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Microbialbiotechnologyinthelaboratoryandinpractce
5.
Othermaterials:serologicalpipettes,automaticpipettes,
steriletipsforautomaticpipettes,Erlenmeyerflasks
(100ml).
Aimoftheexercise
Efectsofoxygencontentandthetypeofculturemediumontheefciencyof
thebiosynthesisofvitaminB
12andorganicacids(lactc,acetcandpropionic)
byPropionibacteriumfreudenreichiissp.shermanii.
Procedure
1.
EstablishingthePropionibacteriumcultures:establish
Propionibacteriumculturesonaliquidmediumwith
yeastextract,mediumwithcaseinhydrolysateandcasein
in100mlflasks.Forthispurpose,inoculate20mlofthe
mediumwitha24hmaterialcollectedwithaloopfrom
a24hbacterialcultureonamediumwithyeastextract.
UseANOXOMATtocreateanaerobicandmicroaerophilic
conditions.Addcobaltsalts(10mg/lCoCl
2
×6H
2
O)toall
cultures.Theculturesshouldbeconductedinanaerobic
jarsandunderaerobicconditionsfor6daysat28°C,on
arotaryshakerat160rpmunderthefollowingconditions:
a)anaerobically,
b)aerobically,
c)
3daysanaerobicallyandthen3daysunderaerobic
conditions,
d)
constantlyinanatmospherewithareducedoxygen
contentof6%,
e)
3daysinanatmospherewithareducedoxygencontent
of6%,andthen3daysunderaerobicconditions.
2.
Preparationofstandardcurves:Standardcurvesof
organicacidsandvitaminB
12
forthedeterminationof
samplestestedbyliquidchromatography(LC-MS)should
bepreparedaccordingtotheconditionsrecommendedby
themanufactureroftheapparatusforindividualanalysed
substrates.Theseparationshouldbeconductedusing
aC18phasechromatographiccolumn.
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