H A N D B O O K O F

Media forEnvironmentalMicrobiologyS E C O N D E D I T I O N

H A N D B O O K O F

Media forEnvironmentalMicrobiologyS E C O N D E D I T I O N

ByR O N A L D M . AT L A S

Published in 2005 byCRC PressTaylor & Francis Group 6000 Broken Sound Parkway NWBoca Raton, FL 33487-2742

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Library of Congress Cataloging-in-Publication Data

Atlas, Ronald M., 1946-Handbook of media for environmental microbiology / Ronald M. Atlas.--2nd ed.

p. cm.Includes index.ISBN 0-8493-3560-4 (alk. paper)1. Microbiology--Cultures and culture media--Handbooks, manuals, etc. 2. Sanitary microbiology

--Handbooks, manuals, etc. 3. Water--Microbiology--Handbooks, manuals, etc. 4. Microbial ecology--Handbooks, manuals, etc. I. Title.

QR66.3.A848 2005

579'.028--dc22 2004065561

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About the Author:Ronald M. Atlas, Ph.D., is dean of the Graduate School, professor of Biology, professor of Public Health, and codirector of the Center for the Deter-rence of Biowarfare and Bioterrorism at the Univer-sity of Louisville. He received his B.S. from the State University of New York at Stony Brook in 1968 and his M.S. and Ph.D. from Rutgers in 1970 and 1972, respectively. After spending a year at the Jet Propulsion Laboratory in Pasadena, California, he joined the faculty at the University of Louisville. He also has served as an Adjunct Professor at the University of Puerto Rico, External Examiner at the National University of Singapore, External Exam-iner at the Chinese University in Hong Kong, and Extraordinary Professor of Microbiology at the Uni-versity of Pretoria. Dr. Atlas has received a number of honors including: The University of Louisville Excellence in Research Award, Johnson and Johnson Fellowship for Biology, the American Soci-ety for Microbiology (ASM) Award in Applied and Environmental Microbiology, the ASM Founders Award, and the Edmund Youde Lectureship Award in Hong Kong. He has taught a variety of courses in microbiology at the University of Louisville and has authored several textbooks in general microbiology and microbial ecology. He has written nearly 300 research papers and authored or edited more than 20 books. He has conducted studies on the fate of oil in the sea. As part of these studies, he has extensively character-

ized marine bacterial populations and examined the diversity of microorganisms. He pioneered the field of bioremediation for marine oil spills. He also has conducted studies on the application of molecular techniques to environmental problems. His studies have included the development of suicide vectors for the containment of genetically engineered micro-organisms and the use of gene probes and the poly-merase chain reaction for environmental monitoring, including the detection of pathogens and indicator bacteria for water quality monitoring.He served as president of the American Society for Microbiology. Additionally, he has served on the National Institutes of Health (NIH) Recombinant Advisory Committee (RAC), as well as on various advisory boards for the Environmental Protection Agency (EPA), Federal Bureau of Investigation, and Department of Homeland Security. He has been chair-person of the Environmental Committee and the Task Force on Biological Weapons of the Public and Scien-tific Affairs Board of the American Society for Microbiology. He has been a national lecturer for Sigma Xi, an American Society for Microbiology Foundation lecturer, and an Australian Society for Microbiology national lecturer. He has served on the editorial boards of Applied and Environmental Micro-biology, Advances in Microbial Ecology, BioScience, Biotechniques, Journal of Environmental Science, Environmental Microbiology, Journal of Industrial Microbiology, and Biosecurity and Bioterrorism. He is editor of Critical Reviews in Microbiology.

Contents

INTRODUCTION .................................................................................................................................................................1Overview ..........................................................................................................................................................................1Organization .....................................................................................................................................................................1Names of Media................................................................................................................................................................1Trademarks .......................................................................................................................................................................1Composition of Media ......................................................................................................................................................1

Agars..........................................................................................................................................................................2Peptones.....................................................................................................................................................................2Meat and Plant Extracts.............................................................................................................................................3Growth Factors ..........................................................................................................................................................4Selective Components ...............................................................................................................................................4Differential Components ...........................................................................................................................................4pH Buffers .................................................................................................................................................................4

Preparation of Media ........................................................................................................................................................4Tyndallization ............................................................................................................................................................5Inspissation ................................................................................................................................................................5Autoclaving .............................................................................................................................................................. 5Filtration ....................................................................................................................................................................5Caution about Hazardous Components .....................................................................................................................5

Uses of Media ...................................................................................................................................................................6References ........................................................................................................................................................................6Web Resources..................................................................................................................................................................6

MEDIA, ALPHABETICAL..................................................................................................................................................7A .......................................................................................................................................................................................7B......................................................................................................................................................................................49C......................................................................................................................................................................................78D ...................................................................................................................................................................................123E....................................................................................................................................................................................195F ....................................................................................................................................................................................210G ...................................................................................................................................................................................231H ...................................................................................................................................................................................240I .....................................................................................................................................................................................273J.....................................................................................................................................................................................278K ...................................................................................................................................................................................278L....................................................................................................................................................................................282M...................................................................................................................................................................................291N ...................................................................................................................................................................................417O ...................................................................................................................................................................................431P ....................................................................................................................................................................................434Q ...................................................................................................................................................................................473R....................................................................................................................................................................................473S ....................................................................................................................................................................................494T....................................................................................................................................................................................552U ...................................................................................................................................................................................612V ...................................................................................................................................................................................614W ..................................................................................................................................................................................620X ...................................................................................................................................................................................623Y ...................................................................................................................................................................................626Z....................................................................................................................................................................................634

INDEX.................................................................................................................................................................................637

Introduction

Overview The second edition of the Handbook of Media forEnvironmental Microbiology includes descriptionsof nearly 2000 media that are used for environmen-tal microbial analyses, e.g., of water quality; for theisolation of microorganisms from soils, waters, andother environmental samples; and for the cultivationand maintenance of environmentally relevant micro-organisms, including plant pathogens. It providesthe formulations for the wide range of media neededto cultivate the ever expanding diversity of microor-ganisms that are culurable.

Organization The media described in the second edition of theHandbook of Media for Environmental Microbiol-ogy are organized alphabetically. Synonyms formedia are listed. The description of each mediumincludes its name(s), composition, instructions forpreparation, commercial sources, safety cautionswhere needed, and uses.

Names of MediaMedia often have numerous names. For the mostpart the second edition of the Handbook of Mediafor Environmental Microbiology retains the originalnames assigned in the literature. In some casesmedia with identical compositions produced by dif-ferent companies have different names. For exampleTrypticase Soy Agar produced as a BBL productof BD Diagnostic Systems, Tryptone Soy Agar pro-duced by Oxoid Unipath, and Tryptic Soy Agar pro-duced as a Difco product of BD Diagnostic Systemshave identical compositions. Many media also areknown by acronyms. TSA, for example, is the com-mon acronym for Trypticase Soy Agar.

TrademarksThe names of some media, components of media,and other terms are registered trademarks. Thetrademarked items referred to in the second editionof the Handbook of Media for Environmental Micro-biology are listed below. American Type Culture Collection and ATCC aretrademarks of the American Type Culture Collection.

Bacto, BiTek, and Difco are trademarks of DifcoLaboratories (registered trademarks owned by BectonDickinson and Company).

Oxoid and LabLemco are trademarks of Unipath Ltd.

Acidase, BBL, Biosate, CTA Medium, DTAMedium, DCLS Agar, Desoxycholate, Desoxycho-late Agar, Desoxycholate Citrate Agar, Enterococ-cosel, Eugonagar, Eugonbroth, GC-Lect, Gely-sate, IsoVitaleX, Mycobactosel, Mycophil,Mycosel, Myosate, Phytone, Polypeptone, Selen-ite-F Enrichment, Thiotone, Trichosel, Trypticase,TSA II, and TSI Agar are trademarks of Becton Dick-inson and Co.

Composition of MediaMedia for the cultivation of microorganisms containthe substances necessary to support the growth ofmicroorganisms. Due to the diversity of microorgan-isms and their diverse metabolic pathways, there arenumerous media. Even slight differences in the com-position of a medium can result in dramatically dif-ferent growth characteristics of microorganisms. When methods for culturing microorganisms werefirst developed in the 19th century, largely by RobertKoch and his colleagues, animal and plant tissueswere principally used as sources of nutrients used tosupport microbial growth. One of the major discov-eries of Fanny Hesse in Kochs laboratory was thatagar could be used to form solidified culture mediaon which microorganisms would grow. Extracts ofplants and animal tissues were prepared as broths ormixed with agar to form a variety of culture media.Virtually any plant, animal, or animal organ wasconsidered for use in preparing media. Infusionswere prepared from beef heart, calf brains, and beefliver, as a few examples. These classic infusions stillform the primary components of many media thatare widely used.

