I

sol
ati
onandScr
eeni
ngofMi
croor
gani
sms
Theeconomi csofaf er
ment ati
onprocesslargel
ydependsupont het
ypeofmi cr
oor gani
sm
used.Iffer
ment at
ionpr ocessistoyiel
dapr oductatacheaperpr i
cethechosenmi croorgani
sm
shouldgivethedesi r
edpr oducti
napr edi
ctableandeconomi cal
lyadequatequanti
ty.The
microorgani
sm wi t
hadesi r
edcharacter
sisgenerall
yisol
atedf r
om natur
alsubstr
atesl i
kesoil
etc.Suchanor ganism i
sgener all
ycall
edasapr oducerst
rain.

Apr
oducerst
rai
nshoul
dpossesst
hef
oll
owi
ngchar
act
ers:

1.I
tshoul
dbeabl
etogr
owonr
elat
ivel
ycheapersubst
rat
es.

2.I
tshoul
dgr
owwel
li
nanambi
entt
emper
atur
epr
efer
abl
yat30-
40°
C.Thi
sreducest
hecool
i
ng
cost
s.

3.I
tshoul
dyi
eldhi
ghquant
it
yoft
heendpr
oduct
.

4.I
tshoul
dpossessmi
nimum r
eact
iont
imewi
tht
heequi
pmentusedi
naf
erment
ati
onpr
ocess.

5.I
tshoul
dpossessst
abl
ebi
ochemi
cal
char
act
eri
sti
cs.

6.I
tshoul
dyi
eldonl
ythedesi
redsubst
ancewi
thoutpr
oduci
ngundesi
rabl
esubst
ances.

7.I
tshoul
dpossessopt
imum gr
owt
hrat
esot
hati
tcanbeeasi
l
ycul
ti
vat
edonal
argescal
e.

Detectionandisol
ati
onofami croor
ganism fr
om anat uralenv
ironmentli
kesoilcontaini
ngl
arge
numberofmi crobi
alpopulati
oniscal
ledasscr eeni
ng.Itisver
yt i
meconsumi ngandexpensive
process.Forexample,El
iLil
ly&Co.Ltddiscoveredthr
eespeci esofanti
biot
icproducing
organismsinaspanof10y earsandaft
erscreening4,00,000organi
sms.

Alt
houghtherearemanyscr
eeni
ngt
echni
ques,
all
oft
hem ar
egener
all
ygr
oupedi
ntot
wobr
oad
cat
egori
es.Theyare:

1.Pr
imar
yscr
eeni
ng

2.Secondar
yscr
eeni
ng.
Pr
imar
yScr
eeni
ngofMi
croor
gani
sms:
Pri
maryscreeningmaybedef i
nedasdet ectionandi sol
ati
onofthedesiredmicroor gani
sm
basedonitsquali
tat
iveabi
li
tytoproducethedesi r
edpr oductli
keant
ibioti
corami noacidoran
enzymeetc.Inthi
sprocessdesir
edmi cr
oor ganism isgeneral
l
yisol
atedf r
om anat ural
envi
ronmentlikesoi
l
,whichcontai
nssev eraldif
ferentspeci
es.Sometimest hedesired
mi
croor
gani
sm hast
obei
sol
atedf
rom al
argepopul
ati
onofdi
ff
erentspeci
esof
mi
croor
gani
sms.

Thef
oll
owi
ngar
esomeoft
hei
mpor
tantpr
imar
yscr
eeni
ngt
echni
ques:

(
i)Thecr
owdedpl
atet
echni
que

(
ii
)Indi
cat
ordy
etechni
que

(
ii
i)Enr
ichmentcul
tur
etechni
que

(
iv)Auxanogr
aphi
ctechni
que

(
v)Techni
queofsuppl
ement
ingv
olat
il
eandor
gani
csubst
rat
es.

(
i)TheCr
owdedPl
ateTechni
que
Thist echni
queispr imaril
yemployedf ordetecti
ngt hosemi croor
ganisms,whichar
ecapableof
produci nganti
biot
ics.Thistechni
quest artswiththesel ect
ionofanat ur
alsubst
rat
um li
kesoil
orot hersourceconsi st
ingofmicroorganisms.Pr ogressiveseri
aldi
luti
onofthesourceismade.
Suitableali
quotoft heserialdi
l
uti
onischosenwhi chisabletoproduce300t o400indiv
idual
colonieswhenpl atedonanagarpl at
e, af
terincubation.Suchapl at
eiscall
edascrowdedpl at
e.

