4.4 Microbiology

8/11/2019 4.4 Microbiology

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4.4 MICROBIOLOGY 1. Bacteria may be round (coccus), rod shaed(baci!!us), sira! shaed (siri!!um). "hae hastraditiona!!y been used #or c!assi$cation a!on%&ith metabo!ic reactions.

'. he shae o# bacteria is due to their ri%id ce!!&a!! &hich

has a uniuestructure.

*. he Gram stain may be used todi+erentiate bet&een Gram ositieand Gram ne%atie bacteria accordin%to &hether the ce!! &a!! retainscrysta! io!et (stains ur!e), or not(stains red by the counterstain).

Gram staining is used to diferentiate betweenGram +ve and Gram –ve Bacteria The diferentstaining properties are due to diferences in thechemical composition o their cell walls. First thesample is Fixed, the r!stal violet is added. Bothretain the r!stal "iolet at this point in time. Then #odine is added and $thanol. The ethanolbrea%s down the &ipopol!saccharide la!er in theGram 'egative bacteria, and so the! become

decolourised. The counter stain – (aarin, isthen added and stains the Gram 'egativebacteria, but the Gram positive bacteria are notcoloured. This leads to the #denti)cation o

whether a bacteria is Gram *ositive'egative.

4. he Gram reaction re-ectsthe more com!e structure o# Gram ne%atie ce!! &a!!s

Gram 'egative Bacteria havemore chemicall! complex wallswhere the peptidogl!can issupplemented b! large moleculeso lipopol!saccharide, whichprotects the cell. The! do 'Tretain the r!stal "iolet stain.Gram negative bacteria areresistant to penicillin and theen-!me l!so-ome.Gram *ositive bacteria lac% thecomplex lipopol!saccharide la!er, and so retain the cr!stal violet stain. Gram*ositive Bacteria are more susceptible to penicillin and the en-!me l!so-ome.

/. Microor%anisms may be %ro&n in the !aboratory i# su!ied &ithsuitab!e hysica! conditions, nutrients and &ater.



0. Or%anisms ary in their reuirements and usua!!y %ro& oer a ran%e o#temeratures and a!ues, &ith an otimum &ithin the ran%e. The ptimum temperature o Bacteria is between /0 and 10, with 23 being theoptimum or mammalian pathogens. The ptimum Temperature is the temperaturewhere the reaction is at its best. ptimum Temperaturesp4 is regulated b!$n-!mes. # the temperature is too high, the en-!me will denature, i it is too lowthe reaction will procede at a ver! slow rate. #n p4 the optimum or most bacteria is

slightl! 5l%aline at 3.1 6 Fungi preer neutral7slightl! acidic range8. The ptimum p4is once again regulated b! en-!mes. This means that at higher al%alinit! or acidit!,the en-!me is again denatured due to shape o the en-!me becoming dis)gured. This cannot be reversed.2. "ome or%anisms are ob!i%ate aerobes, reuirin% oy%en #or metabo!ism,&hi!st others are ob!i%ate anaerobes and can on!y surie in the absenceo# oy%en. Many or%anisms &i!! %ro& in either the resence or absence o#oy%en, #acu!tatie anaerobes.2 T!pes o organisms.

7 bligate 5erobes – x!gen is re9uired or metabolism, The organism cannotsurvive i ox!gen is not present.

7bligate 5naerobes – annot grow in the presence o x!gen7Facultative 5naerobes – Grow better in 5erobic conditions however can

survive in its absence.3. utrients are su!ied in nutrient media and inc!ude5 carbon, usua!!yor%anic such as %!ucose6 nitro%en, or%anic or inor%anic6 %ro&th #actorssuch as itamins and minera! sa!ts.#n the laborator!, 'utrients are supplied in nutrient media such as agar. Thismedium includes 5:B', usuall! in the orm o G&;($. '#T:G$', in bothrganic and #norganic orms. G<T4 F5T:( such as "#T5=#'( and =#'$:5&(5&T(. The 'itrogen us needed or the production o 5mino 5cids in *rotein

(!nthesis.7. G!ucose is norma!!y the source o# ener%y.

18. 9setic techniues ino!e hand!in% cu!tures in such a &ay as toreent their contamination by un&anted or%anisms. o&eer, they haea dua! urose in reentin% the contamination o# ersonne! and theimmediate enironment by the or%anisms bein% cu!tured. The aims o 5septic techni9ue are to prevent contamination to the $'"#:'=$'Tand contamination to the (5=*&$(.11. :uiment and media must be steri!ised be#ore use by aroriatemethods. eat is common!y used, eam!es bein% the use o# an autoc!ae

at a suitab!e temerature (1'1oC) #or 1/minutes, or the heatin% aninocu!atin% !oo in a Bunsen -ame. eat !abi!e !astics are irradiated.Items must be rotected #rom contamination a#ter steri!isin%. To prevent the contamination o pure cultures b! bacteria rom the environment

