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Web Review of Todar's Online Text book of Bact eriology."The Good , t he Bad , and t he Deadly". (SCI ENCE Magazine- June 4 , 2004 - Vol 304 : p. 1421 ).

Tag w ords : bact erial grow t h , grow t h curve, lag phase, exponent ial grow t h , generat ion t im e, viable cell count, cont inuous cult ure. environm ent . I n m ost bact eria, grow t h involves increase in cell m ass and num ber of ribosom es , duplicat ion of t he bact erial chrom osom e, synt hesis of new cell wall and plasm a m em brane , part it ioning of t he t wo chrom osom es, sept um form at ion , and cell division . This asexual process of reproduct ion is calledbin a r y fission.

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1 . Directph ysica l m e a su r e m e n tof dry weight , wet weight , or volum e of cells aft er cent rifugat ion .

2 . Directch e m ica l m e a su r e m e n tof som e chem ical com ponent of t he cells such as t ot al N, t ot al prot ein , or t ot al DNA cont ent .

3 . I ndirectm e a su r e m e n t of ch e m ica l a ct ivit ysuch as rat e of O2product ion or consum pt ion , CO₂product ion or consum pt ion , et c.

4 .Tu r bidit y m e a su r e m e n t sem ploy a variet y of inst rum ent s t o det erm ine t he am ount of light scat t ered by a suspension of cells. Part iculat e obj ect s such as bact eria scat t er light in proport ion t o t heir num bers . The t urbidit y oropt ica l de n sit yof a suspension of cells is direct ly relat ed t o cell m ass or cell num ber, aft er const ruct ion and calibrat ion of a st andard curve . The m et hod is sim ple and nondest ruct ive , but t he sensit ivit y is lim it ed t o about 107cells per m l for m ost bact eria.

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Feat ured Microbe

Web Review of Todar' s Online Text book of Bact eriology."The Good, t he Bad, and t he Deadly". (SCI ENCE Magazine - June 4 , 2004 - Vol 304 : p. 1421 ).

Tag w ords: bact erial grow t h , grow t h curve, lag phase , exponent ial grow t h , generat ion t im e, viable cell count, cont inuous cult ure.

Measuring t echniques involve direct count s, visually or inst rum ent ally , and indirect viable cell count s.

1 .D ir e ct m icr oscopic cou n t sare possible using special slides known as count ing cham bers. Dead cells cannot be dist inguished from living ones. Only dense suspensions can be count ed ( > 107cells per m l) , but sam ples can be concent rat ed by cent rifugat ion or filt rat ion t o increase sensit ivit y.

A variat ion of t he direct m icroscopic count has been used t o observe and m easure grow t h of bact eria in nat ural environm ent s. I n order t o det ect and prove t hat t herm ophilic bact eria were grow ing in boiling hot springs , T . D . Brock im m ersed m icroscope slides in t he springs and wit hdrew t hem periodically for m icroscopic observat ion. The bact eria in t he boiling wat er at t ached t o t he glass slides nat urally and grew as m icrocolonies on t he surface .

2 .Ele ct r on ic cou n t in g ch a m be r scount num bers and m easure size dist ribut ion of cells. For cells t he size of bact eria t he suspending m edium m ust be very clean. Such elect ronic devices are m ore oft en used t o count eucaryot ic cells such as blood cells.

3 .I n dir e ct via ble ce ll cou n t s, also calledpla t e cou n t s, involve plat ing out ( spreading) a sam ple of a cult ure on a nut rient agar surface . The sam ple or cell suspension can be dilut ed in a nont oxic diluent ( e. g . wat er or saline) before plat ing. I f plat ed on a suit able m edium , each viable unit grow s and form s a colony . Each colony t hat can be count ed is called acolon y for m in g u n it ( cfu )and t he num ber of cfu' s is relat ed t o t he viable num ber of bact eria in t he sam ple .

Advant ages of t he t echnique are it s sensit ivit y ( t heoret ically , a single cell can be det ect ed ) , and it allows for inspect ion and posit ive ident ificat ion of t he organism count ed. Disadvant ages are ( 1 ) only living cells develop colonies t hat are count ed; ( 2 ) clum ps or chains of cells develop int o a single colony ; ( 3 ) colonies develop only from t hose organism s for which t he cult ural condit ions are suit able for grow t h. The lat t er m akes t he t echnique virt ually useless t o charact erize or count t het ot a l n u m be r of ba ct e r iain com plex m icrobial ecosyst em s such as soil or t he anim al rum en or gast roint est inal t ract . Genet ic probes can be used t o dem onst rat e t he diversit y and relat ive abundance of procaryot es in such an environm ent , but m any species ident ified by genet ic t echniques have so far proven uncult urable.