 Theant i
bioti
cpr oduci
ngactivi
tyofacol
onyi sindicatedbynogr
owthofanyother
bacter
ialcolonyi ni
tsvi
cini
ty.Thi
sregi
onofnogr owthisi
ndi
cat
edbythefor
mat i
onofa
cl
earandcol orlessareaaroundtheanti
biot
icpr oducingmicr
oor
gani
sm’scol
onyont he
agarplate.Thisregioni
scalledasgrowthinhibitoryzone.

Suchacolonyi
sisol
atedfrom t
heplateandpurif
iedeit
herbymakingrepeatedsub-
cul
tur
ingorbystr
eaki
ngonapl atecontai
ningasuitabl
emedium,beforestockcul
tur
eis
made.Thepuri
fi
edcultur
eisthentest
edforitsanti
biot
icspect
rum.

 However
,thecrowdedplatetechniquehasli
mitedappl
icat
ions,asitwil
lnotgi
ve
i
ndi
cati
onofant i
biot
icpr
oducingor gani
sm agai
nstadesir
edorganism.Hence,thi
s
t
echni
quehasbeeni mprovedlateronbyempl oyi
ngatestorganism t
oknowt hespeci
fi
c
i
nhi
bit
oryacti
vi
tyoftheantibi
oti
c.

i
(i
)Indi
cat
orDy
eTechni
que:
 Microorganismscapableofproduci
ngacidsorami nesf
rom nat
ural sour
cescanbe
detectedusingthismethodbyincorpor
ati
ngcertai
npHi ndi
cat
ordy essuchasneutr
al
redorbr omothymolblueint
onutri
entagarmedium.Thechangeint hecolorofa
parti
culardyeinthevi
cini
tyofacolonywil
lindi
catetheabil
i
tyofthatcolonytopr
oduce
anor ganicaci
dorbase.
 Producti
onofanor ganicacidcanalsobedet
ect
edbyanalter
nati
vemethod.Inthi
s
met hodcalci
um carbonateisi
ncorpor
atedi
ntot
heagarmedium.Theproduct
ionof
organicacidisi
ndicatedbythefor
mat i
onofacl
earzonear
oundthosecoloni
eswhich
rel
easeor gani
cacidintothemedium

 Theidenti
fi
edcoloniesar
eisolat
edandpur
if
iedei
therbyr
epeat
edsub-
cult
uri
ngorby
st
reakingmethodsandast ockcult
urei
smadewhichmaybeusedforfur
therqual
i
tat
ive
orquanti
tat
ivescreeni
ngtests.

i
(i
i)Enr
ichmentCul
tur
eTechni
que:
Thi
st echniquei
sgener al
lyemployedtoi
solatethosemi cr
oorgani
smsthatarever
ylessi
n
numberi nasoilsampleandpossessspecificnutri
entrequi
rementandareimport
ant
i
ndustrial
ly
.Theycanbei solat
edifthenutr
ientsrequi
redbythem isi
ncor
poratedi
ntothe
medium orbyadj ust
ingtheincubati
oncondit
ions.

(
iv)Auxanot
rophi
cTechni
que:
Thistechniqueisemployedforthedetecti
onandi
sol
ati
onofmicr
oorgani
smscapableof
producingcertai
nextr
acell
ularsubst
ancessuchasgrowthst
imul
ati
ngfact
orsli
keaminoacids,
vi
taminset c.Atestor
ganism withadefini
tegr
owt
hrequir
ementf
orthepart
icul
armetabol
i
teis
usedint hi
smet hod

 Forthi
spurpose, spreadasuitablealiquotonthesurfaceofasteri
li
zedagarplateand
al
lowthegr owthofi solat
edcolonies,afteri
ncubat
ion.Asuspensionoftestor
ganism
withgrowthrequirementf ort
hepar ti
cularmetabol
iteisfl
oodedontheabov eplate
contai
ningisol
atedcol oni
es,whichar esubject
edtof urt
heri
ncubati
on.