The composition section of each medium describesthe ingredients that make up the medium, theiramounts, and the pH. It lists those ingredients inorder of decreasing amount. Solids are listed firstshowing the weights to be added, followed by liq-uids showing the volumes to be included in themedium. The composition uses generic terms where these areapplicable. For example, pancreatic digest of caseinis marketed by various manufacturers as trypticase,tryptone, and other commercial product names.While there may well be differences between theseproducts, such differences are undefined. Variationsalso occur between batches of products produced asdigests of animal tissues. Media for the cultivation of microorganisms have asource of carbon for incorporation into biomass. Forautotrophs, the carbon source most often is carbondioxide, which may be supplied as bicarbonatewithin the medium. Carbohydrates, such as glucose,or other organic compounds, such as acetate, variouslipids, proteins, hydrocarbons, and other organiccompounds, are included in media as sources of car-bon for heterotrophs. These carbon sources may alsoserve as the supply of energy. Other compounds,such as ammonium ions, nitrite ions, elemental sul-fur, and reduced iron, may be used as the sources ofenergy for the cultivation of autotrophs. Nitrogenalso is required for microbial growth. It may be sup-plied as inorganic nitrogen compounds for the culti-vation of some microorganisms but more commonlyis supplied as proteins, peptones, or amino acids.Phosphates and metals, such as magnesium and iron,

2 Composition of Media

are also necessary components of microbiologicalmedia. Phosphates may also serve as buffers tomaintain the pH of the medium within the growthtolerance limits of the microorganism being culti-vated. Various additional growth factors may also beincluded in the media. AgarsAgar is the most common solidifying agent used inmicrobiological media. Agar is a polysaccharideextract from marine algae. It melts at 84C andsolidifies at 38C. Agar concentrations of 15.0g/Ltypically are used to form solid media. Lower con-centrations of 7.510.0g/L are used to produce softagars or semisolid media. Below are some agarsused as solidifying agents in various media. Agar Bacteriological (Agar No. 1)

An agar with low calcium and magnesium. Avail-able from Oxoid Unipath.

Agar, Bacto A purified agar with reduced pigmented com-pounds, salts, and extraneous matter. Availablefrom BD Diagnostic Systems.

Agar, BiTek Agar prepared as a special technical grade. Avail-able from BD Diagnostic Systems.

Agar, Flake A technical-grade agar. Available from BD Diag-nostic Systems.

Agar, Grade AA select-grade agar containing minerals. Avail-able from BD Diagnostic Systems.

Agar, GranulatedA high-grade granulated agar that has been fil-tered, decolorized, and purified. Available fromBD Diagnostic Systems.

Agar, PurifiedA very high-grade agar that has been filtered,decolorized, and purified by washing and extrac-tion of refined agars. It has reduced mineral con-tent. Available from BD Diagnostic Systems.

Agar, Technical (Agar No. 3)A technical-grade agar. Available from BD Diag-nostic Systems and Oxoid Unipath.

AgaroseA low-sulfate neutral gelling fraction of agar thatis a complex galactose polysaccharide of nearneutral charge.

IonagarA purified agar. Available from Oxoid Unipath.

Noble AgarAn agar that has been extensively washed and isessentially free of impurities. Available from BDDiagnostic Systems.

Purified AgarAn agar that has been extensively washed andextracted with water and organic solvent. Avail-able from BD Diagnostic Systems and Oxoid Uni-path.

PeptonesMany complex media, that is, media in which not allthe specific chemical components are known, con-tain peptones as the source of nitrogen. Peptones arehydrolyzed proteins formed by enzymatic or acidic

digestion. Casein most often is used as the proteinsubstrate for forming peptones, but other substances,such as soybean meal, also are commonly employed.Below is a list of some of the peptones that are usedas ingredients in various media. Acidase Peptone

A hydrochloric acid hydrolysate of casein. It has anitrogen content of 8% and is deficient in cystineand tryptophan. Available from BD DiagnosticSystems.

Bacto CasitoneA pancreatic digest of casein. Available from BDDiagnostic Systems.

Bacto PeptaminA peptic digest of animal tissues. Available fromBD Diagnostic Systems.

Bacto PeptoneAn enzymatic digest of animal tissues. It has ahigh concentration of low molecular weight pep-tones and amino acids. Available from BD Diag-nostic Systems.

Bacto Proteose PeptoneAn enzymatic digest of animal tissues. It has ahigh concentration of high molecular weight pep-tones. Available from BD Diagnostic Systems.

Bacto SoytoneA enzymatic hydrolysate of soybean meal. Avail-able from BD Diagnostic Systems.

Bacto TripletsAn enzymatic hydrolysate containing numerouspeptides, including those of higher molecularweights. Available from BD Diagnostic Systems.

Bacto TryptoneA pancreatic digest of casein. Available from BDDiagnostic Systems.

Biosate PeptoneA hydrolysate of plant and animal proteins. Avail-able from BD Diagnostic Systems.

Casein HydrolysateA hydrolysate of casein prepared with hydrochlo-ric acid digestion under pressure and neutralizedwith sodium hydroxide. It contains total nitrogenof 7.6% and NaCl of 28.3%. Available fromOxoid Unipath.

GelatoneA pancreatic digest of gelatin. Available from BDDiagnostic Systems.

Gelysate PeptoneA pancreatic digest of gelatin deficient in cystineand tryptophan and which has a low carbohydratecontent. Available from Oxoid Unipath.

Lactoalbumin HydrolysateA pancreatic digest of lactoalbumin, a milk wheyprotein. It has high levels of amino acids. It con-tains total nitrogen of 11.9% and NaCl of 1.4%.Available from BD Diagnostic Systems andOxoid Unipath.

Liver Digest NeutralizedA papaic digest of liver that contains total nitro-gen of 11.0% and NaCl of 1.6%. Available fromOxoid Unipath.

Mycological PeptoneA peptone that contains total nitrogen of 9.5% andNaCl of 1.1%. Available from Oxoid Unipath.

Composition of Media 3

Myosate PeptoneA pancreatic digest of heart muscle. Availablefrom BD Diagnostic Systems.

NeopeptoneAn enzymatic digest of protein. Available fromfrom BD Diagnostic Systems.

Peptone Bacteriological NeutralizedA mixed pancreatic and papaic digest of animaltissues. It contains total nitrogen of 14.0% andNaCl of 1.6%. Available from BD DiagnosticSystems and Oxoid Unipath.

Peptone PA peptic digest of fresh meat that has a high sulfurcontent and contains total nitrogen of 11.12% andNaCl of 9.3%. Available from BD DiagnosticSystems and Oxoid Unipath.

Peptonized MilkA pancreatic digest of high-grade skim milk pow-der. It has a high carbohydrate and calcium con-centration. It contains total nitrogen of 5.3% andNaCl of 1.6%. Available from Oxoid Unipath.

Phytone PeptoneA papaic digest of soybean meal. It has a highvitamin and a high carbohydrate content. Avail-able from BD Diagnostic Systems.

Polypeptone PeptoneA mixture of peptones composed of equal parts ofpancreatic digest of casein and peptic digest ofanimal tissue. Available from BD Diagnostic Sys-tems.

Proteose PeptoneA specialized peptone prepared from a mixture ofpeptones that contains a wide variety of highmolecular weight peptides. It contains total nitro-gen of 12.7% and NaCl of 8.0%. Available fromBD Diagnostic Systems and Oxoid Unipath.

Proteose Peptone No. 2An enzymatic digest of animal tissues with a highconcentration of high molecular weight peptones.Available from BD Diagnostic Systems.

Proteose Peptone No. 3An enzymatic digest of animal tissues. It has ahigh concentration of high molecular weight pep-tones. Available from BD Diagnostic Systems.

Soya PeptoneA papaic digest of soybean meal with a high car-bohydrate concentration. It contains total nitrogenof 8.7% and NaCl of 0.4%. Available from OxoidUnipath.

SoytoneA papaic digest of soybean meal. Available fromBD Diagnostic Systems.

Special PeptoneA mixture of peptones, including meat, plant, andyeast digests. It contains a wide variety of pep-tides, nucleotides, and minerals. It contains totalnitrogen of 11.7% and NaCl of 3.5%. Availablefrom Oxoid Unipath.

