Culture Media, Isolation,Presesrvation

7/24/2019 Culture Media, Isolation,Presesrvation

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REVIEWER

Types of Bacteriological Culture Medium

Types of Bacteriological Culture Medium

Culture media are solutions containing all of the nutrients and necessary physicalgrowth parameters necessary for microbial growth. All microorganisms cannot growin a single culture medium and, in fact, many can't grow in any known culturemedium. Organisms that cannot grow in artificial culture medium are known asobligate parasites. Mycobacterium leprae, rickettsias, Chlamydias, and Treponemapallidum are obligate parasites.

Bacterial culture media can be distinguished on the basis of composition,consistency and purpose.

Classification based on consistency

. !olid mediumsolid medium is media containing agar "at a concentration of .#$%.&( orsome other, mostly inert solidifying agent. !olid medium has physicalstructure and this allows bacteria to grow in physically informati)e or useful

ways "e.g. as colonies or in streaks(.solid medium is useful for isolatingbacteria or for determining the characteristics of colonies.

%. !emisolid mediaThey are prepared with agar at concentrations of &.# or less. They ha)esoft custard like consistency and are useful for the culti)ation ofmicroaerophilic bacteria or for determination of bacterial motility.

*. +iuid "Broth( medium

These media contains specific amounts of nutrients but dont ha)e trace of

gelling agents such as gelatin or agar. Broth medium ser)es )arious

purposes such as propagation of large number of organisms, fermentationstudies, and )arious other tests. eg. !ugar fermentation tests, M-$- broth.

Classification based on the basis of composition

. !ynthetic or chemically defined mediumA chemically defined medium is one prepared from purified ingredients andtherefore whose e/act composition is known.

%. 0on synthetic or chemically undefined medium


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0on$synthetic medium contains at least one component that is neither purified norcompletely characteri1ed nor e)en completely consistent from batch to batch. Oftenthese are partially digested proteins from )arious organism sources. 0utrient broth,for e/ample, is deri)ed from cultures of yeasts. !ynthetic medium may be simple orcomple/ depending up on the supplement incorporated in it. A simple non$synthetic

medium is capable of meeting the nutrient reuirements of organisms reuiringrelati)ely few growth factors where as comple/ non$synthetic medium support thegrowth of more fastidious microorganisms.

Classification based on the basis of purpose/ functional use/ applicationmany special purpose media are needed to facilitate recognition, enumeration, andisolation of certain types of bacteria. To meet these needs, numerous media area)ailable.

On the basis of their application or function, they are classified as follows,

1. General purpose media/ Basic mediaBasal media are basically simple media that supports most non$fastidious bacteria.2eptone water, nutrient broth and nutrient agar considered basal medium. Thesemedia are generally used for the primary isolation of microorganisms.


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2. Enriched medium (dded !ro"th factors#$

Addition of e/tra nutrients in the form of blood, serum, egg yolk etc, to basalmedium makes them enriched media. 3nriched media are used to grow nutritionally

e/acting "fastidious( bacteria. Blood agar, chocolate agar, +oefflers serum slope etc

are few of the enriched media. Blood agar is preparing by adding #$& "by)olume( to a basal medium such as nutrient agar or other blood agar bases.Chocolate agar is also known as heated blood agar or lysed blood agar.

%. &electi'e and enrichment mediaare designed to inhibit unwanted commensalor contaminating bacteria and help to reco)er pathogen from a mi/ture of bacteria.4hile selecti)e media are agar based, enrichment media are liuid in consistency.

Both these media ser)e the same purpose. Any agar media can be made selecti)eby addition of certain inhibitory agents that dont affect the pathogen. arious

approaches to make a medium selecti)e include addition of antibiotics, dyes,chemicals, alteration of p5 or a combination of these.

a. &electi'e medium2rinciple6 7ifferential growth suppression!electi)e medium is designed to suppress the growth of some microorganismswhile allowing the growth of others "i.e., they select for certain microbes(.!olidmedium is employed with selecti)e medium so that indi)idual colonies may be

isolated.


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3/amples of selecti)e media include6Thayer Martin Agar used to reco)er 0.gonorrhoeae contains ancomycin, Colistinand 0ystatin.

Mannitol !alt Agar and !alt Milk Agar used to reco)er !.aureus contain & 0aCl.

2otassium tellurite medium used to reco)er C.diphtheriae contains &.&82otassium tellurite.

McConkeys Agar used for 3nterobacteriaceae members contains Bile salt that

inhibits most gram positi)e bacteria.