 Theproductionoft
hepar ti
cularmetabol
it
erequir
edbythetestor ganism i
sindi
cat
edby
i
tsincreasedgrowt
hadj acenttocol
oniesthathavepr
oducedt her equir
edmetabol
it
e.
Suchcoloniesarei
solat
ed, puri
fi
edandstockcult
uresar
epr eparedwhi chareusedfor
fur
therscreeni
ngprocess.

2.Secondar
yScr
eeni
ngofMi
croor
gani
sms:
Pri
mar yscr
eeninghelpsinthedetect
ionandi solati
onofmi croorganismsf r
om thenatur
al
substr
atesthatcanbeusedf ori
ndustrialf
erment ati
onsforthepr oducti
onofcompoundsof
humanut i
li
ty,buti
tcannotgivet
hedet ail
sofpr oducti
onpot enti
alory i
eldoftheorgani
sm.Such
detai
l
scanbeascer t
ainedbyfur
therexperiment at
ion.Thi
sisknownassecondar yscreeni
ng,
whichcanpr ovi
debroadrangeofinformationper t
aini
ngtot he:

i
.Abi
l
ityorpot
ent
ial
i
tyoft
heor
gani
sm t
opr
oducemet
abol
i
tet
hatcanbeusedasani
ndust
ri
al
or
gani
sm.

i
i
.Thequal
i
tyoft
hey
iel
dpr
oduct
.

i
i
i.Thet
ypeoff
erment
ati
onpr
ocesst
hati
sabl
etoper
for
m.

i
v.El
i
minat
ionoft
heor
gani
sms,
whi
char
enoti
ndust
ri
all
yimpor
tant
.

Toevaluatethetr
uepotenti
aloftheisolat
edmi cr
oorganismsbot hqual i
tati
veandquant
it
ati
ve
anal
ysi
sar egeneral
lyconducted.Thesensit
ivi
tyoft
het estorganism towardsanewly
di
scoveredantibi
oti
cisgenerall
yanalysedduri
ngqualit
ativeanalysis,
whi l
ethequant
um yi
eldof
newl
ydi scov
eredantibi
oti
cisestimatedbythequantit
ativeanalysis.

Ev
aluat
ionofPot
ent
ial
it
iesofMi
croor
gani
sms:

Microor
gani
smsi solat
edi nthepr
imaryscr
eeni
ngar
ecr i
ti
cal
lyeval
uat
edinthesecondar
y
screeni
ngsothatindustr
iall
yimport
antandvi
abl
epotent
ial
it
iescanbeassessed.

Theyi
ncl
ude:

1.Todet
ermi
net
hepr
oductpr
oducedbyanor
gani
sm i
sanewcompoundornot
.

2.Adet
ermi
nat
ionshouldbemadeaboutt
heyieldpot
enti
ali
ti
esofvari
ousi
solat
ed
micr
oor
gani
smsthataredetect
edi
npri
maryscreeni
ngforthatnewcompound.

3.I
tshoul
ddet
ermineaboutt
hev
ari
ousr
equi
rement
soft
hemi
croor
gani
sm suchaspH,
aer
ati
on,t
emperat
ureet
c.

4.I
tshoul
ddet
ectwhet
hert
hei
sol
atedor
gani
sm i
sgenet
ical
l
yst
abl
eornot
.

5.I
tshoul
dr ev
ealwhethertheisol
atedorgani
sm i
sabl
et odest
royoral
terchemical
lythei
rown
fer
mentat
iveproductbyproducingadapt
iveenzy
mesiftheyaccumulat
einhigherquanti
ti
es.

6.I
tshouldr
evealt
hesui
tabi
li
tyofthemedi um ori
tsconst
it
uentchemi
cal
sfort
hegr
owt
hofa
micr
oorgani
sm andit
syi
eldpotent
ial
i
ties.

7.I
tshoul
ddet
ermi
net
hechemi
cal
stabi
l
ityoft
hepr
oduct
.

8.I
tshoul
drev
eal
thephy
sical
proper
ti
esoft
hepr
oduct
.

9.Itshoul
ddetermi
newhet
hert
hepr
oductpr
oducedbyami
croor
gani
sm i
naf
erment
ati
ve
processistoxi
cornot
.