Thiotone E PeptoneAn enzymatic digest of animal tissue. Availablefrom BD Diagnostic Systems.

Trypticase PeptoneA pancreatic digest of casein. It has a very lowcarbohydrate content and a relatively high tryp-tophan content. Available from BD DiagnosticSystems.

TryptoneA pancreatic digest of casein. It contains totalnitrogen of 12.7% and NaCl of 0.4%. Availablefrom Oxoid Unipath.

Tryptone TA pancreatic digest of casein with lower levels ofcalcium, magnesium, and iron than tryptone. Itcontains total nitrogen of 11.7% and NaCl of4.9%. Available from BD Diagnostic Systems andOxoid Unipath.

TryptoseAn enzymatic hydrolysate containing high molec-ular weight peptides. It contains total nitrogen of12.2% and NaCl of 5.7%. Available from BDDiagnostic Systems and Oxoid Unipath.

Meat and Plant ExtractsMeat and plant infusions are aqueous extracts thatare commonly used as sources of nutrients for thecultivation of microorganisms. Such infusions con-tain amino acids and low molecular weight peptides,carbohydrates, vitamins, minerals, and trace metals.Extracts of animal tissues contain relatively highconcentrations of water-soluble protein componentsand glycogen. Extracts of plant tissues contain rela-tively high concentrations of carbohydrates. With regard to infusions, many media list as aningredient infusion from beef heart or another ani-mal tissue. This ingredient is prepared by boiling agiven amount of the animal tissue (e.g., 500.0g), andthen using the liquid or, more commonly, drying thebroth and using the solids from the infusion. Theactual weight of the dry solids extracted from the hotwater used to create the infusion varies, and so theingredient typically is simply listed as 500.0g beefheart infusion, although the actual weight of solidsrecovered from the infusion and used in the mediumis far less.Below is a list of some of the meat and plant extractsthat are used as ingredients in various media.Bacto Beef

A desiccated powder of lean beef. Available fromBD Diagnostic Systems.

Bacto Beef ExtractAn extract of beef (paste). Available from BDDiagnostic Systems.

Bacto Beef Extract DesiccatedAn extract of desiccated beef. Available from BDDiagnostic Systems.

Bacto LiverA desiccated powder of beef liver. Available fromBD Diagnostic Systems.

Lab-LemcoA meat extract powder. Available from OxoidUnipath.

Liver DesiccatedDehydrated ox livers. Available from Oxoid Uni-path.

Malt ExtractA water-soluble extract from germinated graindried by low-temperature evaporation. It has ahigh carbohydrate content. It contains total nitro-gen of 1.1% and NaCl of 0.1%.

4 Preparation of Media

Growth FactorsMany microorganisms have specific growth factorrequirements that must be included in media fortheir successful cultivation. Vitamins, amino acids,fatty acids, trace metals, and blood componentsoften must be added to media. In some cases, spe-cific defined components are used to meet thegrowth factor requirements. Incorporation of growthfactors are used to enrich, that is, to increase thenumbers of particular species of microorganisms.Most often, mixtures of growth factors are used inmicrobiological media. Acid hydrolysates of caseincommonly are used as sources of amino acids.Extracts of yeast cells also are employed as sourcesof amino acids and vitamins for the cultivation ofmicroorganisms. Many media, particularly thoseemployed in the clinical laboratory, contain blood orblood components that serve as essential nutrientsfor fastidious microorganisms. X factor (heme) andV factor (nicotinamide adenine dinucleotide) oftenare supplied by adding hemoglobin, IsoVitaleX, and/or Supplement VX. Selective ComponentsMany media contain selective components thatinhibit the growth of nontarget microorganisms.Selective media are especially useful in the isolationof specific microorganisms from mixed populations.In many media for the study of microorganisms innature, compounds are included in the media as solesources of carbon or nitrogen so that only a fewtypes of microorganisms can grow. Selective toxiccompounds are also frequently used to select for thecultivation of particular microbial species. The iso-lation of a pathogen from a stool specimen, forexample, where there is a high abundance of non-pathogenic normal microbiota, requires selectivemedia. Often, antimicrobics or other selectivelytoxic compounds are incorporated into media to sup-press the growth of the background microbiota whilepermitting the cultivation of the target organism ofinterest. Bile salts, selenite, tetrathionate, tellurite,azide, phenylethanol, sodium lauryl sulfate, highsodium chloride concentrations, and various dyessuch as eosin, Crystal Violet, and Methylene Blueare used as selective toxic chemicals. Antimicrobialagents used to suppress specific types of microor-ganisms include ampicillin, chloramphenicol, colis-tin, cycloheximide, gentamicin, kanamycin, nali-dixic acid, sulfadiazine, and vancomycin. Variouscombinations of antimicrobics are effective in sup-pressing classes of microorganisms, such as entericbacteria. Differential ComponentsThe differentiation of many microorganisms isbased upon the production of acid from various car-bohydrates and other carbon sources or the decar-boxylation of amino acids. Some media includeindicators, particularly of pH, that permit the visualdetection of changes in pH resulting from such met-abolic reactions.

Below is a list of some commonly used pH indica-tors. pH Indicator pH Range Acid Alkaline

Color Colorm-Cresol Purple 0.5 2.5 Red YellowThymol Blue 1.2 2.8 Red YellowBromphenol Blue 3.0 4.6 Yellow BlueBromcresol Green 3.8 5.4 Yellow BlueChlorcresol Green 4.0 5.6 Yellow BlueMethyl Red 4.2 6.3 Red YellowChlorphenol Red 5.0 6.6 Yellow RedBromcresol Purple 5.2 6.8 Yellow PurpleBromothymol Blue 6.0 7.6 Yellow BluePhenol Red 6.8 8.4 Yellow RedCresol Red 7.2 8.8 Yellow Redm-Cresol Purple 7.4 9.0 Yellow PurpleThymol Blue 8.0 9.6 Yellow BlueCresolphthalein 8.2 9.8 Colorless RedPhenolphthalein 8.3 10.0 Colorless Red

pH BuffersMaintaining the pH of media usually is accom-plished by the inclusion of suitable buffers. Becausemicroorganisms grow optimally only within certainlimits of a pH range, the pH generally is maintainedwithin a few tenths of a pH unit. For the phosphagebuffers, the pH is established by using varying vol-umes of equimolar concentrations of Na2HPO4 andNaH2PO4.

pH. Na2HPO4 (mL) NaH2PO4 (mL)5.4 3.0 97.05.6 5.0 95.05.8 7.8 92.26.0 12.0 88.06.2 18.5 81.56.4 26.5 73.56.6 37.5 62.56.8 50.0 50.07.0 61.1 38.97.2 71.5 28.57.4 80.4 19.67.6 86.8 13.27.8 91.4 8.68.0 94.5 5.5

Preparation of MediaThe ingredients in a medium are usually dissolved,and the medium is then sterilized. When agar is usedas a solidifying agent, the medium must be heatedgently, usually to boiling, to dissolve the agar. Insome cases where interactions of components, suchas metals, would cause precipitates, solutions mustbe prepared and occasionally sterilized separatelybefore mixing the various solutions to prepare the

Preparation of Media 5

complete medium. The pH often is adjusted prior tosterilization, but in some cases sterile acid or base isused to adjust the pH of the medium following steril-ization. Many media are sterilized by exposure toelevated temperatures. The most common method isto autoclave the medium. Different sterilization pro-cedures are employed when heat-labile compoundsare included in the formulation of the medium. TyndallizationExposure to steam at 100C for 30 min will kill veg-etative bacterial cells but not endospores. Suchexposure can be achieved using flowing steam in anArnold sterilizer. By allowing the medium to cooland incubate under conditions where endospore ger-mination will occur and by repeating the 100C30min exposure on 3 successive days, the medium canbe sterilized because all the endospores will havegerminated and the heat exposure will have killed allthe vegetative cells. This process of repetitive expo-sure to 100C is called tyndallization, after its dis-coverer, John Tyndall.InspissationInspissation is a heat exposure method that isemployed with high-protein materials, such as egg-containing media, that cannot withstand the hightemperatures used in autoclaving. This processcauses coagulation of the protein without greatlyaltering its chemical properties. Using an Arnoldsterilizer or a specialized inspissator, the medium isexposed to 7580C for 2 hr on each of 3 succes-sive days. Inspissation using an autoclave employsexposure to 8590C for 10 min achieved by hav-ing a mixture of air and steam in the chamber, fol-lowed by a 15 min exposure during which the tem-perature is raised to 121C using only steam underpressure in the chamber; the temperature then isslowly lowered to less than 60C. AutoclavingAutoclaving uses exposure to steam, generally underpressure, to kill microorganisms. Exposure for 15min to steam at 15 psi121C is most commonlyused. Such exposure kills vegetative bacterial cellsand bacterial endospores. Media containing carbo-hydrates often are sterilized at 116118C in orderto prevent the decomposition of the carbohydrateand the formation of toxic compounds that wouldinhibit microbial growth. Below is a list of pressuretemperature relationships.

