184. corres? | Microbial Growsh (@) The bob! saul, £ scolopes Figure’7.24 Squid—Wavio Symbiosis. (9) The bobal qui sa arm.vater 9) Lahtegan rat emai burledin sand curing te dayand ests at right. (ben feacing, use ts lon oman (bod, located ont vant surface) to provide amoung by projecting lant dowd, This the cuine of ne seule apaaars a¢ night the wits surfcete poten predator ooking up traugh theater sutnnduceracumultestathethle concent, geting gh prosucton nado te Benga mode forquoram seeing, smn The ight emaniscnlanitad ny ge numberof Vbno Meche cl, Jotorsip ean Important model othe mast cammon typeof anal bactera associ, celaization of eptrlal surfaces ‘A successful interaction is initined when the bacterium and plant “talk” o each oiber using chemicals they secre. The plant releases molecules calle flavonoids, which ae taken up by the hac- {eriura and bind toa bectril protein ealled NodD. This activates the ‘production of chemicals by the bacterium called Nod factors, Some [Nod factors affect the outer cells of plant root hairs. Ultimately this causes the root hairs to curl, entrapping the bacteria. Other Nod fac {ors tigger formation ofan infection be, through which the bacte- ria enter the plant. Thus by “speaking” to each other, both the plant ‘and bacterium form « relationship that is beneficial 1o both. DPI Rhizabic are symbionts oflguminots plants (section 31.3} Retrieve, Inter, Apply 1. Whatsabionm7Usttwo ways Mein abil advan tagcousfr microbes. 2 What medical challenges dobatims present? 2. Whatis quorum sensing? Descrne naw it occurs, and ety uss Issimportance microorganism. {4 How ie the communicator thatoceursbewween 2 tigdium anda isguminous plat slr tathataccurtinghetween Vivo species? How doos nator? 7.6 Laboratory Culture of Cellular Microbes Requires Media and Conditions That Mimic the Normal Habitat of a Microbe ‘Aer reading this section, you should be able to 1 Describe te importance a atutingmirobes othe study of ‘micoergansms 1 Distinguish dened yh) meeia Fam complex media ar tha Usesof guid romsoll growth media 1 Ustthe charactors of agar that makita particularly useful solyng agont 1 Compare and contast supportive (general purpose), enlehe, salectve andstfererta med, ising examples ofeach and eseribing how each s weed f= scuss te vse of encnmentcutures in olatng microbes 1 Difretiate the ston plate spread pate, and pou late methods for'soating pure cuhures | Use ne terms commonyy used by micobolglts to destribe colony morphology FFor decades studying microbes in their natural habitats has proven to be a significant hurdle for microbiologists to leap. For that ea- son, microbiology research has depended largely on the ability 10 grow and maintain microorganisms in the laboratory. Unfortu ‘lel, it isestimated tha only L to S% ofall microbes are currenly ceulturable. Recently nonculture-based alternatives have become evailable. Yel even with these techniques, am important goal is to ‘eventually isolate microbes from their normal habitat. Thus cultur- ing microbes continues tobe an important tool used by micrebiolo- gists. PPL Microbial biology relies on cules (section 29.1) Culture Media ‘Culturing microbes is possible only if suitable elture media are available. A culture medium is «solid or liquid preparation used to grow, transport, and store microorganisms, To be effective, the mealium must contain all the nutrients the microorganism re- ‘quires for growth. Specialized media are essential in the isolation ‘and identification of microorganisms, the testing of antibiotic sensitivities, water and food analysis, industrial microbiology, and other activities. Although all microorganisms need sources ‘of energy and macro- and micro-aurients, the precise composition76 Laboratory Culture of Collar Microbes Requires Meda and Condlvons That Mimic the Normal Habltat ofa Micobe 155 forea! Pe ‘of a satisfactory medium depends on the species being cultivated ‘duc to the great variety of nutritional zoquisements. Knowledge ‘of microoryanism’s normal habitat often is useful in selecting ‘an spproprite culture medium hecause its nutrient requirements ‘oflect its natural surroundings. Frequently a medium is used to select and grow specific microorganisms or to help identify a particular species. Media can