10.Secondar
yscreeni
ngshoul drevealthatwhethertheproductproducedinfer
ment at
ion
processexi
stsinmorethanonechemi calfor
m.I fso,t
heamountoff ormationofeachchemical
formati
onoftheseaddit
ionalproductsispart
icularl
yimport
antsincethei
rrecover
yandsal eas
byproduct
scangreatl
yimpr ovetheeconomi cstatusofthefer
ment at
ionindust
ry.
11.Thenewor gani
sm shouldbeidenti
fiedtothespeci
esl evel.Thiswil
lhel
pinmaki nga
comparisonofgrowthpatt
ern,y
ieldpotenti
ali
ti
esandot herrequirementsoftestorganism wi
th
thoseal
readydescri
bedinthescient
if
icandpat entl
it
erature,asbei ngabl
etosy nt
hesize
product
sofcommer cial
value.

12.I
tshoul
dselecti
ndust
ri
all
yimpor
tantmi
croor
gani
smsanddi
scar
dot
her
s,whi
char
enot
usef
ulforf
ermentat
ioni
ndust
ry.

13.I
tshoul
ddet
erminetheeconomicstat
usofaf
erment
ati
onpr
ocessunder
takenby
employ
ingnewl
yisol
atedmicr
oorgani
sm.

Met
hodsofSecondar
yScr
eeni
ng:

Secondar yscreeninggivesveryusef
ulinformati
onper tainingt
ot henewlyisolated
mi croorganismst hatcanbeempl oyedinf ermentat
ionpr ocessesofcommer cialval
ue.These
scr eeningtestsareconduct edbyusingpet r
idi
shcont ainingsoli
dmedi aorbyusi ngflasksor
smal l
fermenterscont aini
ngli
quidmedia.Eachmet hodhassomeadv ant
agesand
disadv antages.Somet imesboththemet hodsar eempl oyedsimul t
aneousl
y .

Liqui
dmedi amethodismor esensitiv
et hanagarplatemethodbecauseitprovi
desmor eusef
ul
i
nf or
mat i
onaboutthenutr
it
ional,
phy sicalandproducti
onresponsesofanorganism t
oactual
fermentati
onproduct
ioncondit
ions.Erlenmey erf
laskswithbaff
lescont
aini
nghighlynut
ri
ti
ve
l
iquidmedi aar
eusedf ort
hismet hod.Flasksarefull
yaeratedwithgl
assbaffl
esand
continuousl
yshakenonamechani calshakerinordertohaveoptimum pr
oductyiel
d.

Therearesev eral
techniquesandpr oceduresthatcanbeempl oy
edf orsecondaryscr
eening.
Howev er
,onlyaspeci f
icexampl eofestimati
onofant i
bioti
csubstancepr oducedbyspeciesof
Str
eptomy ces,isdescri
bedinthef ol
lowingparagraph.Simil
armet hodscouldbeusedf orthe
detect
ionandi sol
ationofmi cr
oorganismscapabl eofproducingotherindustr
ialpr
oducts.

(
i)Gi
antCol
onyTechni
que:
Thi
st echni queisusedf ori
solati
onanddet ect
ionofthoseant
ibi
oti
cs,whichdiffuset
hrough
sol
idmedi um.Speci esofStreptomy ces,
iscapableofproduci
nganti
bioti
csdur i
ngprimary
scr
eeni ng.Thei solatedStr
eptomy cesculturei
sinoculat
edint
othecentralareaofasteri
li
zed
pet
ripl at
escont ainingnutr
ientagarmedi um andareselect
ed.Theplat
esar eincubat
eduntil
suf
ficientmi crobi
al growt
ht akesplace.

Cult
uresoftestorgani
sm, whoseanti
biot
icsensi
ti
vit
yistobemeasuredarestr
eakedfrom t
he
edgesofplate’
suptobutnottouchingthegrowthofStr
eptomycesandarefur
theri
ncubatedto
al
lowthegrowt hofthetestor
ganisms.Thenthedist
anceoverwhichthegr
owthofdiff
erent
t
estor
gani
smsi
sinhi
bit
edbyt
heant
ibi
oti
csecr
etedSt
rept
omy
cesi
smeasur
edi
nmi
l
li
met
ers.