2seudosel Agar "Cetrimide Agar( used to reco)er 2.aeruginosa contains cetrimide"antiseptic agent(.Crystal iolet Blood Agar used to reco)er !.pyogenes contains &.&&&% crystal)iolet.+owenstein 9ensen Medium used to reco)er M.tuberculosis is made selecti)e byincorporating Malachite green.

4ilson and Blairs Agar for reco)ering !.typhi is rendered selecti)e by the addition

of dye Brilliant green.

!electi)e media such as TCB! Agar used for isolating . cholerae from fecalspecimens ha)e ele)ated p5 ":.#$#.;(, which inhibits most other bacteria.

b. Enrichment culture/ edium3nrichment medium is used to increase the relati)e concentration of certainmicroorganisms in the culture prior to plating on solid selecti)e medium.


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Certain media are designed in such a way that different bacteria can be recogni1edon the basis of their colony colour. arious approaches include incorporation ofdyes, metabolic substrates etc, so that those bacteria that utili1e them appear asdifferently coloured colonies. !uch media are called differential media or indicatormedia 7ifferential media allow the growth of more than one microorganism of

interest but with morphologically distinguishable colonies.3/amples of differential media include6

Mannitol salts agar "mannitol fermentation > yellow(

Blood agar ")arious kinds of hemolysis i.e.,andhemolysis(

Mac Conkey agar "lactose fermenters, 2ink colonies whereas non$ lactosefermenter produces pale or colorless colonies.TCB! "ibrio cholera produces yellow colonies due to fermentation of sucrose(

+. ,ransport media$Clinical specimens must be transported to the laboratory immediately aftercollection to pre)ent o)ergrowth of contaminating organisms or commensals. Thiscan be achie)ed by using transport media. !uch media pre)ent drying "desiccation(of specimen, maintain the pathogen to commensal ratio and inhibit o)ergrowth of

unwanted bacteria. !ome of these media "!tuarts ? Amies( are semi$solid in

consistency. Addition of charcoal ser)es to neutrali1e inhibitory factors. Cary Blairmedium and enkatraman -amakrishnan "-( medium are used to transport feces

from suspected cholera patients. !achs buffered glycerol saline is used to transport


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feces from patients suspected to be suffering from bacillary dysentery. 2ikes

medium is used to transport streptococci from throat specimens.

-. naerobic media$Anaerobic bacteria need special media for growth because they need low o/ygen

content, reduced o/idationreduction potential and e/tra nutrients.

Media for anaerobes may ha)e to be supplemented with nutrients like hemin and)itamin @. !uch media may also ha)e to be reduced by physical or chemicalmeans. Boiling the medium ser)es to e/pel any dissol)ed o/ygen. Addition of glucose, &. thioglycollate, &. ascorbic acid, &. cysteine or red hot ironfilings can render a medium reduced. Before use the medium must be boiled inwater bath to e/pel any dissol)ed o/ygen and then sealed with sterile liuidparaffin.

-obertson cooked meat that is commonly used to grow Clostridium spps mediumcontain a %.# cm column of bullock heart meat and # ml of nutrient broth.Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast e/tractand casein hydrolysate.

Methylene blue or resa1urin is an o/idation$reduction potential indicator that isincorporated in the medium.


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Other types of medium includes Media for 3numeration of Bacteria, Media forcharacteri1ation of Bacteria, Maintenance media etc.

Techniues of isolation and 3numeration ofBacteria

3numeration of microorganisms is especially important in dairy microbiology, foodmicrobiology, and water microbiology.

*irect icroscopic count/ ,otal cell countdirect microscopic counts are possible using special slides known as counting

chambers,consisting of a ruled slide and a co)er slip. t is constructed in such a

manner that the co)er slip, slide, and ruled lines delimit a known )olume. Thenumber of bacteria in a small known )olume is directly counted microscopically andthe number of bacteria in the larger original sample is determined by e/trapolation.7ead cells cannot be distinguished from li)ing ones. Only dense suspensions canbe counted.

Bacteria can be counted easily and accurately with the petroff$5ausser countingchamber. This is a special slide accurately ruled into suares that are 8&& mm%inarea a glass co)er slip rests #& mm abo)e the slide, so that the )olume o)er asuare is %&,&&& mm*i.e.


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%&, &&&, &&& cm*. f for e/ample, an a)erage of fi)e bacteria is present in eachruled suare, there is # / %&,&&&,&&& or &:, bacteria per milliliter.A suspension ofunstained bacteria can be counted in the chamber, using a phase$contrastmicroscope.

,he formula used for the direct microscopic count is$

The number of bacteria per cc > The a)erage numbers of bacteria per large double$lined suare D The dilution factors of the large suare ",%#&,&&&( D The dilutionfactor of any dilutions made prior to placing the sample in the counting chamber,e.g., mi/ing the bacteria with dye

d'anta!e of *irect icroscopic count

. -apid, !imple and easy method reuiring minimum euipment.%. Morphology of the bacteria can be obser)ed as they counted.