also be specifically designed to fi- litate the growth of one type of microbe present in a sample from nature (p. 158). I44 Bacteria use many mechanisms to ‘bring nutrients iio the cell (section 33) Culture media can be classified based on several parame- ters: the chemical constiwents from which they are made, their ‘physical nature, and their function (table 7.4) The types of me- dia defined by these parameters are described here. Chemical and Physical Types of Culture Media ‘A medium in which all chemical components are known is a defined or synthetle medium. Ican be in aliquid form (broth) or solidified by an agent such as agar. Defined media are often used to ‘culture phoioautotrophs such as cyanobacteria and photosynthetic ‘roils, These microbes use COp as a earbon source and light as ‘an energy source. Thus they can be grown on media containing sodium carbonate of bicarbonate (sources of CO,), nitrate or ie eee ammonia as nitrogen source, sulfate, phosphate, and otber miner als (able 7.5) Many chemoorganobatertrops also can be grown in defined media. These organisms use mdced organic moleculos a carbon and energy sources. Thus @ medium with ghicose asa ‘catfon source and an ammonia sat nitrogen source will sup- prttheir growth. Not all defined media areas simple asthe exam Fes in abl 7.5 some are construc fom dezens of components. Defined mediaare used widely in research, a itis oftendesiabletw Imow exactly what he microorganism is metabolizing. Media that contain some ingredients of unknown chemical ‘composition ae complex media. They ae very useful because a Single complex medium may be able to mect all the wurticnal requirements of many different microorganisms. In addition, ‘complex media often are needed because the mtrtional require. iments of «particular microorganism are unknown, and thus a defined medium cannot be construcied. Complex media are also ued to culture fastidious microbes, microbes with complicated sutritinal or cultural requirements. ‘Most complex media contain undefined components sich as poplones, meat extrac, and yeast exist. Peptones are protein hydrolysates prepared by partial proteclytic digestion of meat, ‘casein, soya meal, gelatin, and othor protein sauces. Thay serve assources of carbon, energy, androgen. Beet extract and yeast ‘extract are aqueous extracts of lean beef and brewer's yeast, re- spectively. Beef extract contains amino acids, peptides, nucleo tides organi acid, vitamins, and minerals. Yeest extract i am ‘excelent scurce of B vitamins as well as nitrogen and carbon ‘compounds: Three commonly used complex media are nutrient broth, tryptic soy broth, and MaeConkey agar (able 7.6). Both liquid and solidified media are routinely wed in tora tories, However, soltfied media ae particularly important be- cause they can be used to isolate different microbes fram each cher o establish pure cultures. As we discuss in chapter this is a ctitical step when using Koch's postulates to demonstrate the ‘Se Dif Rate 7. sacl Gen Medan MSF nda, SS, nck! ourl of Etna Mire ER ‘Teebac mt anos, Ur 0250, M0 Neo, 249,00 SHE neal OHS.186. cxaeren7 | Microbial Growth oe ee Nutvient Broth Amount (ter) Type Soy Broth Peplone geitin 5 ‘Toptone (aancieate hydrovysate) gest of 250i) Beet ntact, 3 Peptone soybean digest Fil p68 ucose Soci chiotde Dipotasstun phosphato Final 6H173 Macconiey Ager ‘Amount (ater) MacConkey Agar Amount ft) 7 Pancreatic digest 70 of gelato 3 Pancreat clgest 15 of casein 25 Peptle digest of 1s anima tissue 5 Lactose 100 28 Sedum chloride 50 Neutral red 03 Ccystal voiet ‘00 Agar a5 | | | ae = | | | | J Fina pH 74 relationship between a microbe and a disease, Agar isthe most com: ‘monly used solidifying agent. It is « sulfated polymer composed mainly of p-galactose, 2,6-anhydro-L-galactose, and D-glucuronie cid It usually is extracted from red algae, Agar is woll suited a8 2 solidifying agent for several reasons. One is tha it melts at about ‘OFC but, once melted, des not hacen unt it reaches about 45°C, “Thus after being melted in boiling water, it cen be cooled 10 ter perature that is tolerated by hurman hands as well as microbes. Fur thermore, microbes growing on azar mem canbe incubated at a wide range of temperatures. Finally, agar isan excellent hardening agent because