HUB i

Keisla Maiie Golez Toiies

Maic Plilipp Sosing
}anelle Velasco
}oan Umaclap
Micvobia! Cu!tivation meilod of muliiplying
bacieiial oiganisms by leiiing ilem iepioduce in
piedeieimined culiuie media undei coniiolled
laboiaioiy condiiions.
Piimaiy diagnosiic meilods of miciobiology used as a
iool io deieimine ile cause of infeciious disease by
leiiing ile ageni muliiply in a piedeieimined medium.
Tle ieim culiuie is used infoimally io iefei io
seleciively giowing a specific lind of miciooiganism
in ile lab.
To piopagaie bacieiium, ii is necessaiy io piovide ile
appiopiiaie bioclemical and bioplysical
enviionmeni.
Tle bioclemical (nuiiiiional) enviionmeni
cu!tuvc mcdium.
Cu!tuvc mcdia aie employed in ile isolaiion and
mainienance of puie culiuies of bacieiia and aie also
used foi ideniificaiion of bacieiia accoiding io ileii
bioclemical and plysiological piopeiiies.
Tlis media is necessaiy io be sieiilized piioi use io
iemove coniaminanis .
Cu!tuvc mcdia aie designed io piovide all ile esseniial
nuiiienis in soluiion foi bacieiial giowil.
Tle nuiiiiional iequiiemenis of a bacieiium sucl as E.
coli aie ievealed by ile cell's elemenial composiiion,
wlicl consisis of C, H, O, N, S. P, K, Mg, Fe, Ca, Mn,
and iiaces of Zn, Co, Cu, and Mo.
Tlese elemenis aie found in ile foim of waiei,
inoiganic ions, small molecules, and maciomolecules
wlicl seive eiilei a siiuciuial oi funciional iole in ile
cells.
Most Ivcc-!iving bactcvia gvow ovcv a pH vangc
oI about thvcc units.
Gvowth vatc vs pH Iov thvcc
cnvivonmcnta! c!asscs oI pvocavyotcs.
Miciooiganisms exlibii a iange of iempeiaiuie ovei
wlicl ii can giow, defined by iliee caidinal poinis :
Mcsophi!cs- oiganisms wiil an opiimum iempeiaiuie
neai degiees (ile body iempeiaiuie of waimblooded
animals)
hcvmophi!cs oiganisms wiil an opiimum T beiween
aboui degiees and o degiees
Hypcvthcvmophi!cs. Some Aiclaea wiil an opiimum
T of o degiees oi liglei and a maximum T as ligl as
ii degiees
Most pvokavyotcs wi!! gvow ovcv a tcmpcvatuvc vangc oI about
o dcgvccs.
hc cuvvcs cxhibit thvcc cavdina! points: minimum, optimum
and maximum tcmpcvatuvcs Iov gvowth.
hcvc is a stcady incvcasc in gvowth vatc bctwccn thc
minimum and optimum tcmpcvatuvcs
Gvowth vatc vs
tcmpcvatuvc Iov
Iivc
cnvivonmcnta!
c!asscs oI
pvocavyotcs
Gvowth Iactovs aie iequiied in small amounis by cells
because iley fulfill specific ioles in biosynilesis. Tle
need foi a giowil facioi iesulis fiom eiilei a blocled
oi missing meiabolic pailway in ile cells. Giowil
faciois aie oiganized inio iliee caiegoiies.
i. puvincs and pyvimidincs: iequiied foi synilesis of
nucleic acids (DNA and RNA)
#. amino acids: iequiied foi ile synilesis of pioieins
. vitamins: needed as coenzymes and funciional
gioups of ceiiain enzymes
iquid mcdia aie used foi giowil of puie baicl
culiuies
o!id mcdia aie used widely foi ile isolaiion of puie
culiuies, foi esiimaiing viable bacieiial populaiions,
and a vaiieiy of oilei puiposes.
gav is ile usual gelling ageni foi so!id oi scmiso!id
mcdium .
a lydiocolloid deiived fiom ied algae.
Agai is used because of iis unique plysical piopeiiies (ii
melis ai ioo
o
C and iemains liquid uniil cooled io o
o
C,
ile iempeiaiuie ai wlicl ii gels) and because ii cannoi
be meiabolized by mosi bacieiia.
As a medium, Agai is ielaiively ineii, ii simply lolds
(gels) nuiiienis ilai aie in aqueous soluiion.
Mcdia Puvposc
Complex Giow mosi leieioiioplic oiganisms
Defined Giow specific leieioiiopls and aie ofien mandaioiy
foi clemoauioiiopls , ploioauioiiopls and foi
miciobiological assays.
