTERPRETATION AND REPORTING attempt is made to transfer smaller and smaller portions of the original fluid to each successive vial. Discard syringe. considered acceptable for oilfield situations. Do not touch any object (other bacterium from Vial 2. Theoretically. after incubation of a set of inoculated vials. In this case. 5. and the more precise the estimate. Adding to the confusion is that bacterial media SAMPLING inherently underestimate bacterial populations. but presumably no more than IO. when growth occurs in Syringe 1 Fill syringe with 2 mL of sample.000 or > I O 6 bacteria/mL. This is original inoculum to these concentrations. Shake vials or aspirate fluid vigorously with the 3. indicating growth. This is Following incubation.`. Shake vials.`. no bacteria are transferred. the results operation. This is important..``. NACE International 15 --``. Remove 1 mL from 1. Therefore..`. Sometimes..000. it into General-Count Vial 1. bacterial populations shown in Table 1 are actually too narrow.````. Record the population as t1. severely infected waters produce growth in all vials of a test series..`. Discard syringe. each vial contains 9 mL of growth Steps Label General-Count Vials (heterotrophic medium. and use Table 1 of the transferred bacteria. and inject into Sulfate-Reducer Vial 2. visually examine all vials and report accomplished by a stepwise 1 : l O dilution scheme until.`-`-`. Vial 3 must have received at least 1 syringe. COMMON GROWTH INTERPRETATION PROBLEMS Syringe 3 Repeat as above from Vial 2 to Vial 3. the upper limit number of Syringe 2 Remove 1 mL from General-Count Vial 1 and this range is reported as the estimation result. with the the dilution factor gives the approximate number of bacteria duplicate testing prescribed in this standard. This indicates that a true end point Syringe 5 Repeat as above from Vial 4 to Vial 5. The growth medium in growth that has settled to the bottom. find Paragraph 3.000 or more Syringe 6 Repeat as above from Vial 5 to Vial 6. Count the number of the serial-dilution vials provides nutrients for prolific growth positive vials (the ones that show growth). However. the sample is thereby diluted tenfold. One might.`--- Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. Multiplying by illustrated in Tables D I through D3. On transferring 1 mL of fluid to Vial 2. was not reached. Sulfate-Reducer Vial 1. Inject 1 mL Vials 1. ¡. by the results. 10/24/2005 13:11:09 MDT . For example. By convention. by convention.2. and 1 mL into Sulfate-Reducer follows that no bacteria were transferred into Vial 4 from Vial Vial 1. than the vials' rubber septums) with the needle during this Because three tenfold dilutions were involved. the values reported in Table 1 are Collect samples according to Section 2. Turn the vials over several times to resuspend theory.`.``. be the vial that received between 1 and 10 bacteria and The more replicate samples done.e. TMOI 94-2004 APPENDIX D BACTERIAL CULTURING BY SERIAL DILUTION In the serial-dilution approach to bacterial culturing.. a tenfold dilution of Vial 1 Label Sulfate-Reducer Vials 1 through 6. NOTE: Table 1 gives a simplistic approximation of vials and a black precipitate in sulfate-reducer vials. inject into General-Count Vial 2.. per mL. is effected.`.000. GENERAL CULTURING PROCEDURE SERIAL DILUTION-TO-EXTINCTION THEORY (Performed in duplicate) The basis for estimating the bacterial population is as Preliminary follows: As supplied. of a set. if all the vials in a series of six show growth. A growing bacterial population to approximate the number of bacteria in the original causes turbidity (cloudiness) in general-count heterotrophic sample. then the population is 1. indicate a range of 100 to 999 bacteria per mL in the original sample. the ranges of per mL present in the original sample (See note below).. each vial in the series is a tenfold dilution of the preceding one. All Vials Show Growth Syringe 4 Repeat as above from Vial 3 to Vial 4. the tighter the statistical represents the dilution factor necessary to reduce the distribution. for example. and 3. For example. Occasionally. a vial that is clear (no growth) may be followed by one that is NOTE: See alternative inoculation procedure described in turbid. The the bacterial population. but not in Vials 4.000 bacteria/mL are reported.2. When 1 mL of a water sample is added to Vial 1 medium) 1 through 6.2. Estimating bacterial populations by final vial of a dilution series to show these conditions should the serial-dilution method is a subject for statistical analysis.4.and 6 of a series. Incubation Incubate the dilution series at a temDerature There Is a Gap in the Positive Vials approximating the field temperatures (within f5"C' [f9"F]). the using small-volume serial-dilution testing is an inherently chances that contamination has occurred are more likely. Those experiencing problems in assessing the could have been picked up in the 1 mL of fluid transferred bacterial populations in a system may wish to try this latter from Vial 4 to Vial 5. 3.`.. Precision can be increased further by using even Duplicates Show Different Results more replicate samples.000 per mL. Both results may be tabulated for the sample. this standard specifies this case. It should not be.