The Evo­lu­tion of Bac­te­r­ial Growth Me­dia

by Ananya Sen

In the fall of 1818, the Ital­ian chemist and phar­ma­cist Bar­to­lo­meo Bizio stud­ied the com­mon oc­cur­rence of po­len­ta (corn meal mush) some­times tak­ing on a "bril­liant red color." The red color was be­lieved by the pop­u­lace to be blood, and thus a mirac­u­lous por­tent. For many decades, farm­ers had lived in fear of this "bloody po­lenta" and would be­seech their priests to ex­or­cise the malef­i­cent be­ings from their homes. Bizio, through a se­ries of de­light­ful ex­per­i­ments, proved that the red­den­ing was due to nat­ural causes, when the air was damp and warm. Thus, the ap­pear­ance of Ser­ra­tia marcescens on po­lenta is an early record­ing of bac­te­ria grow­ing on solid sur­faces.

Fig­ure 1. "Bloody bread": Ser­ra­tia marcescens grow­ing on bread. CC BY-SA 3.0 Dbn

How­ever, cul­tur­ing bac­te­ria dates back to 1776 when Laz­zaro Spal­lan­zani, in his at­tempt to dis­prove the "spon­ta­neous gen­er­a­tion" of mi­crobes, grew them in in­fu­sions of kid­ney beans, bar­ley, maize, etc. The dis­ad­van­tage of liq­uid cul­tures even­tu­ally be­came ap­par­ent ‒ they could not be used to make pure cul­tures of bac­te­ria. Bac­te­ria grew to­gether, which made it im­pos­si­ble to dis­tin­guish be­tween dif­fer­ent kinds. Fifty years later Robert Koch laid the foun­da­tion for cul­tur­ing bac­te­ria on solid me­dia. Koch at­tempted to iso­late pathogens in pure cul­ture by grow­ing them on potato slices. This tech­nique turned out to be un­suit­able, as the slices would fre­quently be­come con­t­a­m­i­nated by fungi. Fur­ther­more, potato slices had rel­a­tively few avail­able nu­tri­ents and most bac­te­ria did not grow well on them. As an im­prove­ment, Koch used gelatin as a so­lid­i­fy­ing agent. Al­though gelatin was ef­fec­tive, a ma­jor draw­back was that it did not re­main solid at 37°C (the op­timum growth tem­per­a­ture for most hu­man pathogens) and would frus­trate fur­ther stud­ies of pure cul­tures. A bet­ter al­ter­na­tive was re­quired.

Agar to the Res­cue!

In 1881, Walther Hesse joined Koch's lab to study air qual­ity. He trapped air­borne mi­crobes (us­ing fil­ters) and at­tempted to study them on potato slices or on gelatin-con­tain­ing me­dia. He faced the same tech­ni­cal chal­lenges that plagued his col­leagues. When Walther voiced his con­cerns to his wife and lab tech­ni­cian, New York City-born Fanny Hesse, she sug­gested the use of agar-agar. This was a com­mon gelling agent in warmer coun­tries, and a neigh­bor in her for­mer home­town, who had im­mi­grated from Java, had sug­gested it as a trick for so­lid­i­fy­ing jel­lies and thick­en­ing soups. This house­hold hint trans­formed the tech­nique of ob­tain­ing pure cul­tures. Agar is the per­fect gel­ling agent ‒ it is solid at 37°C, not de­graded by the great ma­jor­ity of bac­te­ria, and can be eas­ily poured into ster­ile con­tain­ers.

