Antibiotics

By : Bijaya Kumar Uprety

Historical development of antibioticsPaulVuilemin(1889) wasthe first and foremostscientistwhovehemently promulgated the very concept ofactivity to introducethe terminology(or antibiotic influences)in order to describethe prevailingnegativeinteractionsamongstthe animalsandplants.

Later on, Walksman (1940s) eventually coined the termand alsointroduceda plausibledefinition as

chemicalsubstancederived from microorganismswhich hasthe capacityof inhibiting growth, and evendestroying,othermicroorganismsin dilute .

Another school of thought advocatesthat the natural product antibioticsessentially comprise of a specific category of chemical entities invariablytermedasthe secondarymetabolites.

Besides, on a rather broader perspective such substances may becharacterizedfor possessingchemicalstructureswhich are found to be quiteunusualwhencomparedwith thoseof the intermediarymetabolites.

Nevertheless,such natural product antibiotics, are being generatedat anextremelylow ebb (below the usual condition or standard) specificgrowthrates,and alsosupportedby the fact that theseare not absolutelyessentialfor the growth of the in a pureculturemedium.

In fact, the are observedto be of highly critical nature withrespect to the producing organisms in their usual natural environmentbecausetheir presenceis an absolutemust not only for the survivalbut alsofor the competitiveadvantage.

TerminologiesSecondary metabolitesareorganic compoundsthat are not directly involved in the normalgrowth,development, orreproductionof an organism.Unlikeprimary metabolites, absence of secondary metabolities does not result in immediate death, but rather in long-term impairment of the organism'ssurvivability,fecundity, or aesthetics, or perhaps in no significant change at all. Secondary metabolites are often restricted to a narrow set of species within aphylogeneticgroup.Secondary metabolites often play an important role in plant defense against herbivoryand other interspecies defenses. Humans use secondary metabolites as medicines, flavorings, and recreational drugs. For example : Small "small molecules"

Alkaloids(usually a small, heavily derivatized amino acid):

Hyoscyamine, present inDatura stramonium

Atropine, present inAtropa belladonna, Deadly nightshade

Cocaine, present inErythroxylon cocathe Cocaplant.

Terpenoids(come fromsemiterpene oligomerization):Azadirachtin (Neemtree)

Artemisinin, present inArtemisia annuaChinese wormwood tetrahydrocannabinol, present in Cannabis

Steroids(Terpeneswith a particularring structure)

Saponins(plantsteroids,often glycosylated)

Glycosides(heavilymodifiedsugarmolecules):

Nojirimycin

Glucosinolates

Naturalphenols:

Resveratrol, etc.

Big "small molecules", produced by large, modular, "molecular factories"

Polyketides:

Erythromycin

Discodermolide

Fatty acidsynthaseproducts:

FR-900848

U-106305

phloroglucinols

Nonribosomalpeptides:

Vancomycin

A primary metabolite is a kind of metabolite that is directlyinvolvedin normalgrowth,development,andreproduction.

Alcohol is an exampleof a primary metabolite producedinlarge-scaleby industrialmicrobiology.

Someantibioticsuseprimarymetabolitesasprecursors,suchas actinomycin which is created from the primarymetabolite,tryptophan.

Examples of primary metabolites produced by industrial microbiology:

Class Example

Alcohol Ethanol

Amino acids Glutamic acid, aspartic acid

Nucleotides 5' guanylic acid, etc

However, the most widely accepteddefinition of anpromulgated by the scientific jargonsis chemicalsubstanceproduced by a microorgansims, that has the capacity, in lowconcentration,to inhibit or kill, selectively,other .

Importantly,the aforesaiddefinition laysparticularemphasison theterminologieslike or that explicitelysuggeststhat the substanceeither checksthe growth of pathogensor exertsa bactericidalactionon the microbeswithout displayingasimilaractionon the hostorganismsi.e., the humans.

Interestingly,We can observe from the above cited definition(s)that it excludesthe large number of non-medicinal compoundsessentiallyhavingthe puresyntheticgenesis(origin).