`. Tables D I through D3 (below) showthe impact more often. and the other 1O0 to 999. In inaccurate process.. 3. by W.`--- transferred into Vial 4 from Vial 3. Only a few living bacterial cells may have been the conditions that the field bacteria are accustomed to --``. duplicate testing as a minimum requirement for all growth testing..`.2.000 69.`-`-`.000.450 1:1.000 69 to 14. whereas the bacteria transferred to Vial 5 did. The bacteria left in Vial 4 did not survive for unknown RESULTS INTERPRETATION reasons.. it is usual to ignore the odd positive. A variety of bacterial media formulations are offered by TABLE D I SINGLE SERIAL DILUTION Number of Positive Vials Actual Dilution of Sample Estimated Range of Bacteria per mL 1 1:lO 1 to 145 2 1:lOO 7 to 1.5. The result would be growth in Vial 5. McCance.500 1:10. costs associated with increased replicate testing (including testing time) must be Quite often duplicate serial dilutions provide different weighed against the practical value derived from the estimates for the bacterial population in a water sample. What is equally 1.E. only the higher population range is reported. For this reason. example. These tables were derived from BACTERIAL GROWTH MEDIA Laboratory Methods in Food and Dairy Microbiology. sampling large-volume oilfield systems and then would be 10. and these same cells seeing. Perhaps critical. Harrigan and M.000 to 14. one serial dilution reports 10 to 99. of increased sample replication in serial-dilution testing on the precision of the results. Some effort at assessing several There are several likely explanations: of these media in each individual system to find the best medium to use generally pays dividends. granted.F. Accidental contamination of Vial 5 occurred. and 5 of a set. in the system water. is that the total dissolved the syringe needle touched some contaminated object in solids (TDS) in the media must approximate the TDS found the process of transferring fluid from Vial 4 to Vial 5. 2. or To illustrate. not 100. a generalized table is The interpretation when a gap occurs is still based on the routinely used to report the results of serial dilution testing. formulations.500.. but not in Vial 4. For increased precision. ¡. If more than estimating the bacterial population in those systems by one negative vial occurs between positive vials.``.000 per mL.000 6.`. TM0194-2004 turbidity in Vials 1. However.900 to 1.``. various media suppliers. and often overlooked.3).`.7 It should also be noted The selection of the proper growth media to analyze that these tables are based on statistical estimates and are bacterial populations in oilfield systems is often taken for not empirical.e.. the population in this example However.000 1:100..000 690 to 145. media preparation technique with alternative media but not in Vial 4.000 16 NACE International Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale.000 1:1. As noted earlier (Paragraph 3. Media selection is often critical.`.450. number of positive vials.````. 10/24/2005 13:11:09 MDT . Some vendors prepare the bacterial media in the actual field water to approximate more closely 2. components marked below a re of isobutyric acid. 0.`. 0.O.O 0.O.``..1 mL (from autoclaved stock Sterilize by autoclaving.O. NiCI.4H. caproic acid.````.).`. CaC1.2H. vitamin B. 10/24/2005 13:11:09 MDT .000 1.O 0.`.SH.4 e.5 g. and/or 7% propionic acid). 1.). NACE International 17 Copyright NACE International Provided by IHS under license with NACE Licensee=Occidental Chemical Corp New sub account/5910419101 No reproduction or networking permitted without license from IHS Not for Resale. p-aminobenzoic acid. 2-methylbutyric acid. Adjust pH to 7. 120 mg.300 to 33. Trace elements. 20% sodium acetate tri- hydrate. NaHCO.000 330 to 3.`-`-`. 2. Selenite..000 APPENDIX E ALTERNATIVE SRB GROWTH MEDIUM FORMULATION Postgate Medium GI1 g/L c. Additions to Postgate Medium G: succinic acid.. 1 mg.000 15. valeric acid. 60 mg.36 g (3 mL of 12% w/v solution Na.000 330. autoclaved stocks (e. thiamine.000 1:1. NaMo04. 10 mg/100 mL).PO.. 5 --``.000.000 1:100.15 MgCI.500 to 66.300. 3- added aseptically later.Se + 0.BO..000 1:100.`--- KCI 0.0 autoclaved under N.CI 0..600. 3 g (from autoclaved stock of 3 mg Carbon sources.000 33.`. 15 mg.O. Distilled water 970 mL f.2 “. a. 1 mL (from autoclaved stock of FeCI.3 NaCI 1.6H. 0. Vitamins.2 mg.5 g/L NaOH)..000 TABLE D3 FIVE REPLICATE SERIAL DILUTION Number of Positive Vials Actual Dilution of Sample Estimated Range of Bacteria per mL 1 1:lO 1 to 33 2 1:lOO 33 to 330 1:1.000 3. MnCI.000.O. 0. 70 mg.S.0.6H.3 d. 0.. H.1 mL (from filter-sterilized stock of biotin..``.000 1:1. 5 mg.000 to 660. fi Iter-steriI¡zed after satu ration with CO.. TMOI 94-2004 TABLE D2 DUPLICATE SERIAL DILUTION Number of Positive Vials Actual Dilution of Sample Estimated Range of Bacteria per mL 1:lO 1 to 66 1:lOO 15 to 660 1:1.2 g.`.55 mg (30 mL of 8.000 to 6. 0.O.000 150.000 to 330.. 100 mg.4H..2 with 2 N HCI. CuCIi2H20.SO.000 to 3.. Na. ZnCI.600 1:10.`.. methylbutyric acid. 25 mg/L).5% w/v solution.. 3. 25 mg.’GH.’2H. KH.g. CoCI. b. 1 mU100 mL final medium of Na.300 1:10.6 g/100 mL NaOH to pH 9).5 g of each. 0.000 150 to 6. Growth stimulants.