Pressurepsi TemperatureC0 100.01 101.92 103.63 105.34 106.95 108.46 109.87 111.38 112.6

9 113.910 115.211 116.412 117.613 118.814 119.915 121.016 122.017 123.018 124.019 125.020 126.021 126.922 127.823 128.724 129.625 130.4

FiltrationFiltration is commonly used to sterilize media con-taining heat-labile compounds. Liquid media arepassed through sintered glass or membranes, typi-cally made of cellulose acetate or nitrocellulose,with small pore sizes. A membrane with a pore sizeof 0.2mm will trap bacterial cells and, therefore,sometimes is called a bacteriological filter. By pre-venting the passage of microorganisms, filtrationrenders fluids free of bacteria and eukaryotic micro-organisms, that is, free of living organisms, andhence sterile. Many carbohydrate solutions, antibi-otic solutions, and vitamin solutions are filter steril-ized and added to media that have been cooled totemperatures below 50C. Caution about Hazardous ComponentsSome media contain components that are toxic orcarcinogenic. Appropriate safety precautions mustbe taken when using media with such components.Basic fuchsin and acid fuchsin are carcinogens, andcaution must be used in handling media with thesecompounds to avoid dangerous exposure that couldlead to the development of malignancies. Thalliumsalts, sodium azide, sodium biselenite, and cyanideare among the toxic components found in somemedia. These compounds are poisonous, and stepsmust be taken to avoid ingestion, inhalation, or skincontact. Azides also react with many metals, espe-cially copper, to form explosive metal azides. Thedisposal of azides must avoid contact with copper orachieve sufficient dilution to avoid the formation ofsuch hazardous explosive compounds. Media withsulfur-containing compounds may result in the for-mation of hydrogen sulfide, which is a toxic gas.Care must be used to ensure proper ventilation.Media with human blood or human blood compo-nents must be handled with great caution to avoidexposure to human immunodeficiency virus andother pathogens that contaminate some blood sup-plies.

6 Uses of Media

Uses of MediaThe Handbook of Media for Environmental Micro-biology, Second Edition contains the media used forthe testing of waters and wastewaters recommendedby the USEPA for the standard methods examinationof water and food. It also includes the formulationsof media used to cultivate the microorganisms ofenvironmental significance that are held in the majorculture collections of the world.

ReferencesBelow is a list of references that can be consulted forfurther information about media used for the isola-tion, cultivation, and differentiation of microorgan-isms. Archaea: A Laboratory Manual. 1995. F.T. Robb,A.R. Place, K.R. Sowers, H.J. Schreier, C. Das-Sarma, and E.M. Fleischmann. Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York. Archaea: A Laboratory Manual: Halophiles. 1995.DasSarma, S. and E.M. Fleischmann. Cold SpringHarbor Laboratory Press. Cold Spring Harbor, NewYork.Archaea: A Laboratory Manual: Methanogens.1995. Sowers, K.R. and H.J. Schreier. Cold SpringHarbor Laboratory Press. Cold Spring Harbor, NewYork.Archaea: A Laboratory Manual: Thermophiles.1995. Robb, F.T. and A.R. Place. Cold Spring Har-bor Laboratory Press, Cold Spring Harbor, NewYork.Difco & BBL Manual: Dehydrated Culture Mediaand Reagents for Microbiology. 2003. Becton, Dick-inson and Co., Sparks, Maryland.The Oxoid Manual. 1990. E.Y. Bridson, ed. UnipathLtd. Basingstoke, Hampshire, England.The Prokaryotes: An Evolving Electronic Resourcefor the Microbiological Community. 1999. M.Dworkin, Springer-Verlag Inc. New York. Standard Methods for the Examination of Water andWastewater. 1998. Clesceri, L.S., A.E. Greenberg,and A.D. Eaton, American Public Health Associa-tion Publications, Washington D.C.

Web ResourcesBelow is a list of Web sites that provide informationabout media and microbial cultures.

American Type Culture Collection (ATCC), a globalbiological resource in the United States with many ser-vices. http://www.atcc.org/catalogs/catalogs.html

Belgian Coordinated Collections of Microorganismsof the Laboratorium Voor Microbiologie at the Uni-versity of Gent (LMG). http://www.belspo.be/bccm/db/media.htm

Czechoslovokian Collection of Microorganisms.(CCM). http://www.sci.immuni.cz/ccm

Finish Culture Collection, Valtion Teknillinen Tutkimuskeskus (VTT). http://www.inf.vtt.fi

German Collection of Microorganisms (DSMZ).http://www.gbf-braunschweig.de

Japanese Collection of Microorganisms and Microbial Cultures. http://www.jcm.riken.go.jp

Netherlands Centraalbureau voor Schimmelcultures(CBS). http://www.cbs.know.know.nl/databases.

Russian Specialized Collection of Alkanotrophs.http://www.ecology. psu.ru/iegmcol

Spanish Collection of Microorganisms (ColeccinEspaola de Cultivos Tipo Catalogo de Cepas).http://www.cect.org/english/index.htm

United Kingdom National Collection of Yeast Cultures. http://www.ifr.bbsrc.ac.uk/ncyc

United Kingdom National Culture Collection Microbiological Resources. http://www.ukncc.co.uk

United States Food and Drug Administration Bacteriological Analytical Manual. http://vm.cfsan.fda.gov/~embam/bam-toc.html.

World Federation of Culture Collections. http://www. wfcc.info

Media, Alphabetical 7

A 1 BrothComposition per liter:Pancreatic digest of casein .......................................... 20.0gLactose .......................................................................... 5.0gNaCl .............................................................................. 5.0gSalicin ........................................................................... 0.5gTriton X-100 ..........................................................1.0mL

pH 6.9 0.1 at 25C

Source: This medium is available as a premixed powderfrom BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.Gently heat and bring to boiling. Distribute into test tubescontaining an inverted Durham tube. Autoclave for 10 minat 15 psi pressure121C.

Use: For the detection of fecal coliforms in treated waste-water, and seawater by a most-probable-number (MPN)method. Multiple dilutions of samples (3, 5, or 10 replicatesper dilution) are added to tubes containing A 1 broth. Afterincubation, test tubes with gas accumulation in the Durhamtubes are scored positive and those with no gas as negative.A MPN table is consulted to determine the most probablenumber of fecal coliforms.

Acanthamoeba MediumComposition per liter:Proteose peptone ......................................................... 15.0gGlucose ....................................................................... 15.0gKH2PO4......................................................................... 0.3gL-Methionine............................................................ 14.9mgThiamine .................................................................... 1.0mgBiotin ......................................................................... 0.2mgVitamin B12 .................................................................1.0gSalt solution ...............................................................1.0mL

pH 5.5 0.2 at 25CSalt Solution:Composition per 100.0mL:MgSO47H2O ............................................................. 2.46gCaCl22H2O................................................................. 0.15gFeCl3 ........................................................................... 0.02g

Preparation of Salt Solution: Add components to dis-tilled/deionized water and bring volume to 100.0mL. Mixthoroughly.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.Adjust pH to 5.5. Filter through Whatman filter paper to re-move particles. Distribute into screw-capped tubes orflasks. Autoclave for 15 min at 15 psi pressure121C.

Use: For the cultivation of Acanthamoeba species.

ACC MediumComposition per liter: Proteose peptone ......................................................... 20.0gAgar ............................................................................ 12.0gGlycerol ........................................................................ 1.5gK2SO4............................................................................ 1.5gMgSO47H2O ................................................................ 1.5gAntibiotic solution ...................................................10.0mL

pH 7.2 0.2 at 25C.Antibiotic Solution:Composition per 10.0mL: Cycloheximide .......................................................... 0.075gAmpicillin ................................................................... 0.05gChloramphenicol..................................................... 0.0125g

Preparation of Antibiotic Solution: Add componentsto distilled/deionized water and bring volume to 10.0mL.Mix thoroughly. Filter sterilize.