In reality and actual practice, theseare virtually treatedat par with the hostof

natural compounds together with their respectivederivativesunderthe terminology thatcould be further sub-divided predominantly into twocategories namely : antifungals and antibacterialsdepending on the specific type(s) of microbeundergoinginhibition.

Therefore,in order to circumventthe practicalaspects,both the terminologies, viz., and

may be used effectively andinterchangeablyirrespectiveof the specificsourceofthe chemicalentity.

In general,the are producedon a largescale by three well-known and definedmethodologies,suchas : (a) fermentation process;(b) semi-syntheticprocess; and(c)syntheticprocess.

A tremendousquantumleapandqualifiedsuccessfuldiversificationin the specificfield ofhashelpedthe first two processes(i.e., and inaccomplishinganenormousenhancementin the rateof production as well as improved upon their yieldandpurity.

Antibiotic Development:Thelatest progressivetrend in the logisticaspectsof antibiotic developmentmaybe

observedvividly by the under mentionedsequenceof goalsand objectives,suchas:

To screen and evaluate different types of sources of microorganismsfor thedetectionof purposefulantagonism.

To identify and select modified versionsof microbial mutants, establishoptimalenvironmentaland nutritional conditions,and to developsuitabletechnique(s)forthe recoveryof antibiotics from cultures,

Toinducethe productionof particulardesiredmetabolites,

To improve upon and modify the fermentative metaboliteseither by the aid ofbiological and chemical manipulations to accomplishmore useful antibioticsubstances,

To developan elaboratedmethodsfor the of antibiotics from abinitio for a feasibleeconomicadvantage,and

Tomakeuseof an to distinctlyenhancethe impact or availability ofan antibiotic.

Antibiotics

Theseare naturally occurringantimicrobialagentsproducedbymicroorganismsthat in smallquantitiesinhibit the growth or killunrelatedspeciesof microorganisms.

Sourcesof antibiotics

Main sources of antibiotics are bacteria, fungi andactinomycetes. Depending on the sources antibiotics areclassifiedas those obtained from fungi, those obtained frombacteria, those synthesized in the laboratory and thoseproducedby the microorganismsandmodifiedin the laboratory.

From Fungi Cephalosporinsfrom Cephalosporium, Pencillinfrom Pencillium.

FromBacteria Bacitracinfrom Bacillussubtilis,Chorophenicolfrom Streptomycesvenezuela.

Synthetic Etambutol, Nitrafurans

Semi-synthetic Rifampin from Rifampicin (Streptomyces mediterranei), Amikacin (from Kanamycin).

Classification of Antibiotics

Classification of Antibiotics is based on different criteria:

a) Based on their spectrum of activity:-

Group-1: Active against Gram-positive bacteria and gram-negative cocci

Ex: Pencillins, Erythromycin

Group-2: Mainly active against Gram-negative bacilli.

a) For systemic infection: - Aminoglycosides, Polymyxins.

b) For urinary tract infections:- Nitrofurantoin, Nalidixic acid

Group-3: Broad spectrum antibiotics

Ex: Sulphanamides, Tetracycline

Group-4: Specific antibacterials

a) Active against anaerobic organisms.

Ex: metronidazole, lincomycin

b) For tuberculosis

Ex: Streptomycin, Isoniazid

c) for Chlamydia, Rickettsia, Mycoplasma infections

Ex: Erythromycin, Tetracyline

Group-5: Antifungal, antiviral agents

a) Antifungal agents: Polyenes, Nystatin

b) Antiviral: Idoxuridine, Amantadine

c) Antimalignancy antibiotics: Actinomycin, Mitomycin

b) Based on the structure:Aminoglycosides, Macrolides, Tetracyclines, Polyenes, Nitrofurans.

c) Based on their source:

Natural, Synthetic and Semi-synthetic

Based on whether they are cidal or static:

Ex: Bacteriocidal: Penicillin, Aminoglycosides

Bacteriostatic: Sulphanamides, Tetracyclines.

d) Based on their mechanism of actions:

1) Inhibitors of cell wall synthesis.

2) Inhibitors of cell membrane.

3) Inhibitors of protein synthesis.