most microorganisms cannot degrade i Functional Types of Mecia ‘Media such as tryptic soy broth and tryptic soy agar are called ‘general purpose or supportive media because they sustain the B-movie colony (0) Blood ager Figure 7.22 Enriched Meda. (9) Blood a cause of ts chocoiate brown colo. ‘growth of many microorganisms, Blood and other nutrients may be added to supportive media to encourage the growth of fastidi- ‘ous microbes. These fortified media (eg, blood agar) ae called ‘enriched media (gure 7.22). ‘Selective media allow the growth of particular microorgan- isms, while inhibiting the growth of others (table7.7) Forinstance, ‘Gram negative bacteria will grow on media containing bile sais ot clyes such as asc fuchsin and erysal vole; however, the growth ‘of Gram-positive bacteria is inhibited. Eoxin methylene blue agar and MacConkey agar Gable 77) are widely used forthe detection of E, coli and related bacteria in water supplies and elsewhere, These media suppress the growrh of Gram-positive bacteria. Differential media are media that distinguish among differ: ‘ent groupsof microbes and even permit tentative idemification of ‘microorganisms based on theit biological characteristics. Blood (0) Chocolate ager ‘ature of actor om the human toa. a} Chaclat agar e uted to grow fastsluserganéme sen Neisseve generhaeve. The brawn olor fthe esi of heating el blondce'san sing the befere addingthem othe mes Its called ehsesate agar76 Laboratory Culture of Cellular Microbes Requires Madia and Cordions That Mimic the Normal Habitat of a Microbe 157 ae ee “Mechanism of Action Functional Type “Two dyes ecan and netfee bu, nb a roto Garp bac They so react th acide products released by certain Gam-negaive bacteria ee ae = ies roeateapes oe ae ‘use mannitol as a car “anes y products soca comers gars both a differential medium and an enriched one. It dstin- ‘uishes between hemolytic and nonhemolytic bacteria. Some he- ‘molytic bacteria (eg. many streptococci and staphylococci Isolated from throats) produce clear zones around their colonies ‘because of red blood cell destruction (figure 7.222). Blood agar is ‘an enriched growth medium in that blood (usually sheep blood) provides protein, carbohydrate, lipid, ron, onda numberof growth factors and vitamins necessary forthe cultivation of fastidious or ‘ganisms, MacConkey agar is both differential and selective, Be- eause it contains Iaetose and neutral red dye, hacteria that catabolize laciose by fermenting it release acidic waste products that make colonies appear pink to red in color. These are easly distinguished from colonies of bacteria that do not ferment lactose, Cultivation of Aerobes and Anaerobes Because aerobes eed Ovand anaerobes are killed by i radically different approaches must be used when they are cultivated. When large volumes of serobic microorganisms are cultured, sithr they must beshaken to seratetheculture mediumor rile Ar ust be pursped through the culture vessel Without aeration, the low solubility of O» in liquid would prevent them from ob- taining an adequate supply. Precisely the opposite problem arses with anaerobes: all Oy snuste exclide. This i complished in several ways. (1) Anaero- tic media containing reducing agents such a thioglycollate or cyse= ‘ne may be used, The medium is boiled ding preparation dissolve its components and dive af oxygen. The recing agents eliminate any residual dsolved O, inthe medium so that anserobes can grow beneath its suse. (2) Oxygen alo may be eliminated from an enclosed werk area, ten cll an anaerobic chamber or anaerobic workstation. Most ofthe aris removed with 2 vacuum pump fol lowes by purges with nitrogen pas (gure 7.23). gas mix contain ‘ng hydrogen is then introduced into the workstation. In the presence cof apallaiim catalyst, the hydrogen and lst remaining molecules of COsreactt form wales, creating an anoxic environment, Often COs is axed to the chamber because many anaerobes require a small amount of COs for best growth. (@) A method for culmying small ‘numbers of anaerobes isthe GasPak sytem, which alsouses hydrogen and s palladium catalyst to remove O» (igure 724). (4) A similar spprosch uses plastic hags or pouches containing caleium carbonste ana a catalyst, which produce an anoxic, extbon dioxide-rch