Preparation of Medium: Add components, except an-tibiotic solution, to distilled/deionized water and bring vol-ume to 990.0mL. Mix thoroughly. Gently heat and bring toboiling. Autoclave for 15 min at 15 psi pressure121C.Cool to 4550C. Aseptically add sterile antibiotic solu-tion. Mix thoroughly. Pour into sterile Petri dishes or dis-tribute into sterile tubes.

Use: For the selective isolation and cultivation of fluores-cent Pseudomonas species.

Acetamide AgarComposition per liter:Agar .............................................................................15.0gAcetamide....................................................................10.0gNaCl ..............................................................................5.0gK2HPO4 .........................................................................1.0gNH4H2PO4 .....................................................................1.0gMgSO47H2O.................................................................0.2gBromothymol Blue ......................................................0.08g

pH 6.9 0.2 at 25C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.Gently heat and bring to boiling. Adjust pH. Distribute intotubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Cool tubes in a slanted position to produce a longslant.Use: For the differentiation of nonfermentative Gram-neg-ative bacteria, especially Pseudomonas aeruginosa. Can beused as a confirmatory test for water analysis. Bacteria thatdeamidate acetamide turn the medium blue.

Acetamide AgarComposition per liter:Agar .............................................................................15.0gAcetamide....................................................................10.0gNaCl ..............................................................................5.0gK2HPO4 .......................................................................1.39gKH2PO4 .......................................................................0.73gMgSO47H2O.................................................................0.5gPhenol Red ................................................................0.012g

pH 6.9 0.2 at 25C

Source: This medium is available as a premixed powderfrom BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.Gently heat and bring to boiling. Adjust pH. Distribute intotubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Cool tubes in a slanted position to produce a longslant.Use: For the differentiation of nonfermentative Gram-neg-ative bacteria, especially Pseudomonas aeruginosa. Can beused as a confirmatory test for water analysis. Bacteria thatdeamidate acetamide turn the medium blue.

Acetamide BrothComposition per liter:Acetamide....................................................................10.0gNaCl ..............................................................................5.0gK2HPO4 .......................................................................1.39gKH2PO4 .......................................................................0.73g

8 Acetate Agar

MgSO47H2O ................................................................ 0.5gPhenol Red................................................................ 0.012g

pH 6.9 0.2 at 25C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.Adjust pH. Autoclave for 15 min at 15 psi pressure121C.Use: For the differentiation of nonfermentative Gram-neg-ative bacteria, especially Pseudomonas aeruginosa. Can beused as a confirmatory test for water analysis. Bacteria thatdeamidate acetamide turn the broth purplish red.

Acetate AgarComposition per liter:Agar ............................................................................ 15.0gYeast extract .................................................................. 2.0gSodium acetate .............................................................. 1.0gPancreatic digest of casein ............................................ 1.0g

pH 7.4 0.2 at 25C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.Gently heat and bring to boiling. Distribute into tubes orflasks. Autoclave for 15 min at 15 psi pressure121C. Pourinto sterile Petri dishes or leave in tubes.Use: For the cultivation and maintenance of Caryophanonlatum.

Acetate Differential Agar(Sodium Acetate Agar)

(Simmons Citrate Agar, Modified)Composition per liter:Agar ............................................................................ 20.0gNaCl .............................................................................. 5.0gSodium acetate .............................................................. 2.0g(NH4)H2PO4.................................................................. 1.0gK2HPO4......................................................................... 1.0gMgSO47H2O ................................................................ 0.2gBromothymol Blue ..................................................... 0.08g

pH 6.8 0.2 at 25C

Source: This medium is available as a premixed powderfrom BD Diagnostic Systems.Preparation of Medium: Add components to cold dis-tilled/deionized water and bring volume to 1.0L. Mix thor-oughly. Gently heat and bring to boiling. Distribute intotubes to produce a 1 cm butt and 30 cm slant. Autoclave for15 min at 15 psi pressure121C. Cool tubes in a slanted po-sition.Use: For the differentiation of Shigella species from Es-cherichia coli and also for the differentiation of nonfer-menting Gram-negative bacteria. Bacteria that can utilizeacetate as the sole carbon source turn the medium blue.

Acetivibrio cellulolyticus MediumComposition per 1170.0mL:Cellobiose or cellulose (MN 300, Whatman

CF II, Kleenex tissue paper, or HCl-treated cotton)............................................ 3.0g

NaHCO3 ........................................................................ 2.0gL-CysteineHCl............................................................ 0.25gNa2S9H2O.................................................................. 0.25gFeSO47H2O................................................................ 0.02gResazurin .................................................................. 0.001gMineral solution 1....................................................75.0mLMineral solution 2....................................................75.0mLCellobiose solution ..................................................50.0mLTrace elements solution ...........................................10.0mL

Vitamin solution ...................................................... 10.0mLReducing agent solution .......................................... 10.0mL

pH 7.2 0.2 at 25CMineral Solution 1:Composition per liter:K2HPO4 .........................................................................3.9g

Preparation of Mineral Solution 1: Add K2HPO4 todistilled/deionized water and bring volume to 1.0L. Mixthoroughly.

Mineral Solution 2:Composition per liter:(NH4)2SO4 .....................................................................6.0gK2HPO4 .........................................................................2.4gMgSO47H2O.................................................................1.2gCaCl22H2O .................................................................0.72gNaCl ............................................................................0.59g

Preparation of Mineral Solution 2: Add componentsto distilled/deionized water and bring volume to 1.0L. Mixthoroughly.

Cellobiose Solution:Composition per 50.0mL:D-Cellobiose ..................................................................5.0g

Preparation of Cellobiose Solution: Add cellobioseto distilled/deionized water and bring volume to 50.0mL.Mix thoroughly. Sparge under 100% N2 gas for 3 min. Filtersterilize. Store under N2 gas.

Trace Elements Solution:Composition per liter:MgSO47H2O ................................................................3.0gNitrilotriacetic acid........................................................1.5gCaCl22H2O...................................................................1.0gNaCl ..............................................................................1.0gMnSO42H2O ................................................................0.5gCoSO47H2O ...............................................................0.18gZnSO47H2O ...............................................................0.18gFeSO47H2O..................................................................0.1gNiCl26H2O ...............................................................0.025gKAI(SO4)212H2O ......................................................0.02gCuSO45H2O ...............................................................0.01gH3BO3 .........................................................................0.01gNa2MoO42H2O ..........................................................0.01gNa2SeO35H2O ...........................................................0.3mg

Preparation of Trace Elements Solution: Add ni-trilotriacetic acid to 500.0mL of distilled/deionized water.Dissolve by adjusting pH to 6.5 with KOH. Add distilled/deionized water to 1.0L. Add remaining components. Mixthoroughly. Adjust pH to 7.0 with 1N KOH.

Vitamin Solution:Composition per liter:PyridoxineHCl.........................................................10.0mgCalcium DL-pantothenate ...........................................5.0mgLipoic acid..................................................................5.0mgNicotinic acid .............................................................5.0mgp-Aminobenzoic acid .................................................5.0mgRiboflavin...................................................................5.0mgThiamineHCl .............................................................5.0mgBiotin ..........................................................................2.0mgFolic acid ....................................................................2.0mgVitamin B12.................................................................0.1mg

Preparation of Vitamin Solution: Add components todistilled/deionized water and bring volume to 1.0L. Mixthoroughly.

Acetobacter diazotrophicus Agar 9

Reducing Agent Solution:Composition per 110.0mL:L-CysteineHClH2O ..................................................... 2.5gNa2S9H2O.................................................................... 2.5g

Preparation of Reducing Agent Solution: Add110.0mL of distilled/deionized water to a 250.0mL flask.Boil under N2 gas for 1 min. Cool to room temperature. AddL-cysteineHClH2O and dissolve. Adjust to pH 9 with 5NNaOH. Add washed Na2S9H2O and dissolve. Distributeunder N2 gas in 10.0mL volumes into tubes. Autoclave for10 min at 15 psi pressure121C.

Preparation of Medium: Add components, except cel-lobiose solution and reducing agent solution, to distilled/deionized water and bring volume to 940.0mL. Gently heatand bring to boiling. Continue boiling for 3 min. Cool toroom temperature under 80% N2 + 20% CO2. Adjust pH to7.6 by gassing. Distribute anaerobically under 80% N2 +20% CO2. Autoclave for 15 min at 15 psi pressure121C.After autoclaving, the pH of the medium will be 7.2. Priorto inoculation of cultures, aseptically and anaerobically add0.1mL of sterile reducing agent solution and 0.5mL of ster-ile cellobiose solution to each tube containing 9.4mL ofsterile basal medium.