7(terilise all apparatus and media – prevent initial contamination.7;se sterile loops > 4andle cultures careull! – prevent subse9uent

contamination To prevent contamination to the $'"#:'=$'T

7 (terilise wor% suraces beore and ater an experiment. 7 isinectant7 ;se correct handling techni9ues

74old the ap with !our little )nger and '$"$: place cap on the bench7Flame the mouth o the bottle or /72 (econds74old the inoculating loop in the ?ame until the wire goes red – red hot.7&it the lid o the petri dish @;(T $';G4 to allow entr! o the

inoculating loop



7(ecure the lid with / pieces o adhesive tape – 'T cover with tapeas this induces 5naerobic conditions – *athogenic Bacteria.

7#ncubate at around /0 egrees7o 'T open petri dishes ater incubation

5utoclaves A preerred method o sterilisation in a lab7/ degrees in steam under pressure or 0 minutes7 $nsure that resistant $'(*:$( are destro!ed.7 *lastic *etri dishes can be placed in autoclavable bags and then disposed o

aterwards

1'. ;irect ce!! counts may be tota! counts, &hich inc!ude both !iin% anddead ce!!s, and iab!e counts, &hich count !iin% ce!!s on!y.irect cell counts can be divided into7Total counts – ead and 5live cells7"iable counts – 5live cells onl!

5 4aemoc!tometer ma! be used. This produces a Total count as dead and alivecells cannot be diferentiated. Turbidimetr! is a method that uses a colorimeter to measure the cloudiness o the

culture as cell numbers are increased.1*. <or a iab!e count a =no&n o!ume o# or%anisms is added to a%ar!ates, incubated and the co!onies counted. It is assumed that one ce!!%ies rise to one co!ony. his ma=es no a!!o&ance #or c!umin% o# ce!!s somay cause an underestimate o# numbers.For a viable count, lumping ma! account or an underestimation o numbers. Theseparate colonies are presumed to have arisen rom bacteria. (o to )nd the Total"iable ount, count the number o colonies present, then times this b! the dilutionactor to give an estimate number o bacteria.14. In both cases the ori%ina! cu!ture usua!!y reuires di!ution by ten #o!d

stes, seria! di!ution, in order to roide a $na! number &ithin a countab!eran%e. Ten Fold ilution 6 (erial ilution8 is used so that the number o cells that arise whenincubated is within a countable range – not too ew or too man!.cm2 o the original solution is added to Ccm2 o sterile medium and mixed up. Thencm2 o this solution is ta%en and added to another Ccm2 o sterile medium andmixed up. This is repeated until the spread o bacteria on the agar plates is in acountable range.1/. 9 ure cu!ture o# an or%anism is needed #or the #ormation andharestin% o# a ure roduct durin% and a#ter %ro&th in a #ermenteresse!. he or%anism must be su!ied &ith suitab!e conditions #or %ro&th

and &ithout cometition #or maimum e>ciency.

10. he esse! shou!d be steri!isedbe#orehand and an aroriate steri!emedium used. <i!ters are used to reentcontamination throu%h the esse!?s oenin%s.9setic conditions and hand!in% are reuiredto maintain urity.

12. <orced aeration may be needed #ormaimum %ro&th o# aerobes and thisaeration may a!so mi the cu!ture to imroecontact &ith nutrients. Miin% may beimroed by a searate mier.



13. emerature monitorin% and contro! are reuired to maintain constantconditions and &ater @ac=ets remoe ecess heat roduced durin% thecu!ture rocess.

17. Commercia!!y, sohisticated monitors are used to imroe contro! o#temerature and , and air in!ets may use sar%ers or other deices toimroe aeration.

'8. <or the commercia! roduction o# enici!!in, the #un%us Penicilliumnotatum is %ro&n in a batch cu!ture and the antibiotic is roduced a#terthe %ro&th hase, &hen %!ucose is de!eted. his re-ects the need #or theor%anism, &hen #ree !iin%, to reduce cometition &hen #ood sources arede!eted.*enicillin is produced in a batch ermenter. #t ta%es roughl! 2D hours or penicillinproduction to begin. *enicillin is secreted b! the ungus and accumulates in themedium.*enicillin is a ($'5:E =$T5B&#T$. This means that it is produced b! theungus 5FT$: the exponential phase is complete, when glucose is depleted.5ntibiotic production is a ($'5:E =$T5B&#(=. This means that the antibiotic

is produced at a period in the lie o the ungus when there is a change 5<5E romits *T#=;= conditions. This re?ects the need or the organism, when ree living, to:$;$ =*$T#T#' when ood sources are depleted. '1. he myce!ium is remoed by $!tration o# the cu!ture -uid and theantibiotic uri$ed #rom the residua! !iuid.5ter about da!s, the culture ?uid mixture is )ltered and penicillin is extracted andpuri)ed.

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4.4 Microbiology