Ther el
ativei
nhibit
ionofgr owthofdi
fferenttestorgani
smsbyt heant i
biot
ici
scall
edinhi
bit
ion
spectrum.Thoseor gani
smswhosegr owthisi nhi
bit
edtoaconsi der
abledist
anceare
consideredmor esensi t
ivetotheant
ibioti
cthant hoseorgani
sms, whichcangrowclosetothe
anti
bioti
c.Suchspeci esofSt rept
omyces,whi chhavepotenti
ali
tyofinhi
bit
ingmicr
oorgani
sms
i
spr eservedforfurthertesti
ng.

(
ii
)Fi
lt
rat
ionMet
hod:
Thismethodisemployedf
ortesti
ngt
hoseant i
biot
icswhi
charepoorl
ysolublei
nwaterordo
notdi
ffuset
hroughthesol
idmedium.TheSt r
eptomycesi
sgrowninabr ot
handi t
smycelium i
s
separ
atedbyfil
tr
ati
ontogetcult
uref
il
trat
e.Vari
ousdil
uti
onsofanti
biot
icfi
l
trat
esareprepared
andaddedtomol t
enagarplat
ingmedium andall
owedtosoli
dif
y.

Lateroncul
tur
esofvari
oustestorganismsar est
reakedonparall
ell
inesont hesoli
dif
ied
medium andsuchpl
atesareincubated.Theinhi
bit
oryeff
ectofanti
bioti
cagai nstt
hetest
organi
smsismeasuredbytheirdegreeofgrowthindiff
erentant
ibi
oti
cdilut
ions.

(
ii
i)Li
qui
dMedi
um Met
hod:
Thi
smet hodi
sgeneral
l
yemploy
edforf
urtherscreeni
ngtodet
ermi
net
heexactamountof
ant
ibi
oti
cproducedbyami
croor
gani
sm l
ikeStreptomyces.

Erl
enmey erconi
calfl
askscont
aini
nghi ghl
ynutrit
ivemedium ar
einocul
atedwi t
hStrept
omy
ces
andincubatedatroom temper
ature.Theyarealsoaeratedbyshaki
ngcontinuousl
yand
vi
gorouslyduri
ngincubati
onperi
odt oall
owStreptomy cest
oproducetheantibi
oti
cinan
opti
mum quant i
ty

Samplesofcult
uref
lui
dsar
eper
iodi
cal
l
ywi
thdr
awnasept
ical
l
yforunder
taki
ngt
hef
oll
owi
ng
rout
inechecks:

1.Tocheckt
hesui
tabi
l
ityofdi
ff
erentmedi
aformaxi
mum ant
ibi
oti
cpr
oduct
ion.

2.Todeterminetheval
ueofpHatwhi
cht
her
ewi
l
lbemaxi
mum gr
owt
hoft
hemi
croor
gani
sm
andanti
bioti
cproducti
on.

3.Tocheckf
orcont
ami
nat
ion.

4.Todet
ermi
newhet
hert
heant
ibi
oti
cpr
oducedi
snewornot
.

5.Tocheckt
hest
abi
l
ityoft
heant
ibi
oti
catv
ari
ouspHl
evel
sandt
emper
atur
es.

6.Todet
ermi
net
hesol
ubi
l
ityoft
heant
ibi
oti
cinv
ari
ousor
gani
csol
vent
s.

7.Tocheckaboutt
het
oxi
cit
yoft
heant
ibi
oti
cagai
nstt
heexper
iment
alani
mal
s.
Aftercarr
yingoutt
heabovementionedr
out
inet
est
sfur
therst
udi
esar
eal
soconduct
edt
oknow
thefoll
owingaddit
ional
inf
ormat
ion:

1.Ef
fectofi
ncubat
iont
emper
atur
eandant
if
oami
ngagent
sonf
erment
ati
on.

2.Rat
eofr
esi
stancedev
elopedamongt
het
estor
gani
sms.

3.Checki
ngtheant
ibi
oti
cfori
tsbact
eri
ostat
icorbact
erici
dalpr
oper
ti
es.I
tsabi
l
ityt
opr
eci
pit
ate
ser
um protei
nst
ocausehemolysi
sofbloodortoharm phagocyt
es.

4.Checki
ngf
orpossi
bil
i
tyofi
ncl
usi
onofpr
ecur
sorchemi
cal
oft
heant
ibi
oti
cpr
oduct
ioni
nthe
medium.

5.Sui
tabi
l
ityoft
heor
gani
sm f
ormut
ati
onandot
hergenet
icst
udi
es.