Use: For the cultivation and maintenance of Acetivibriocellulolyticus, an anaerobic, cellulolytic bacterium.

Acetivibrio Desulfovibrio Medium(LMG Medium 105)

Composition per liter:Solution A ..............................................................869.0mLSolution C ..............................................................100.0mLSolution D ................................................................10.0mLSolution E ................................................................10.0mLSolution F.................................................................10.0mLSolution B ..................................................................1.0mL

pH 7.7 0.2 at 25C

Solution A:Composition per 869.0mL:Na2SO4.......................................................................... 3.0gNaCl .............................................................................. 1.0gKCl................................................................................ 0.5gMgCl26H2O.................................................................. 0.4gNH4Cl............................................................................ 0.3gKH2PO4......................................................................... 0.2gCaCl22H2O................................................................. 0.15g

Preparation of Solution A: Add components to dis-tilled/deionized water and bring volume to 869.0mL. Mixthoroughly. Prepare and autoclave part A under 80% N2 +20% CO2. Autoclave for 15 min at 15 psi pressure121C.Cool to room temperature.

Solution B:Composition per liter:FeCl24H2O ................................................................... 1.5gCoCl26H2O ................................................................ 0.19gMnCl24H2O.................................................................. 0.1gZnCl2........................................................................... 0.07gH3BO3 ......................................................................... 0.06gNa2MoO42H2O .......................................................... 0.04gNiCl26H2O ................................................................. 0.02gCuCl22H2O ................................................................ 0.02gHCl, 25% .................................................................10.0mL

Preparation of Solution B: Add the FeCl24H2O to theHCl. Add distilled/deionized water and bring volume to1.0L. Add remaining components. Mix thoroughly. Auto-

clave under 100% N2 for 15 min at 15 psi pressure121C.Cool to room temperature.

Solution C:Composition per 100.0mL:NaHCO3 ........................................................................5.0g

Preparation of Solution C: Add the NaHCO3 to dis-tilled/deionized water and bring volume to 100.0mL. Mixthoroughly. Filter sterilize. Gas with 80% N2 + 20% CO2 toremove residual O2.

Solution D:Composition per 10.0mL:Sodium butyrate ............................................................0.7gSodium caproate ............................................................0.3gSodium octanoate ........................................................0.15g

Preparation of Solution D: Add components to dis-tilled/deionized water and bring volume to 10.0mL. Mixthoroughly. Autoclave under 100% N2 for 15 min at 15 psipressure121C. Cool to room temperature.

Solution E:Composition per 10.0mL:Yeast extract ..................................................................1.0gThiamineHCl ......................................................... 100.0gp-Aminobenzoic acid ............................................... 40.0gD(+)-Biotin............................................................... 10.0gPreparation of Solution E: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thor-oughly. Autoclave under 100% N2 for 15 min at 15 psi pres-sure121C. Cool to room temperature.

Solution F:Composition per 10.0mL:Na2S9H2O ....................................................................0.4g

Preparation of Solution F: Add Na2S9H2O to dis-tilled/deionized water and bring volume to 10.0mL. Mixthoroughly. Autoclave under 100% N2 for 15 min at 15 psipressure121C. Cool to room temperature.

Preparation of Medium: To 869.0mL of sterile cooledPart A, aseptically add the remaining sterile solutions in thefollowing order: solution B, solution C, solution D, solutionE, and solution F. Mix thoroughly. Adjust pH to 7.7. Anaer-obically distribute under 90% N2 + 10% CO2 into steriletubes or flasks.

Use: For the cultivation of Acetivibrio ethanolgignens andDesulfovibrio sapovorans which reduces sulfate.

Acetobacter diazotrophicus AgarComposition per liter:Glucose........................................................................50.0gCaCO3..........................................................................30.0gAgar .............................................................................25.0gYeast extract ................................................................10.0g

pH 5.5 0.2 at 25C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughlyto evenly distribute CaCO3. Bring pH to 5.5. Gently heatand bring to boiling. Distribute into tubes or flasks. Auto-clave for 15 min at 15 psi pressure121C. Cool rapidly to5055C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of Acetobacterdiazotrophicus, a nitrogen fixing endophyte.

10 Acetobacterium dehalogenans Medium

Acetobacterium dehalogenans Medium (DSMZ Medium 787)

Composition per liter:NaHCO3 ...................................................................... 10.0gYeast extract .................................................................. 2.0gNH4Cl............................................................................ 1.0gK2HPO4....................................................................... 0.45gKH2PO4....................................................................... 0.33gMgSO47H2O ................................................................ 0.1gResazurin ................................................................... 1.0mgNaHCO3 solution .....................................................30.0mLNa2CO3 solution.......................................................20.0mLTrace elements solution ...........................................20.0mLVitamin solution.......................................................20.0mLNa-syringate solution ...............................................10.0mLCysteine solution......................................................10.0mL

pH 7.4 0.2 at 25CNaHCO3 Solution:Composition per 100.0mL:NaHCO3 ...................................................................... 10.0gPreparation of NaHCO3 Solution: Add NaHCO3 todistilled/deionized water and bring volume to 100.0mL.Mix thoroughly. Sparge with 80% N2 + 20% CO2. Filtersterilize.Cysteine Solution:Composition per 10.0mL:L-CysteineHClH2O ..................................................... 0.3gPreparation of Cysteine Solution: Add L-cys-teineHClH2O to distilled/deionized water and bring vol-ume to 10.0mL. Mix thoroughly. Sparge with 100% N2.Autoclave for 15 min at 15 psi pressure121C. Cool to25C. Na-syringate Solution:Composition per 20.0mL:Na-syringate.................................................................. 1.2g

Preparation of Na-syringate Solution: Add Na-sy-ringate to distilled/deionized water and bring volume to20.0mL. Mix thoroughly. Sparge with 100% N2. Filter ster-ilize.Trace Elements Solution:Composition per liter:MgSO47H2O ................................................................ 3.0gNitrilotriacetic acid ....................................................... 1.5gNaCl .............................................................................. 1.0gMnSO42H2O ................................................................ 0.5gCoSO47H2O ............................................................... 0.18gZnSO47H2O ............................................................... 0.18gCaCl22H2O................................................................... 0.1gFeSO47H2O.................................................................. 0.1gNiCl26H2O ............................................................... 0.025gKAl(SO4)212H2O....................................................... 0.02gH3BO3 ......................................................................... 0.01gNa2MoO44H2O .......................................................... 0.01gCuSO45H2O ............................................................... 0.01gNa2SeO35H2O........................................................... 0.3mgPreparation of Trace Elements Solution: Add ni-trilotriacetic acid to 500.0mL of distilled/deionized water.Dissolve by adjusting pH to 6.5 with KOH. Add remainingcomponents. Add distilled/deionized water to 1.0L. Mixthoroughly. Vitamin Solution:Composition per liter:Pyridoxine-HCl ........................................................ 10.0mgThiamine-HCl2H2O.................................................. 5.0mg

Riboflavin...................................................................5.0mgNicotinic acid .............................................................5.0mgD-Ca-pantothenate ......................................................5.0mgp-Aminobenzoic acid .................................................5.0mgLipoic acid..................................................................5.0mgBiotin ..........................................................................2.0mgFolic acid ....................................................................2.0mgVitamin B12.................................................................0.1mg

Preparation of Vitamin Solution: Add components todistilled/deionized water and bring volume to 1.0L. Mixthoroughly. Sparge with 80% N2 + 20% CO2. Filter steril-ize.

Preparation of Medium: Prepare and dispense mediumunder 80% N2 + 20% CO2 gas atmosphere. Add compo-nents, except NaHCO3 solution, Na2CO3 solution, vitaminsolution, Na-syringate solution, and cysteine solution, todistilled/deionized water and bring volume to 900.0mL.Mix thoroughly. Gently heat and bring to boiling. Boil for 5min. Cool while sparging with 80% N2 + 20% CO2. Distrib-ute 9.0mL aliquots into serum bottles. Autoclave under80% N2 + 20% CO2 for 15 min at 15 psi pressure121C.Aseptically and anaerobically add approximately 0.20mLsterile Na2CO3 solution to each 9.0mL of medium so thatpH is adjusted to 7.4. For every 9.0mL of medium inject1.0mL NaHCO3 solution, 0.15mL Na-syringate solution,0.2mL vitamin solution, and 0.17mL cysteine solution.