4) Inhibitors of nucleic acid synthesis.

ANTIMICROBIAL SPECTRUM AND METHODS USED FOR THEIRSTANDARDIZATION

Microbiology, in particular clinical medical microbiology, is a scientific discipline chiefly concerned with the isolation and subsequent identification of causative disease-producing microorganisms (or pathogens) : bacteria, fungi (including yeast), viruses, rickettsia, and parasites.

In general, there are well-defined specific as well as non-specific techniques available with regard to the as stated under :

e.g., mouse, embryonated egg, tissue culture and the like,

.

Specific Tests to identify pathogens

There are three specific test that may be used to identify the pathogens, namely :

(a) Enzymatic and Immunological Tests : The introduction of rapid manual enzymatic and immunological test kits have enormously enabled to identify the presence of the cerebrospinal fluid (CSF) analysis.

(b) CoagglutinationTests : In this specific tests, the particular antibody is bound to protein A on the surface of a staphylococcal cell, and the very presence of antigen causes agglutination, and

(c) Latex-Agglutination Tests : In this particular tests a specific antibody gets coated onto the latex particles and when an antigen is present, the latex particles are visible distinctly.

Types of antibiotic spectrum

An antibiotic's spectrum can be broad or narrow.

Narrow spectrum antibioticsact against a limited group of bacteria, either gram positive or gram negative, for example sodium fusidate only acts against staphylococcal bacteria.

Broadspectrum antibioticsact againstgrampositiveand gramnegativebacteria, for example amoxicillin, ampicillin, tetracycline,chloramphenicol,etc. Broadspectrumantibioticsareproperlyusedin thefollowing medicalsituations: empirically(i.e., basedon the experienceofthe practitioner), prior to the formal identification of the causativebacteria and when there is a wide range of possible illnessesand apotentiallyseriousillnesswould result if treatment isdelayed.

Broadspectrumantibioticsare alsousedfor drug resistantbacteriathatdo not respond to other, more narrow spectrumantibiotics and in thecaseof superinfections, wherethere aremultiple typesof bacteriacausingillness,thus warrantingeither a broad-spectrumantibiotic or combinationantibiotic therapy.

https://www.boundless.com/microbiology/definition/superinfection/

Antimicrobial Susceptibility Tests : It may be defined asdetermination of the least amount of an antimicrobial chemotherapeuticagentthat will inhibit the growth of a microorganismin vitro, using a tube-dilutionmethod,agar-cupmethod,or disk-diffusion .

OR

Antibiotic SensitivityMethods

Antibiotic sensitivitytests are done to determine the degreeof sensitivityor resistanceof the pathogenisolatedfrom the patient to an appropriaterangeof antimicrobialdrugs. It is best studied as 1) antibiotic sensitivityfor fast growing facultatively anaerobic organisms, 2) for anaerobicorganisms3) for M. tuberculosis.

Theantibioticsensitivitytest in vitro isdoneby:

1) Dilutiontestsand2) Diffusiontests.

Dilutiontest maybeagardilution test or broth dilution test

Dilution test

Various dilutions of antibiotic are prepared in agar or broth and themicroorganismis inoculatedandincubatedat 37Cfor 18-24hours.

Theminimumconcentrationof the antibiotic requiredto inhibit the visiblegrowth isMIC(minimuminhibitory concentration)of the antibiotic.

Theminimumamountof the antibiotic requiredto kill the microorganismis the MBC(minimumbactericidalconcentration)of the antibiotic. MBCisobtained by sub culturing the organisms from tubes containing theantibiotic and no visible growth onto an antibiotic free medium anddeterminingthe numberof survivors.

Advantagesof dilution methods:

1) Todeterminethe MICandMBCof anantibiotic.

2) Theypermit a quantitative result to be reported, indicatingthe amountofa givendrugnecessaryto kill or inhibit the microorganism.

Disadvantages: Laboriousandtime consuming,costly.

http://www.medschool.lsuhsc.edu/micro

biology/Flash/MICMBC.htm

http://www.medschool.lsuhsc.edu/microbiology/Flash/MICMBC.htm

Diffusion Test-PrincipleAntibiotics are applied to the inoculatedsurfaceof test platesinto holesor ditchescut in the medium or to hollow cylinders(Heatleycups)placed on the medium or in the form of filterpaperdiscs.