Ta ble 1 . Som e M e t h ods u se d t o m e a su r e ba ct e r ia l gr ow t h

Met hod Applicat ion Com m ent s

Direct m icroscopic count Enum erat ion of bact eria in_{m ilk or cellular vaccines} Cannot dist inguish living from_{nonliving cells}

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I n t he laborat ory, under favorable condit ions, a grow ing bact erial populat ion doubles at regular int ervals. Growt h is by geom et ric progression: 1 , 2 , 4 , 8 , et c. or 20, 21, 22, 23_{. . . 2}n_{( where n = t he num ber of generat ions) . This is called}_{e x pon e n t ia l gr ow t h}_. I n realit y, exponent ial growt h is only part of t he bact erial life cycle, and not

represent at ive of t he norm al pat t ern of grow t h of bact eria in Nat ure.

When a fresh m edium is inoculat ed wit h a given num ber of cells, and t he populat ion growt h is m onit ored over a period of t im e, plot t ing t he dat a w ill yield at ypica l ba ct e r ia l

Four charact erist ic phases of t he grow t h cycle are recognized .

1 .La g Ph a se. I m m ediat ely aft er inoculat ion of t he cells int o fresh m edium , t he populat ion rem ains t em porarily unchanged. Alt hough t here is no apparent cell division occurring , t he cells m ay be grow ing in volum e or m ass, synt hesizing enzym es, prot eins, RNA , et c. , and increasing in m et abolic act ivit y.

The lengt h of t he lag phase is apparent ly dependent on a wide variet y of fact ors including t he size of t he inoculum; t im e necessary t o recover from physical dam age or shock in t he t ransfer ; t im e required for synt hesis of essent ial coenzym es or division fact ors ; and t im e required for synt hesis of new ( inducible) enzym es t hat are necessary t o m et abolize t he subst rat es present in t he m edium .

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during t he st at ionary phase t hat spore- form ing bact eria have t o induce or unm ask t he act ivit y of dozens of genes t hat m ay be involved in sporulat ion process.

4 .D e a t h Ph a se. I f incubat ion cont inues aft er t he populat ion reaches st at ionary phase, a deat h phase follow s, in which t he viable cell populat ion declines. ( Not e, if count ing by t urbidim et ric m easurem ent s or m icroscopic count s, t he deat h phase cannot be observed . ) . During t he deat h phase, t he num ber of viable cells decreases geom et rically ( exponent ially ) , essent ially t he reverse of grow t h during t he log phase.

Gr ow t h Ra t e a n d Ge n e r a t ion Tim e

As m ent ioned above, bact erial grow t h rat es during t he phase of exponent ial grow t h, under st andard nut rit ional condit ions ( cult ure m edium , t em perat ure , pH, et c. ) , define t he bact erium 's generat ion t im e. Generat ion t im es for bact eria vary from about 12 m inut es t o 24 hours or m ore . The generat ion t im e forE. coliin t he laborat ory is 15 - 20 m inut es, but in t he int est inal t ract , t he coliform ' s generat ion t im e is est im at ed t o be 12- 24 hours. For m ost known bact eria t hat can be cult ured , generat ion t im es range from about 15 m inut es t o 1 hour . Sym biont s such asRhizobiumt end t o have longer generat ion t im es . Many lit hot rophs, such as t he nit rifying bact eria, also have long generat ion t im es. Som e bact eria t hat are pat hogens , such asMycobact erium t uberculosisandTreponem a pallidum, have especially long generat ion t im es, and t his is t hought t o be an advant age in t heir virulence. Generat ion t im es for a few bact eria are are shown in Table 2. Ta ble 2 . Ge n e r a t ion t im e s for som e com m on ba ct e r ia u n de r opt im a l con dit ion s of gr ow t h.