Use: For the cultivation of Acetobacterium dehalogenans,which dehalogenates methyl chloride.

Acetobacterium tundrae Medium (DSMZ Medium 900)

Composition per liter:Yeast extract ..................................................................1.0gMgCl26H2O ................................................................0.52gKCl ..............................................................................0.33gNH4Cl ..........................................................................0.33gKH2PO4 .......................................................................0.33gCaCl22H2O ...................................................................0.3gResazurin ....................................................................0.5mgFructose solution ..................................................... 30.0mLNaHCO3 solution..................................................... 20.0mLL-Cysteine solution.................................................. 10.0mLNa2S9H2O solution ................................................ 10.0mLTrace mineral solution SL-10.................................... 1.0mLSeven vitamin solution .............................................. 1.0mL

pH 7.0 0.2 at 25CL-Cysteine Solution:Composition per 10.0mL:L-CysteineHClH2O......................................................0.3g

Preparation of L-Cysteine Solution: Add L-cys-teineHClH2O to distilled/deionized water and bring vol-ume to 10.0mL. Mix thoroughly. Sparge with 100% N2.Autoclave for 15 min at 15 psi pressure121C.

NaHCO3 Solution:Composition per 20.0mL:NaHCO3 ........................................................................5.0g

Preparation of NaHCO3 Solution: Add NaHCO3 todistilled/deionized water and bring volume to 20.0mL. Mixthoroughly. Sparge with 80% N2 + 20% CO2. Filter steril-ize.

Na2S9H2O Solution:Composition per 10mL:Na2S9H2O ....................................................................0.3g

Acetogen Medium 11

Preparation of Na2S9H2O Solution: Add Na2S9H2Oto distilled/deionized water and bring volume to 10.0mL.Mix thoroughly. Autoclave under 100% N2 for 15 min at 15psi pressure121C. Cool to room temperature.

Fructose Solution:Composition per 30.0mL:Glucose ......................................................................... 5.0g

Preparation of Fructose Solution: Add glucose todistilled/deionized water and bring volume to 30.0mL. Mixthoroughly. Sparge with 100% N2. Filter sterilize.

Seven Vitamin Solution:Composition per liter:Pyridoxine hydrochloride ...................................... 300.0mgThiamine-HCl2H2O.............................................. 200.0mgNicotinic acid ......................................................... 200.0mgVitamin B12 ............................................................ 100.0mgCalcium pantothenate ............................................ 100.0mgp-Aminobenzoic acid ............................................... 80.0mgD(+)-Biotin ............................................................... 20.0mg

Preparation of Seven Vitamin Solution: Add com-ponents to distilled/deionized water and bring volume to1.0L. Sparge with 100% N2. Mix thoroughly. Filter sterilize.

Trace Elements Solution SL-10:Composition per liter:FeCl24H2O ................................................................... 1.5gCoCl26H2O ...........................................................190.0mgMnCl24H2O...........................................................100.0mgZnCl2........................................................................ 70.0mgNa2MoO42H2O ....................................................... 36.0mgNiCl26H2O .............................................................. 24.0mgH3BO3 ........................................................................ 6.0mgCuCl22H2O ...............................................................2.0mgHCl (25% solution) ..................................................10.0mL

Preparation of Trace Elements Solution SL-10:Add FeCl24H2O to 10.0mL of HCl solution. Mix thorough-ly. Add distilled/deionized water and bring volume to 1.0L.Add remaining components. Mix thoroughly. Sparge with80% N2 + 20% CO2. Autoclave for 15 min at 15 psi pres-sure121C.

Preparation of Medium: Prepare and dispense mediumunder 80% N2 + 20% CO2 gas atmosphere. Add compo-nents, except seven vitamin solution, NaHCO3 solution,fructose solution, L-Cysteine solution, and Na2S9H2O so-lution, to distilled/deionized water and bring volume to929.0mL. Mix thoroughly. Sparge with 80% N2 + 20%CO2. Distribute into anaerobe tubes or bottles. Autoclavefor 15 min at 15 psi pressure121C. Aseptically and anaer-obically add per liter of medium, 1.0mL seven vitamin so-lution, 20.0mL NaHCO3 30.0mL fructose solution, 10.0mLL-Cysteine-HClH2O solution, and 10.0mL Na2S9H2O so-lution. Mix thoroughly. The final pH should be 7.0.

Use: For the cultivation of Acetobacterium tundrae, a psy-chrophilic, acetigenic bacterium from tundra soils.

Acetogen MediumComposition per 421.8mL:NaHCO3 ........................................................................ 2.4gNH4Cl............................................................................ 0.2gYeast extract .................................................................. 0.2gStock salts solution #1 .............................................40.0mLPotassium phosphate buffer .....................................20.0mLClarified rumen fluid ...............................................20.0mLStock salts solution #2 ...............................................4.0mLTrace minerals............................................................4.0mL

Vitamin solution ........................................................ 4.0mLReducing agent .......................................................... 4.0mLTungstate solution ..................................................... 0.4mLResazurin (0.1% solution) ......................................... 0.4mLPotassium Phosphate Buffer:Composition per 830.0mL:K2HPO4 .....................................................................15.68gKH2PO4 .......................................................................4.72gPreparation of Potassium Phosphate Buffer: Dis-solve K2HPO4 in 600.0mL of distilled/deionized water andKH2PO4 in 230.0mL of distilled/deionized water. Mix thetwo solutions together and use.Stock Salts Solution #1:Composition per liter:KCl ................................................................................1.6gNaCl ..............................................................................1.4gMgSO47H2O.................................................................0.2gPreparation of Stock Salts Solution #1: Add compo-nents to distilled/deionized water and bring volume to 1.0L.Mix thoroughly.Stock Salts Solution #2:Composition per liter:CaCl22H2O ...................................................................0.1gPreparation of Stock Salts Solution #2: Add compo-nents to distilled/deionized water and bring volume to 1.0L.Mix thoroughly.Trace Minerals:Composition per liter:Nitrilotriacetic acid........................................................1.5gMgSO47H2O.................................................................3.0gMnSO4H2O...................................................................0.5gNaCl ..............................................................................1.0gNiCl26H2O ...................................................................0.1gFeSO47H2O ..................................................................0.1gCoCl26H2O...................................................................0.1gCaCl2 .............................................................................0.1gZnSO47H2O..................................................................0.1gNa2SeO35H2O ............................................................0.01gCuSO45H2O................................................................0.01gAlK(SO4)212H2O .......................................................0.01gH3BO3..........................................................................0.01gNa2MoO42H2O...........................................................0.01gPreparation of Trace Minerals: Add nitrilotriaceticacid to 500.0mL of distilled/deionized water. Dissolve byadjusting pH to 6.5 with KOH. Bring volume to 1.0L withdistilled/deionized water. Add remaining components. Mixthoroughly. Vitamin Solution:Composition per liter:PyridoxineHCl.........................................................10.0mgAscorbic acid..............................................................5.0mgCalcium pantothenate .................................................5.0mgCholine chloride .........................................................5.0mgLipoic acid..................................................................5.0mgi-Inositol .....................................................................5.0mgNiacinamide ...............................................................5.0mgNicotinic acid .............................................................5.0mgp-Aminobenzoic acid .................................................5.0mgPyridoxalHCl.............................................................5.0mgRiboflavin...................................................................5.0mgThiamineHCl .............................................................5.0mgBiotin ..........................................................................2.0mgFolic acid ....................................................................2.0mgVitamin B12.................................................................0.1mg

12 Acetohalobium Medium

Preparation of Vitamin Solution: Add components todistilled/deionized water and bring volume to 1.0L. Mixthoroughly. Store frozen.

Tungstate Solution:Composition per liter:Na2WO42H2O......................................................... 99.0mg

Preparation of Tungstate Solution: Add componentsto distilled/deionized water and bring volume to 1.0L. Mixthoroughly.

Reducing Agent:Composition per 110.0mL:L-CysteineHClH2O ..................................................... 2.5gNa2S9H2O.................................................................... 2.5g

Preparation of Reducing Agent: Add 110.0mL of dis-tilled/deionized water to a 250.0mL round-bottomed flask.Boil under N2 gas for 1 min. Cool to room temperature. AddL-cysteineHCl and dissolve. Adjust to pH 9 with 5NNaOH. Add washed Na2S9H2O and dissolve. Distribute inamounts needed into tubes or flasks. Autoclave for 10 minat 15 psi pressure121C.