Eiilei one of ile above caiegoiies supplemenied wiil giowil
inlibiiing oi piomoiing addiiives:
Seleciive Suppiess unwanied miciobes, oi encouiage
desiied miciobes
Diffeieniial Disiinguisl colonies of specific miciobes fiom
oileis
Eniiclmeni Similai io seleciive media bui designed io inciease
ile numbeis of desiied miciooiganisms io a
deieciable level wiiloui siimulaiing ile iesi of ile
bacieiial populaiion
Reducing Giowil of obligaie anaeiobes
A clemically complex medium (also lnown as a bosol
oi unJefineJ meJium) is a medium ilai coniains:
a caibon souice sucl as glucose foi bacieiial giowil
waiei
vaiious salis needed foi bacieiial giowil
Coniains exiiacis of naiuial subsiances of unlnown
conceniiaiion
Nuiiieni Agai Example of a complex, geneial giowil
media
onstituent Amount
Pepione .og Souice of amino acids, N, S, and P
Beef exiiaci .o g Souice of viiamins and oilei giowil faciois
Sodium Clloiide .o g Na
+
souice foi lalopliles and inlibiioiy
io nonlalopliles
Agai i.o g
Waiei i iiiei
A clemically defined medium (also lnown as JefineJ
meJium oi synileiic medium) is a medium in wlicl :
all ile clemicals used aie lnown
no yeasi, animal oi plani iissue is pieseni
all ile ingiedienis of a culiuie medium aie lnown, boil
qualiiaiively and quaniiiaiively,
Glucose Minimal Salis Bioil Example of clemically
defined medium designed foi ile giowil of E.coli.
onstituent Amount
Glucose .o g C and eneigy souice
Ammonium plosplaie monobasic i.o g pH buffei, N and
P souice
Sodium clloiide .o g Na
+
souice foi lalopliles and
inlibiioiy io nonlalopliles
Magnesium sulfaie o.# g S and Mg
++
souice
Poiassium plosplaie, dibasic i.og pH buffei, P and K
souice
Waiei i liiei
scptic cchniqucs
Befoie inoculaiion, miciobiological media and all maieiials
coming inio coniaci wiil ii musi be sieiile.
Sieiilizaiion implies ile compleie desiiuciion of all
miciooiganisms including spoies, ilis is accomplisled by
ile use of :
Heai
Clemicals
Radiaiion
Filiiaiion
Hcat Denaiuies and coagulaies viial pioieins. Tleie aie vaiious
foims of leai sieiilizaiion.
cd Hcat
noculaiing wiies oi loops aie sieiilized by lolding ilem in a Bunsen
flame uniil iley aie ied loi.
Moist Hcat (Auioclave)
Bacieiia aie moie ieadily desiioyed by moisi leai (sieam) ilan diy
leai.
sieiilizaiion of culiuie media, aqueous soluiions and ile
desiiuciion of discaided culiuies.
i#i C ai i psi foi i minuies
Uiilizes sieam undei piessuie
vy Hcat (oven)
Usually employed foi maieiials wlicl could eiilei be coiioded by
sieam oi musi iemain diy befoie use. Tlese include meial
insiiumenis, glass peiii disles, flasls and pipeiies and coiion wool.
n piaciice, diy leai sieiilizaiion iequiies longei iime inieivals and
liglei iempeiaiuies ilan sieam sieiilizaiion
ioC foi i#o minuies.
Chcmica!
Usually employed foi delicaie equipmeni sucl as opiical
insiiumenis and eleciiical devices wlicl would be damaged by
leai. Due io ile ioxiciiy of ile clemicals used, ilis is noi ile
mosi populai foim of sieiilizaiion.
adiation
Employed foi leaisensiiive maieiials and foi enviionmenial
samples sucl as soil and sedimeni wleie siiuciuial clanges
caused by leai need io be avoided.
i!tvation
Filiiaiion sieiilizaiion opeiaies iliougl ile exclusion iailei
ilan desiiuciion of miciooiganisms. i is safe foi ile usei and is
employed foi sensiiive liquids and gases.
Auioclave is basically a luge sieam coolei.
Sieam enieis inio a jaclei suiiounding ile
clambei.
Tle piessuie will go up ovei i pounds pei
squaie incl (psi): ai ilis poini ile iimei
begins io couni down usually foi i
minuies, depending on ile iype of media.
Tle ligl piessuie in a closed coniainei
allows ile iempeiaiuie io go above ile
liglesi iempeiaiuie one could gei by jusi
boiling, aiound i#i degiees C.
Tleiefoie, ile paiameieis foi sieiilizaiion
wiil an auioclave aie i#i C ai >i psi foi i
minuies.
Fifieen minuies is ile ileimal
deail iime foi mosi oiganisms (excepi
some ieally laidy spoiefoimeis).
i uses an eleciiic elemeni io geneiaie leai.