stmo- sphere. (5) Inreasingly in clinical laboratories, the GasPak systom is being replaced by bacterial enzyme that when aie to broth e- ‘moves oxygen from the broth andthe headspace ofthe containet =~ fick or ester were Gaol tect inabior oe Yon. ‘po ute chen io Peociociene (Bing those gover. Figure 7.23 An Anserobic Workstation andincubator. Thissytem Contalns an aayoen tee wotk area andan|ncutator.Thelnterchange crmparmenton the igh ofthe workarea alows maioralste be transfered Insite without exasing he nieve axygen.Treanoacatmesphere is aint lage wha vacuum pur ar trogen pages he rmalning crygon romDved by 8 paladun clayton hydrogen. The oxygen reacts ‘wth hydrogen to form wat, whch is absorbed by desiccant8 cnarren7 | Microbial Growth Figure7.24 The GasPak Anaerobic System, Hidiogen and carbon lie am generatad bya GasPak ‘envelope. The paladin catalyst inthe chamberid catayes the formation ot uae rom hydrogen and xygen, uber gaskot seal Ccatast chamber (oreans pala patos ‘Oxigan's removed to chamber by ‘ambinng wit ykogen to fr wate. ‘Tis rotons cate yod by he thereby removing xygen rom the paladum pele. ‘ened chamber as generator rmetape 7 Water is added te chemicals eile aetna ethylene due Secomes coloress in Fh emlopo to ganorate Hy and CO,, slaw, ‘Garbo dx se promotes more apd ‘goth ofmioworaniens, Enrichment and Isolation of Pure Cultures {In natural habitats, miertorganisms often grow in complex, micro- bial communities containing many different species, This presents ‘ problem for microbiologists because a single typeof microorgan- {sm cannot be studied adequately in a mixed culture. One neods & pure or axenic eulture, a population of cells arising from a single cel to characterize an individual species. When isolating microbes {rom soil or aquatic habitats, microbiologists often begimby seting up conditions that favor growth of the desired microbes; that is, they enrich for the microbes of interes. This is followed by using bother methods to obtain an axenic culture arising fom asingle cell Enrichment Cultures ‘The enrichment culture technique is « powerful tool used to en courage the growih of microbes having particular characteristis, ‘while atthe same time inhibiting the grow of other microbes. It ‘has been used by microbiologists since it was first developed by Sergei Winogradsky and Martinus Beijerinck in the late 1800s, ‘They used enrichment cultures to isolate numerous bacteria with interesting metaholic capabilities such a nitrogen fixation andthe use of inorganic molecules (e.g, ammonia) as energy sources. To enrich for these organisms, they considered three factors: (1) 2 suitable source of the microbes, (2) nutrients that should and should not be included in the culture medium, and (3) environ- ‘mental conditions provided during the incubation perio. ‘Consider the following. Suppese you wanted to isolate an oil- degrading bacterium and study tin puse culture. A logical source ofthe bacterium (Le., an inoculum) would be soil contaminated with oi. The assumption isthat such a source would have exposed the indigenous bacteria to oil and selected for those able to use all as a sole source of energy and curbon. To select forthe oil- degrading bacteria, a medium would be used that contained inor- ‘ganic nitrogen, sulfur, and phosphorus sources but no source of carbon and energy other than the types of molecules typically ‘ound in oil. Finally, a temperature, pH, and oxygen level similar to that of the soil environment would be used during incubation. Despite its lity, the enrichment culture technique is not with ‘out problems. Iris sometimes dificult to create the proper selective conditions to successfully isolate the desired organisms. Further- sore, oven if microbet are successfully cultivated, they may nat adecuatoly represent the population as a whole at a sampling ste ‘enrichment cultures yield the organisms that row fastest, ‘and these may not he the predominant organisms in the habitat. Finally, enrichment cultures ate not pure cultures. They usually con ‘ain more than one species having similar characteristics, To obtain ‘4 pure culture, one ofthe methods described next must be used, Streak Plate “The streak plate method and similar methods for isolating pure cul- tures were developed by