Preparation of Medium: Add components, exceptNaHCO3 and reducing agent, to distilled/deionized waterand bring volume to 417.8mL. Mix thoroughly. Gently heatand bring to boiling under 80% N2 + 20% CO2. Cool to 4550C. Add NaHCO3 and reducing agent. Distribute intotubes or flasks under 80% N2 + 20% CO2. Autoclave for 15min at 15 psi pressure121C. After inoculation, exchangeheadspace with 80% H2 + 20% CO2.

Use: For the cultivation and maintenance of acetogenicanaerobes such as some Clostridium species.

Acetohalobium MediumComposition per 1025.0mL:NaCl .......................................................................... 150.0gMgCl26H2O.................................................................. 4.0gCaCl22H2O................................................................. 0.33gKCl.............................................................................. 0.33gKH2PO4....................................................................... 0.33gNH4Cl.......................................................................... 0.33gResazurin ................................................................... 1.0mgTrace elements solution ...........................................10.0mLVitamin solution.......................................................10.0mLTrimethylamineHCl solution ..................................10.0mLYeast extract solution .................................................5.0mLNaHCO3 solution .......................................................5.0mLNa2S9H2O solution...................................................5.0mL

pH 7.6 0.2 at 25C

Trace Elements Solution:Composition per liter:MgSO47H2O................................................................ 3.0gNitrilotriacetic acid ...................................................... 1.5 gCaCl22H2O ................................................................. .1.0gNaCl .............................................................................. 1.0gMnSO42H2O................................................................ 0.5gCoSO47H2O............................................................... 0.18gZnSO47H2O............................................................... 0.18gFeSO47H2O ................................................................. 0.1gNiCl26H2O............................................................... 0.025gKAI(SO4)212H2O ...................................................... 0.02gCuSO45H2O............................................................... 0.01gH3BO3......................................................................... 0.01gNa2MoO42H2O.......................................................... 0.01gNa2SeO35H2O........................................................... 0.3mg

Preparation of Trace Elements Solution: Add ni-trilotriacetic acid to 500.0mL of distilled/deionized water.Dissolve by adjusting pH to 6.5 with KOH. Add distilled/deionized water to 1.0L. Add remaining components. Mixthoroughly. Adjust pH to 7.0 with KOH.Vitamin Solution:Composition per liter:PyridoxineHCl.........................................................10.0mgCalcium DL-pantothenate ...........................................5.0mgLipoic acid..................................................................5.0mgNicotinic acid .............................................................5.0mgp-Aminobenzoic acid .................................................5.0mgRiboflavin...................................................................5.0mgThiamineHCl .............................................................5.0mgBiotin ..........................................................................2.0mgFolic acid ....................................................................2.0mgVitamin B12.................................................................0.1mgPreparation of Vitamin Solution: Add components todistilled/deionized water and bring volume to 1.0L. Mixthoroughly. Filter sterilize.TrimethylamineHCl Solution:Composition per 10.0mL:TrimethylamineHCl......................................................2.4g

Preparation of TrimethylamineHCl Solution: AddtrimethylamineHCl to distilled/deionized water and bringvolume to 10.0mL. Mix thoroughly. Sparge under 100% N2gas for 3 min. Autoclave for 15 min at 15 psi pressure121C. Store under N2 gas. One may use 4.5g of glycine be-taine in place of trimethylamineHCl.Yeast Extract Solution:Composition per 5.0mL:Yeast extract ................................................................0.05g

Preparation of Yeast Extract Solution: Add yeastextract to distilled/deionized water and bring volume to5.0mL. Mix thoroughly. Sparge under 100% N2 gas for 3min. Autoclave for 15 min at 15 psi pressure121C. Storeunder N2 gas.

NaHCO3 Solution:Composition per 5.0mL:NaHCO3 ........................................................................0.5gPreparation of NaHCO3 Solution: Add NaHCO3 todistilled/deionized water and bring volume to 5.0mL. Mixthoroughly. Sparge under 100% N2 gas for 3 min. Autoclavefor 15 min at 15 psi pressure121C. Store under N2 gas.Na2S9H2O Solution:Composition per 5.0mL:Na2S9H2O ....................................................................0.5g

Preparation of Na2S9H2O Solution: Add Na2S9H2Osolution to distilled/deionized water and bring volume to5.0mL. Mix thoroughly. Sparge under 100% N2 gas for 3min. Autoclave for 15 min at 15 psi pressure121C. Storeunder N2 gas.Preparation of Medium: Add components, except vita-min solution, trimethylamineHCl solution, yeast extractsolution, NaHCO3 solution, and Na2S9H2O solution, todistilled/deionized water and bring volume to 1.0L. AdjustpH to 7.27.4 with NaOH. Mix thoroughly. Gently heat andbring to boiling. Boil for a few minutes. Allow to cool toroom temperature under 100% N2. Distribute into tubes orflasks under 100% N2. Autoclave for 15 min at 15 psi pres-sure121C. Cool to room temperature. Before inoculation,aseptically and anaerobically add vitamin solution, trimeth-ylamineHCl solution, yeast extract solution, NaHCO3 solu-

Acid Tomato Broth 13

tion, and Na2S9H2O solution. If necessary, adjust pH 7.6 towith sterile anaerobic Na2CO3 solution.

Use: For the cultivation and maintenance of Acetohalobi-um arabaticum, which is capable of chemolithotrophicgrowth on H2 + CO2 or CO, of methylotrophic growth ontrimethylamine, and of organotrophic growth on betaine,lactate, etc.

Acetylglucosamine Medium(N-Acetylglucosamine Medium)

Composition per liter:N-Acetylglucosamine.................................................. 20.0gBeef extract ................................................................. 10.0gPeptone........................................................................ 10.0gYeast extract .................................................................. 5.0gK2HPO4......................................................................... 2.0gTriammonium citrate..................................................... 2.0gMgSO47H2O ................................................................ 0.2gMnSO44H2O .............................................................. 0.05gTween 80................................................................1.0mL

pH 6.2 0.4 at 25C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.Adjust pH to 6.2. Distribute into tubes or flasks. Autoclavefor 15 min at 15 psi pressure121C.

Use: For the cultivation of bacteria that can utilize N-acetylglucosamine.

Acid Glucose Salts MediumComposition per liter:Glucose ......................................................................... 5.0gMgSO47H2O ................................................................ 0.5g(NH4)2SO4................................................................... 0.15gKH2PO4......................................................................... 0.1gKCl........................................................................... 50.0mgCa(NO3)2.................................................................. 10.0mg

pH 3.0 0.2 at 25C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.Distribute into tubes or flasks. Autoclave for 15 min at 15psi pressure121C.

Use: For the cultivation of Thiobacillus organoparus.

Acid Rhodospirillaceae MediumComposition per 1050.0 mL:Ammonium acetate ....................................................... 1.5gKH2PO4......................................................................... 0.5gMgSO4.7H2O ............................................................... 0.4gNaCl .............................................................................. 0.4gNH4Cl............................................................................ 0.4gDisodium succinate..................................................... 0.25gYeast extract .................................................................. 0.2gCaCl22H2O................................................................. 0.05gFerric citrate solution .................................................5.0mLTrace elements solution SL-6 ....................................1.0mLVitamin B12 solution ..................................................0.4mLNeutralized sulfide solution .................................... variable

pH 5.7 0.2 at 25CFerric Citrate Solution:Composition per 10.0mL:Ferric citrate .............................................................10.0mg

Preparation of Ferric Citrate Solution: Add ferriccitrate to distilled/deionized water and bring volume to10.0mL. Mix thoroughly. Sparge under 100% N2 gas for 3

min. Autoclave for 15 min at 15 psi pressure121C. Storeunder N2 gas.

Trace Elements Solution SL-6:Composition per liter:MnCl24H2O ..................................................................0.5gH3BO3............................................................................0.3gCoCl26H2O...................................................................0.2gZnSO47H2O................................................................. 0.1gNa2MoO42H2O.......................................................... 0.03gNiCl26H2O..................................................................0.02gCuCl22H2O.................................................................0.01g

Preparation of Trace Elements Solution SL-6: Addcomponents to distilled/deionized water and bring volumeto 1.0L. Mix thoroughly.

Vitamin B12 Solution:Composition per 100.0mL:Vitamin B12...............................................................10.0mg

Preparation of Vitamin B12 Solution: Add VitaminB12 to distilled/deionized water and bring volume to100.