Fig­ure 2. A Bell jar (glass). Source. B Petri dishes. Source

The next ques­tion that arose was ‒ what ster­ile con­tain­ers to use? The state of the art sys­tem at the time was Koch's flat plate tech­nique. This en­tailed pour­ing the agar based me­dia into plates, stack­ing them, and cov­er­ing them with a bell jar. When­ever the plates needed to be stud­ied, the bell jar would be lifted, which would lead to fre­quent con­t­a­m­i­na­tion. The fi­nal tweak in this sys­tem was made in 1887 by Richard Petri (also Koch's as­sis­tant). He de­signed the Petri dish which, as every­one knows, con­sists of two flat cir­cu­lar dishes where the up­per dish serv­ing as an over­hang­ing lid. This sim­ple mod­i­fi­ca­tion has sev­eral ad­van­tages: the larger dish serves as a bar­ri­er against con­t­a­m­i­na­tion, colonies on the sur­face of the plate are fully ex­posed to air and can be eas­ily ex­am­ined. Plus, the plates can be stacked and ster­il­ized sep­a­rately from the medium. With the help of the agar plates, mi­cro­bi­ol­o­gists of the day (many of whom were Koch's stu­dents) went on to iso­late the pathogens re­spon­si­ble for tu­ber­cu­lo­sis, diph­the­ria, ty­phoid, many pneu­mo­nias, menin­gi­tis, tetanus, and bubonic plague.

Se­lec­tive Growth ‒ Sur­vival of the Fittest

The next ad­vance­ment was the de­vel­op­ment of se­lec­tive me­dia. Se­lec­tive me­dia fa­vor the growth of cer­tain bac­te­ria based on spe­cific meta­bolic char­ac­ter­is­tics, thereby al­low­ing them to out­grow other bac­te­ria. This tech­nique is use­ful when bac­te­ria must be iso­lated from nat­ural en­vi­ron­ments where their num­bers are too small to be stud­ied di­rectly. The de­vel­op­ment of se­lec­tive me­dia was pi­o­neered by Sergei Wino­grad­sky in 1888. He was study­ing sul­fate re­duc­tion by Beg­gia­toa and was able to grow these or­gan­isms in pure cul­ture from en­vi­ron­men­tal sam­ples. Al­most si­mul­ta­ne­ous­ly, Mar­t­i­nus Bei­jer­inck drew from Winogradsky's ex­per­i­ments and iso­lated ni­tro­gen-fix­ing bac­te­ria by grow­ing them in a "poor nu­tri­ent medium at room tem­per­a­ture." Here, the only or­gan­isms that grew were those that could use at­mos­pheric ni­tro­gen. Bei­jer­inck also used en­rich­ment me­dia to iso­late a plethora of mi­croor­gan­isms with widely dif­fer­ent meta­bolic prop­er­ties ‒ sul­fate-re­duc­ing bac­te­ria, den­i­tri­fy­ing bac­te­ria, lac­tic acid, and acetic acid bac­te­ria.

Dif­fer­en­ti­ate and Con­quer

Fig­ure 3. Strep­to­coc­cus agalac­tiae on Granada agar, anaer­o­bic in­cu­ba­tion. CC BY-SA 4.0 43trevenque

Iso­lat­ing bac­te­ria and grow­ing pure cul­tures from en­vi­ron­men­tal sam­ples was ex­tremely help­ful in cha­rac­te­riz­ing bac­te­ria. How­ever, closely re­lated groups of bac­te­ria could not be eas­ily dis­tin­guished just us­ing se­lec­tive me­dia. To this end, Al­fred Mac­Conkey de­vel­oped Mac­Conkey medium in 1905. Among other in­gre­di­ents, this medium con­tained bile salts (that in­hibit cer­tain groups of bac­te­ria) and a pH-sen­si­tive dye, neu­tral red. The ra­tio­nale was that if the sug­ars in the medium were bro­ken down by the bac­te­ria, the acid re­leased would turn the dye red. If the bac­te­ria were in­ca­pable of this re­ac­tion, the colonies would re­main col­or­less. Other ex­am­ples of dif­fer­en­tial me­dia are EMB agar (Holt-Har­ris & Teague, 1916) that dif­ferentiates be­tween lac­tose fer­menters and lac­tose non-fer­menters, man­ni­tol salt agar (Chap­man, 1945) that dif­fer­en­ti­ates man­ni­tol fer­ment­ing and man­ni­tol non-fer­ment­ing species of Staphy­lo­coc­cus, and Granada medium (De La Rosa et al., 1983) that dif­fer­en­ti­ates be­tween he­molytic and non-he­molytic Group B Strep­to­coc­cus strains.