*. ery dense suspensions can be counted if they are diluted appropriately.

0imitations of *irect icroscopic count

. 7ead cells are not distinguished from li)ing cells.

%. !mall cells are difficult to see under the microscope, and some cells areprobably missed.

*. 2recision is difficult to achie)e

8. A phase contrast microscope is reuired when the sample is not stained.

#. The method is not usually suitable for cell suspensions of low density i.e. E&F Cells per ml, but samples can be concentrated by centrifugation orfiltration to increase sensiti)ity.

Viable count/ plate count techniue

*ilution$


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with both the spread plate and pour plate methods, it is important that the numberof colonies de)eloping on the plates not be too large because on crowded platessome cells may not form colonies and some colonies may fuse, leading toerroneous measurements. t is also essential that the number of colonies not be toosmall, or the statistical significance of the calculated count will be low. The usual

practice, which is the most )alid statistically, is to count colonies only on plates thatha)e between *& and *&& colonies. The number of bacteria in a gi)en sample maybe usually too great to be counted directly. To obtain the appropriate colonynumber, the sample be counted must almost always diluted in such a manner

thatsingle isolated bacteria form )isible isolated colonies, the number of colonies

can be used as a measure of the number of )iable "li)ing( cells in that knowndilution. !e)eral &$fold dilutions of the sample are commonly used. n most cases,serial dilutions are employed to reach the final desired dilution.

5owe)er, if the organism normally forms multiple cell arrangements, such aschains, the colony$forming unit may consist of a chain of bacteria rather than asingle bacterium. n addition, some of the bacteria may be clumped together. Thede)elopment of one colony can occur e)en when the cells are in aggregates. i.e.cocci in clusters "staphylococci(, chains "streptococci(, or pairs "diplococci(, theresulting counts will be lower than the number of indi)idual cells. 3ach colony that

can be counted is called acolony forming unit "C= #&,&&&,&&& C=


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plate count, or colony count. This method is used to enumerate bacteria in milk,water, foods, soils cultures etc and the number of bacteria are e/pressed ascolony$forming units "C=


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The cultures are inoculated into melted agar that has been cooled to 8#oC. The

liuid medium is well mi/ed then poured into a petri dish"or)ice )ersa) Colonies

form within the agar matri/ rather than on top as they do when streaking a plate.

2our plates are useful for uantifying microorganisms that grow insolid medium.

Because the Hpour plateH embeds colonies in agar it can supply a sufficientlyo/ygen deficient en)ironment that it can allow the growth and uantification ofmicroaerophiles.

&pread 3late ,echniue

!pread plate techniueis an additional method of uantifying microorganisms

onsolid medium. 4ith the spread plate method, a )olume of an appropriately

diluted culture usually no greater than &. ml is spread o)er the surface of an agarplate using a sterile glass spreader. The plate is than incubated until the coloniesappear, and the number of colonies counted. nstead of embedding microorganisms

into agar, as is done with the pour plate method, liuid cultures arespreadon the

agar surface.
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An ad)antage of spreading a plate o)er the pour plate method is thatcultures are

ne)er e/posed to 8#oC "i.e. melted agar temperatures(.0ote6 !urface of the plate must be dry, so that the liuid that is spread soaks in.)olume greater than &.ml are rarely used because the e/cess liuid does not soakin and may cause the colonies to coalesce as they from, making them difficult to

count.

&trea5 3late ,echniue=or organisms that grow well on agar plate, streak plate is the method of choice for

obtaining pure culture.The key principles of this method is that, by streaking, a dilution gradient "numberof cells decrease as they mo)e across the agar and away from the point ofinoculation( is established across the face of the plate as bacterial cells aredeposited on the agar surface. Because of this dilution gradient, confluent growthoccurs on part of the plate where the bacterial cells are not sufficiently separated inother regions of the plate where few bacteria are deposited separate macroscopiccolonies de)elop that can easily be seen with naked eye. 3ach well isolated colonyis assumed to arise from a single bacterium and therefore to represent a clone of apure culture.

3urpose of &trea5 3late ,echniue$The purpose of the streak plate is to obtain

isolated colonies from an inoculum by creating areas of increasing dilution on a

single plate.solated colonies represent a clone of cells, being deri)ed from a

single precursor cell.


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Many different streaking patterns can be used to separate indi)idual bacterial cells

on the agar surface.

ethods of ma5in! a strea5 plate to obtain pure cultures.. +oop is sterili1ed, and then a loopful of inoculums is remo)ed from tube

%. A loopful of bacterial cells is streaked across the surface of a sterile solidified

nutrient medium.