Ba ct e r iu m M e diu m Ge n e r a t ion Tim e ( m in u t e s)

Escherichia coli Glucose - salt s 17

Bacillus m egat erium Sucrose- salt s 25

St rept ococcus lact is Milk 26

St rept ococcus lact is Lact ose brot h 48

St aphylococcus aureus Heart infusion brot h 27- 30

Lact obacillus acidophilus Milk 66- 87

Rhizobium j aponicum Mannit ol- salt s - yeast ext ract 344- 461 Mycobact erium t uberculosis Synt het ic 792- 932

Treponem a pallidum Rabbit t est es 1980

Ca lcu la t ion of Ge n e r a t ion Tim e

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Ex a m ple: W h a t is t h e ge n e r a t ion t im e of a ba ct e r ia l popu la t ion t h a t in cr e a se s fr om 1 0 , 0 0 0 ce lls t o 1 0 , 0 0 0 , 0 0 0 ce lls in fou r h ou r s of gr ow t h?

G = t _____ 3 . 3 log b / B G = 240 m inut es 3 . 3 log 107/ 104 G = 240 m inut es 3 . 3 x 3 G = 24 m inut es

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The cult ures so far discussed for grow t h of bact erial populat ions are calledba t ch cu lt u r e s. Since t he nut rient s are not renew ed, exponent ial grow t h is lim it ed t o a few generat ions. Bact erial cult ures can be m aint ained in a st at e of exponent ial grow t h over long periods of t im e using a syst em ofcon t in u ou s cu lt u r e( Figure 4 ) , designed t o relieve t he condit ions t hat st op exponent ial grow t h in bat ch cult ures. Cont inuous cult ure, in a device called ach e m ost a t, can be used t o m aint ain a bact erial populat ion at a const ant densit y , a sit uat ion t hat is, in m any ways, m ore sim ilar t o bact erial grow t h in nat ural environm ent s.

I n a chem ost at , t he grow t h cham ber is connect ed t o a reservoir of st erile m edium . Once growt h is init iat ed, fresh m edium is cont inuously supplied from t he reservoir . The volum e of fluid in t he grow t h cham ber is m aint ained at a const ant level by som e sort of overflow drain. Fresh m edium is allowed t o ent er int o t he grow t h cham ber at a rat e t hat lim it s t he growt h of t he bact eria. The bact eria grow ( cells are form ed ) at t he sam e rat e t hat bact erial cells ( and spent m edium ) are rem oved by t he overflow. The rat e of addit ion of t he fresh m edium det erm ines t he rat e of grow t h because t he fresh m edium always cont ains a lim it ing am ount of an essent ial nut rient . Thus, t he chem ost at relieves t he insufficiency of nut rient s, t he accum ulat ion of t oxic subst ances, and t he accum ulat ion of excess cells in t he cult ure, which are t he param et ers t hat init iat e t he st at ionary phase of t he growt h cycle. The bact erial cult ure can be grown and m aint ained at relat ively const ant condit ions , depending on t he flow rat e of t he nut rient s.

Figu r e 4 . Sch e m a t ic dia gr a m of a ch e m ost a t , a de vice for t h e con t in u ou s cu lt u r e of ba ct e r ia . Th e ch e m ost a t r e lie ve s t h e e n vir on m e n t a l con dit ion s t h a t r e st r ict gr ow t h by con t in u ou sly su pplyin g n u t r ie n t s t o ce lls a n d r e m ovin g w a st e su bst a n ce s a n d spe n t ce lls fr om t h e cu lt u r e m e diu m .

Syn ch r on ou s Gr ow t h of Ba ct e r ia

St udying t he grow t h of bact erial populat ions in bat ch or cont inuous cult ures does not perm it any conclusions about t he grow t h behavior of individual cells, because t he dist ribut ion of cell size ( and hence cell age) am ong t he m em bers of t he populat ion is com plet ely random . I nform at ion about t he growt h behavior of individual bact eria can, how ever, be obt ained by t he st udy ofsyn ch r on ou s cu lt u r e s. Synchronized cult ures m ust be com posed of cells which are all at t he sam e st age of t heba ct e r ia l ce ll cycle. Measurem ent s m ade on synchronized cult ures are equivalent t o m easurem ent s m ade on individual cells.

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rapidly lose synchrony because not all cells in t he populat ion divide at exact ly t he sam e size, age or t im e .

Figu r e 5 . Th e syn ch r on ou s gr ow t h of a ba ct e r ia l popu la t ion . By ca r e fu l se le ct ion of ce lls t h a t h a ve j u st divide d , a ba ct e r ia l popu la t ion ca n be syn ch r on iz e d in t h e ba ct e r ia l ce ll division cycle . Syn ch r on y ca n be m a in t a in e d for on ly a fe w ge n e r a t ion s.

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