Note on LB

LB is a widely used growth medium for many di­verse stud­ies with E. coli. Since many labs use it, I thought it would be in­ter­est­ing (while run­ning the risk of ap­pear­ing to be an E.coli snob) to learn a lit­tle more about this medium's ori­gins. It was de­vel­oped in 1951 by Giuseppe Bertani who was study­ing lysogeny (hence Lysogeny Broth, or LB). The com­po­nents of this medium are yeast ex­tract, sodium chlo­ride and a pep­tone. Pep­tone is the generic term for prod­ucts made by en­zy­ma­tic cleav­age of pro­tein. In the case of LB, the pep­tone that is used in called tryp­tone, the re­sult of di­gest­ing the milk pro­tein ca­sein with the pro­tease trypsin. Al­though it is un­clear when the in­cor­po­ra­tion of yeast ex­tract in me­dia came to be, the ad­di­tion of pep­tones to growth medium can be traced back to Friedrich Lo­ef­fler in 1887. Pep­tones are a rich source of amino acids and other ni­tro­genous com­pounds that helped the growth of Corynebac­terium diph­the­riae. In the same pa­per, he also dis­cussed adding salt for os­motic bal­ance. LB is now used as a stan­dard medium in ma­ny dif­fer­ent labs, from mi­cro­bi­ol­ogy to bio­chem­istry to cell bi­ol­ogy (all thanks to LB's pre­do­mi­nant con­sumer and won­der bug, E. coli ).

Fig­ure 4. LB medium: A liq­uid, B solid (agar) in Petri dish. GFDL Ma­sur

A word of cau­tion: LB does have sev­eral draw­backs ‒ nei­ther tryp­tone nor yeast ex­tract have de­fined com­po­si­tions, caus­ing batch-to-batch vari­abil­ity. Thus, the re­sults in LB are of­ten hard to re­produce ac­cu­rately. In ad­di­tion, it has only a low amount of fer­mentable car­bo­hy­drates (un­less ad­ded), and is poor in mag­ne­sium. The mi­nus si­d­es of LB have been neatly sum­ma­rized by Hi­roshi Ni­ka­i­do. To counter the things that are wrong with LB, Neid­hardt, Bloch, and Smith de­vel­oped in 1974 a ba­sic mi­ni­mal medium that can be en­riched at will by the ad­di­tion of other nu­tri­ents. This medium has de­fined com­po­nents , thus mak­ings its chem­i­cal com­po­si­tion re­pro­ducible with­out com­pro­mis­ing the abil­ity to sup­ple­ment ex­tra de­sired nutrients.Thus, it can be used to grow bac­te­ria at a va­ri­ety of rates.

This is not the end of the story on mi­cro­bial growth me­dia. De­vel­op­ment of new growth me­dia to meet spe­cific needs is a very im­por­tant on­go­ing ef­fort in the mi­cro­bial sci­ences. Wit­ness, for ex­am­ple, the re­cent pub­li­ca­tion of a nearly uni­ver­sal me­dium that per­mits the growth of anaer­o­bic bac­te­ria in nor­mal at­mos­phere con­di­tions.

Mi­cro­bial art

Hav­ing all these dif­fer­ent me­dia is well and good for ad­vanc­ing the march of sci­ence. But what about man's in­nate need to see art in every­thing? Marc Cha­gall once said "Great art picks up where na­ture ends." Painters of the mi­cro­bi­ol­ogy sort use bac­te­ria to make fine art. The can­vases are a med­ley of agar plates onto which mi­crobes have been 'painted.' From Van Gogh's Starry Night to a hum­ble mug of beer, the range of mi­cro­bial art can be found here. En­joy the art and re­mem­ber the cen­turies of work that led to it.

 

Ananya is a grad­u­ate stu­dent in the De­part­ment of Mi­cro­bi­ol­ogy at the Uni­ver­sity of Illi­nois at Ur­bana-Cham­paign. She works in the lab of James A. Im­lay. Ananya has re­cently started a blog of her own called " The His­tory of Sci­ence."