the German bactriologist Robert Koch. ‘He used these techniques ina set of steps that now bear his name: ‘Kach’s postulates, As we describe in chapter 1, Kack’s postulates ‘ane used o establish that a microbe isthe causative agent of par Hiculardiseise, The use of these methods ienstorred micrabial- ‘ogy, and within 20 years of their development, most pathogens responsible for major bacterial diseases had been isolated and iden lified. Koch reasoned tha if cells fom a mixture of micrabes could be spatially isolated from each ether, each cell would give rise to a ‘completely separate colony—a macroscopically visible cluster of ‘microorganisms in oF on a solid medium, Because each colony arises from a single cell each colony represents a pure culture. ‘One method for separating cells isthe streak plate. In this technigue, cells are transferred to the edge of an agar plate with ‘an inoculating loop or swab and then streaked across the surface im one of several pattems (figure 7.25). After the frst sector is. ‘seaked, the inoculating loop is sterilized and an inoculum for the second sector is obtained from the frst sector. A similar ‘process is followed for streaking the third sector, except thatthe ‘inoculum is from the second sector: Thus this is essentially di- lution process. Eventually very few cells willbe on the loop, and single cells will drop from i as it maves across the agar surface. ‘These develop into separate colonies. ‘Spread Plate and Pour Plate ‘Spread-pate and pourlate techniques are similar in that they hoth dilute » sample of cells before separating them spatially "They difer im tha the spread plate spreads the calls om the su fe ofthe agar, whereas the pour plate embeds the cells within ‘the agar. Both methods can be used to determine the number of ‘microorganisms ina sample (p. 166). For the spread plate, a small volume of a dilued mixxure ‘containing around 25 250 cells is transferred to the center of ‘agar plate and spread evenly everthe surface with sterile bent od (figure 7.26). "The dispersed cells develop ino isolated colonies.76 Laboratory Culture of Celluiar Microbes Requitas Media and Condtions That Mimic the Normal Habitat of a Micobe 159 Note: Tia method on} works it he spreading tuo) (usually an inoculating oop) is vstorias shor each ot steps 4 50990 (e) Steps na streak late ® Figure 7.28 SteaicPate Technique. typical soaking patio shown (as wea: an example of asta at ® ‘Aamall amount of he eamole € ppatod io the cater of ‘solid macium ry taming oy Figure 7.26 Sprend-Piate Technique. () the preparation of sprent pat.) Type result sf spread plate technique "The dilutions for «spread plate are sully made inthe same way 1s fora pour plate—by preparing serial dilutions (figure 7.27), ‘The pour plate is oxtensively used with becteria, archaea, and fungi. lis particularly useful when sampling a heterogenecus popiiation of microbes, some of which might produce colonies ‘that spread aver an agar surface if isolated by the streak-plate or spread-plate methods. Inthe pour-plate method, the original sam- ple is serially diluted to reduce the microbial population sufti- Gently 10 oblain separate colonies when plating (figure 7.27), ‘Then small yolumes of several diluted samples are mixed with liquid agar that has been cooled to about 4$°C, and the mixtures tre poured immediately ito sterile culture dishes. Mast microbes survive a brief exposine to the warm agar. Each cell becomes fixed in place to form an individual colony after the agar hardens. “Although the preparation of serial dilutions is the bane of ‘many microbiology students, it has many applications other than the spread plate and pour-plate methods. The nurbers of cells in ‘Solution can be dilued tothe point where no cells are present in ‘the dilution tube. If replicate dilaions are prepared and the mum- hor of bes yielding growth determined, then the number of vi thle cells in the sample can be estimated by the most probable umber metho! (MPN; se figure 29.3) Similarly, the number of viruses in-a solution or the number of antibodies in a blood sam- Me can he determined hy first diluting the sample and then test- ing for the presence of viruses or antibodies. [44 Cultivation ‘. “he glass prado steiizad by doping Itt ethanol and te “The