Cuiieni flows iliougl ile wiie in ile ceniei of
ile iesisiance elemeni, wleie ile eleciiical
eneigy is iuined inio leai. Tle leai is
conducied iliougl ile insulaioi ilai
suiiounds ile iesisiance elemeni, inio ile
meial jaclei, ilen io ile suiiounding aii.
Eleciiic ovens lave a ileimosiai, wlicl is used
io coniiol ile iempeiaiuie of ile oven.
leais ile aii io piovide a unifoim iempeiaiuie
in ile oven
Diffeieniiaie clemicallydefined fiom clemically
complex media
Opeiaie an auioclave and a meclanical oven
Undeisiand effeci of sieiilizaiion on medium pH
Recognize impoiiance of complex media foi
miciooiganism giowil
i) oo ml eilenmeyei flasl
#) Culiuie media
a. Nuiiieni agai
b. Glucose Minimal Sali
agai
) Disiilled waiei
) Analyiical balance
) Aluminum foil
) Siiiiing iod
) Spaiula
) Bioil Culiuies
a. E.coli,
b. P.aeiuginosa,
c. S.auieus,
d. B.subiilis
) Pan
io) pH meiei
ii) ioo ml bealei
i#) Biomilymol blue
i) io ml pipei
i) NaOH
i) HCL
i) Disinfeciani
i) Tesi iubes
i) Siove
A. Nuiiieni Agai piepaiaiion (Clemicallycomplex
medium)
Add ioo ml waiei
io oo ml flasl
Add Nuiiieni Agai
Siii
Fill io oo ml
mail
Boil
Conienis weie
iiansfeied io
iesiube ( ml
eacl)
Add ioo ml
waiei io ooml
flasl
GMSA was
added
Waiei added
io oo ml
mail
Siii
Subdivded io flasls:
Auioclave
oven
unsieiilized
B. Glucose Minimal Sali Agai (GMSA)piepaiaiion (
Clemically defined medium)
C. Sieiizaiion by Heai
i. Auioclave
a) Placed ile media inside ile auioclave ,clecled foi ile
waiei volume inside and locled.
b) Time and iempeiaiuie was sei ai iminuies and i#i deg C
and iuinedon ile cycle.
c) Upon compleiion of ile cycle, ile media was ieiiieved
using leaipioof gloves.
C. Sieiizaiion by Heai
#. Oven
a) Placed ile media inside ile oven.
b) Time and iempeiaiuie was sei ai ili and ii deg C and
iuinedon ile cycle.
c) Upon compleiion of ile cycle , ile oven was opened and
lefi io cool io o deg C.
d) Tle media was ieiiieved using leaipioof gloves.
D. Adjusiing pH of ile Media
i. ioml of of sieiilized medium was placed in a iooml
bealei and pH was clecled.Tlen, io diops of
biomilymol blue was added.
#. Adjusied ile pH of ile medium using o.oN NaOH oi
o.oN HCL io aclieve pH.
. Adjusied ile pH of ile iemaining bioil as well using
ile appiopiiaie amouni of NaOH oi HCL.
. Sieps i was iepeaied using an unsieiile medium.
E. Agai slanis and plaies piepaiaiion
i. Placed iubes in a slaniing boaid and sei aside
#. Placed ile flasl of media in a waiei bail of o degC
io cool.
. Afiei disinfeciing ile woil aiea, ile coiion plug was
iemoved and ile mouil of ile flasl flamed.
. Tle media was allowed io solidify in a cool , level
suiface.
E. Effecis of Sieiilizaiion on Bacieiia Giowil
i. Reiiieved ile NA plaie sieiilized in auioclave and
divided inio foui paiis.
#. noculaied E.coli , P.aeiuginosa,S.Auieus and B.
subiilis inio eacl paii and incubaied ai degC foi
lis.
. Repeai sieps i# using NA plaie sieiilized in oven and
unsieiile NA plaie.
. Obseived ile piesence of bacieiial giowil and
measuied ile iaie of giowil in mm]li.
O diffeieni oiganisms suivive ai diffeieni enviionmenis
and iequiies diffeieni nuiiienis based on iis cellulai
needs.
O Tlis expeiimeni esiablisled ilai nuiiieni agai and
glucose minimal sali agai as culiuie mediums weie
able io susiain giowil of culiivaied bacieiia.
O Tlis expeiimeni esiablisled ilai pH is an impoiiani
paiameiei foi oiganism suivival
O Tlis expeiimeni esiablisled ilai ile piopei
sieiilizaiion ieclniques musi be done io ensuie
oiganism suivival in ile culiuie media.
actcvia Minimum pH Optima! pH Maximum pH
E. Coli .
P. Aeiuginosa . .
S. Aeieus .# . .
B. subiilis
. ii
actcvia Minimumtcmp
(dcg C)
Optima! tcmp
(dcg C)
Maximum tcmp
(dcg C)
E. Coli io
P. Aeiuginosa
Known ai i
#
S. Aeieus io o
B. subiilis i o
Obscvvation and iscussion
i. Diffeieniiaie clemically defined medium fiom a clemically
complex medium based on ile ingiedienis and ile iype of
bacieiia culiivaied.