*. =ollowing the initial streak, subseuent streaks are made at angles to it, theloop being sterili1ed between streaks.

8. The plates are than incubated under fa)orable conditions to permit growth ofthe bacteria.

#. After incubation coloniesappear along the points of the streak. t is from suchwell isolated colonies that pure cultures can usually be obtained.


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Maintenance and 2reser)ation of 2ure Culturesof Bacteria

Once a microorganism has been isolated and grown in pure culture, it

becomes necessary to maintain the viability and purity of the

microorganism by keeping the pure cultures free from contamination.

Normally in laboratories, the pure cultures are transferred periodically onto

or into a fresh medium (sub culturing) to allow continuous growth and

viability of microorganisms. The transfer is always subject to aseptic

conditions to avoid contamination.

ince repeated sub culturing is time consuming, it becomes difficult tomaintain a large number of pure cultures successfully for a long time. !n

addition, there is a risk of genetic changes as well as contamination.

Therefore, it is now being replaced by some modern methods that do not

need fre"uent sub culturing. These methods include refrigeration, paraffin

method, cryopreservation, and lyophili#ation (free#e drying).

3eriodic ,ransfer to 6resh edia

!trains can be maintained by periodically preparing a fresh culturefrom the pre)ious stock culture. The culture medium, the storagetemperature, and the time inter)al at which the transfers are made)ary with the species and must be ascertained beforehand. Thetemperature and the type of medium chosen should support a slowrather than a rapid rate of growth so that the time inter)al betweentransfers can be as long as possible. Many of the more commonheterotrophs remain )iable for se)eral weeks or months on a

medium like nutrient agar. The transfer method has thedisad)antage of failing to pre)ent changes in the characteristics of astrain due to the de)elopment of )ariants and mutants.

Refri!eration

$ure cultures can be successfully stored at %&' either in refrigerators or

in cold&rooms. This method is applied for short duration (&* weeks for

bacteria and *&' months for fungi) because the metabolic activities of themicroorganisms are greatly slowed down but not stopped. Thus their


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growth continues slowly, nutrients are utili#ed and waste products released

in medium. This results in, finally, the death of the microbes after

sometime.

3araffin ethod/ preser'ation b o'erlain! cultures "ithmineral oilThis is a simple and most economical method of maintaining pure cultures

of bacteria and fungi. !n this method, sterile li"uid paraffin in poured over

the slant (slope) of culture and stored upright at room temperature. The

layer of paraffin ensures anaerobic conditions and prevents dehydration of

the medium. This condition helps microorganisms or pure culture to

remain in a dormant state and, therefore, the culture can be preserved form

months to years (varies with species). The advantage of this method is thatwe can remove some of the growth under the oil with a transfer needle,

inoculate a fresh medium, and still preserve the original culture. The

simplicity of the method makes it attractive, but changes in the

characteristics of a strain can still occur.

Cropreser'ation

ryopreservation (i.e., free#ing in li"uid nitrogen at &+- or in the gasphase above the li"uid nitrogen at &+%) helps survival of pure cultures

for long storage times.

!n this method, the microorganisms of culture are rapidly fro#en in li"uidnitrogen at &+- in the presence of stabili#ing agents such as glycerol or


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/imethyl ulfo0ide (/1O) that prevent the cell damage due to

formation of ice crystals and promote cell survival. This li"uid nitrogen

method has been successful with many species that cannot be preserved by

lyophili#ation and most species can remain viable under these conditions

for +% to *% years without undergoing change in their characteristics,however this method is e0pensive.

0ophili7ation(2ree#e&/rying)

!n this method, the culture is rapidly fro#en at a very low temperature (&

3%) and then dehydrated by vacuum. 4nder these conditions, the

microbial cells are dehydrated and their metabolic activities are stopped5 as

a result, the microbes go into dormant state and retain viability for years.6yophili#ed or free#e&dried pure cultures and then sealed and stored in the

dark at ' in refrigerators. 2ree#e&drying method is the most fre"uently

used techni"ue by culture collection centers. 1any species of bacteria

preserved by this method have remained viable and unchanged in their

characteristics for more than *% years.

Advantage of Lyophilization

7Only minimal storage space is re"uired5 hundreds of lyophili#ed cultures

can be stored in a small area


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7 small vials can be sent conveniently through the mail to other

microbiology laboratories when packaged in a special sealed mailing

containers

7 6yophili#ed cultures can be revived by opening the vials, adding li"uid

medium, and transferring the rehydrated culture to a suitable growthmedium.

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Culture Media, Isolation,Presesrvation