spreaders cotled ae ten tted'o spread the sample vey ‘ver the suface ofthe medium, o ‘and enumeration of viruses (section 6.5) PPA Antibodies are proteins that bind to specific 3-D molecules (section 347) Microbial Growth on Solid Media Colony development on agar surfaces aids microbiologists in idomiifying microorganisms because individual species often form colonies of characteristic size and appearance (figure 7.28; also figure 7.26b). When a mixed population has been plated propedy, it sometimes is possible to identify the desired colony based onitsoverall appearance and use it toobiain a pureclture. ‘Kis obvious from the colonies pictured in figure 7.28 that bacteria growing on solid surfaces such as agar can form quite complex colony shapes. These patterns and colony size depend fon many factors, including nutrient diffusion and availability, bacterial chemotaxis, the presence of liquid on the surface, and Frdness of the agar. Cell-cell communication is important as160 curree | Mcrobial Growth “Tre original sare is dist sever! tines. tom! 10m 40m) toni wll Much esearch fs curently focused on understanding ths foesaton of bacterial coknied, c™ ZNO New Approaches to Culturing Microbes ‘The importance of curing even those microbes thal are the most recalcitrant to prot in the Lb tas ed micrbiologis 0 devise new echniqs [As we have noted traditional methods for clu ing microbes have depended cn belng able fo Ce ee ee) construct artificial habitats in a flask or peti Ss grFaliey ould) geTuiten) cP dish that mimic the microbes’ natural environ mH eat oe me ‘ments However, itis often difinl to know all, the environmental conditions require for Some of te cuter ten he ‘om hom ‘est a) ae mixed wit ‘warm agar and pour ‘nde the patos, growth, One way 10 solve this problem is t0 ng part of the natural environment into the Jab. For instance, an aquarium containing seawa- ter, beach sand, marine flora, and macroscopic ‘organisms collected from a natural habitat ean be used 0 culture marine microbes. This works very ‘well for maintaining # mixed population in the lab, but it tll leaves the probiem of isolating particular ‘microbes from the mixture. One solution i to create an enclosure within the man-mde “natura habitat that al- lows free diffusion of nutrients and other factors from the sur- @ ‘sola o's grow rt colonies on tho surface (appear round) ssid wituathe maclum [aapearles-shaped. The sole colonies ‘Gav be counted or ud to extaboh puro culture, roundings but keeps the microbes in the enclosure. For our aquarium example, a very dilute suspension ofthe seawater theo- resically conttining a single cell would be used to inoculate the ‘enclosure. Thus an axenic cult: would form in the enclosure Another approach is useful for microbes that require the pres- lence ofa different species in order to survive. In this cae the mi= crobe of interest is co-cultured with the required organism, Sometimes the required organism is an animal, animal Ussue, a plat, or plant iste, The microbe must be maintained growing in ‘ron that organism, Another example of co-culturing occurs in 8 petri dish on which a difute suspension of the desired microbe has ‘heen spread and then the helper microbe spotted onto the m fin ey aan moe coe a oo thon OD <> ale Be Ee Unhista——Lobate « Flare Figure7.27 PourPiete Technique, atone locaton, The factors produced by the helper microbe diffuse info the medium and support the growth of the microbe of interest. ‘This method is particularly useful for long-term maintenance of ‘eltees once the microbe has heen ised in axenic culture “Microbiologists are explering even more methods for culturing the unculmrable. Inthe funue itis expected that with these new techniques microbiologists will gain a heuer understanding of the diversity of microbes and their roles in many envitonments. PPL aicrobial biology relies on cultares (section 29.1) 0 Re = a Untomee » Figure’7.2B BacteialColony Morphology. (2)Varatins in baciealclary morphalogy seen wiht raed eye The general fom ofthe exionyanatne ‘shape othe aege or margin can ue deterin by eekng down at et ofthe clory, The nate of olny elevations apparent when vowed fam the size ase pate Is neld at eye evel. ()Parion ofa Pet ash showing some commonly observe colony morons