A clemically complex medium (also lnown as a bosol oi unJefineJ
meJium) is a medium ilai coniains:
a caibon souice sucl as glucose foi bacieiial giowil
waiei
vaiious salis needed foi bacieiial giowil
Coniains exiiacis of naiuial subsiances of unlnown conceniiaiion
Giow mosi leieioiioplic oiganisms
A clemicallydefined medium(also lnown as JefineJ meJium oi synileiic
medium) is a medium in wlicl :
all ile clemicals used aie lnown
no yeasi, animal oi plani iissue is pieseni
all ile ingiedienis of a culiuie medium aie lnown, boil qualiiaiively
and quaniiiaiively,
Giow specific leieioiiopls and aie ofien mandaioiy foi
clemoauioiiopls, , ploioauioiiopls and foi miciobiological assays.
#. Fill in iable below . Effeci of sieiilizaiion piocess on
pH of medium.
Culture medium
sterilized in
Initial pH Final pH
Autoclave 7.5 6.38
Oven 7.5 6.3
Control(unsterile) 7.5 6.43
. Based on iable fiom quesiion #,explain ile effeci of
sieiilizaiion piocess on pH of ile medium.
Based on ile iable, ile pH of ile media decieased, maling ile media
sliglily acidic. Tle pH of a soluiion will clange as iempeiaiuie clanges.
Howevei, ileie is no geneial ielaiionslip beiween iempeiaiuie and pH .
Tlis is dependeni on ile composiiion of ile soluiion. Tleiefoie , ile veiy
small clange in pH due io inciease in iempeiaiuie was biougli aboui by ile
ieaciion of ile clemicals in ile media wiil iempeiaiuie.
pH is ile negaiive common (base io) logaiiilm of ile lydiogen ion (H+)
aciiviiy in a soluiion. Tle lydiogen ion aciiviiy depends on all ile possible
clemical ieaciions ilai pioduce oi consume lydiogen ions. Tle exieni of a
clemical ieaciion depends on iis equilibiium consiani, and ile equilibiium
consiani of any ieaciion depends on ile iempeiaiuie. Some ieaciions
become moie lilely as iempeiaiuie incieases, some less.
Tle mosi basic clemical ieaciion affeciing pH is ile dissociaiion of waiei:
H#O > H+ + OH
Ovei ile iempeiaiuie iange o io o degiees Celsius, ilis ieaciion becomes
moie favoiable. Tlis means ilai neuiial waiei will coniain a liglei
conceniiaiion of H+ as ile iempeiaiuie incieases, so ile pH will deciease.
. Fill in iable below. Effeci of vaiying sieiilizaiion
piocess on giowil of bacieiia.
acteria
Growth rate (mm/hr)
Unsterile Media
Autoclave-
sterilized media
Oven-sterilized
media
Mlcrococcus luLeus 17 mm (00334) 3mm (00623) 4mm (00833)
Saccharomyces
cerevlslae
1 mm (00208) 4mm (00833) 3mm (00623)
roLeus vulgarls 2 mm (00417) 37mm (01188) 7mm (01438)
SLaphylococcus
aureus
18 mm (00373) 4mm (00833) 2mm (00417)
Average 00339 00870 00833
. Based on iable fiom quesiion , wlai is
ile effeci of subjeciing ile medium in
ligl iempeiaiuie on giowil of bacieiia ?
Based on ile iable, subjeciing ile medium io ligl
iempeiaiuie incieases ile giowil iaie of bacieiia. Since
mosi miciooiganisms do noi suivive ai ligl
iempeiaiuies (above ioo deg C) , ile leaiing oi
sieiilizaiion ieclniques (auioclave and oven) used,
eliminaies coniaminanis ilai may compeie wiil
inoculaied bacieiia foi nuiiienis. Tlis slould allow
liglei giowil iaie ilan on ile unsieiilized media.
. Compuie foi ile quaniiiy of eacl ingiedieni in medium X.
Piepaie i agai plaies, #o agai slanis and io bioils.
Med|um We|ght (grams)
!eptone
15 !lates
03
Assumption:
1 plate 20ml
1 slant 8 ml
1 broth 10 ml
ThereIore , required qty oI medium x Ior :
15 plates 6.96 g
20 slants 3.72 g
10 broths 0.16 g
20 Slants
016
10 broths
003
Sodium
Chloride
15 !lates
048
20 Slants
0236
10 broths
008
BeeI Extract
15 !lates
018
20 Slants
0096
10 broths
003
Agar
15 !lates
6
20 Slants
32
10 broths
none