The antibiotic absorbswater and diffusesacrossthe medium.Thereis a graduallychanginggradientof drug concentrationinthe agarsurroundingeachdisc.

Theorganismthat isnot inhibited by the antibioticwill growandform a layerof growth, whereasthoseinhibited will fail to growandform a zoneof inhibition aroundthe disc.

The diameters of the zone of inhibition are measured anddependingon the methodused,it is classifiedasbeingresistant,intermediateor sensitive.

Severalforms of discdiffusion testshavebeenadvocatedwhichvaryin their methodsof standardization,readingandcontrol.

Diskdiffusion test. A standardizedinoculum of Streptococcuspneumoniaeis swabbedonto thesurface of Mueller-Hinton agarwith 5%sheepblood. Filter paperdisks impregnated withantimicrobialagentsareplacedonthe agar and after overnightincubation, the diameter of thezone of inhibition is measuredaround each disk.In "strain 11" good susceptibilityto all tested antibiotics (widezonesof growth inhibition arounddisks)can be seen. "Strain 23" isresistantto penicillin (diskon thetop) and sulfonamides(growth tothe disks).

information

Mller-Hinton agar isan microbiologicalgrowth mediumthat iscommonly used for antibioticsusceptibility testing. It is also used toisolate and maintain NeisseriaandMoraxellaspecies.

It typicallycontains(w/v)

30.0%beef infusion

1.75%caseinhydrolysate

0.15%starch

1.7%agar

pHadjustedto neutralat 25 C.

Five percent sheep blood may also beaddedwhen susceptibilitytesting is doneon Streptococcusspecies. Thistype is alsocommonly used for susceptibility testingof Campylobacter.

http://en.wikipedia.org/wiki/Percentage_solution

Laboratory Diagnosis of Viral Infections :In actual practice, the laboratory diagnosis of several viralinfections is exclusivelybasedupon the following five cardinalfactors,namely:

1. Examinationof the infectedtissuesfor actualpathognomonicchangesor for the presenceof viral material.

2. Isolationandidentificationof the viralagent.

3. Demonstration of an appreciable enhancement in thetiter to a given virus in the span of the

illness.

4. Detectionof viral antigenspresent in lesionsby employingfluorescein-labeledantibodies.

5. Electron microscopic examination of either the tissueextractsor the vesicularfluids.

Serological Tests :It isa commonpracticeto use for carryingout the serologicaltests,but quite rarelyfor virusisolation.

However, it is absolutely important and vital that both acute andconvalescent-phaseblood specimensshould be examinedthoroughly inparallel to estimate precisely whether have appeared,loweredor enhancedin the in the spanof the disease.

enumerated under :

e.g. Adenovirus group)

e.g., Polio and Coxsackie viruses of the picornavirus group)

Clinical Parasitology :It is indeeda well-definedsciencethat isexclusivelyconcernedwiththe parasitic protozoa (amoeba), the helminths (cestodes,tapeworms; trematodes,flukes; nematodes,roundworms),and thearthopods.

Identification of ProtozoanOva : It is based upon the detailedmicroscopicmorphologicalstudies(includingnuclei)by makinguseof wet mounts(e.g., salineor iodine)or stainedpreparations(e.g.,iron, hematoxylin) obtained from fecal specimens (fresh orpreserved with polyvinyl alcohol) that have been adequatelyconcentrated by sedimentation, centrifugation, or floatationtechniques.

Example: AmebicDysentry: Specifically,in the fecalspecimensthepresenceof trophozoite and/or cystic stagescould be detectedalong with intestinal protozoa,as in the caseof amebicdysentryusuallycausedby Entamoebahistolytica.

Serodiagnosisof ParasiticDiaseases: Serodiagnosisessentiallyconcernswith the diagnosisby observingthe reactions of blood serum. Importantly, theserodiagnosisof parasitic diseases includes thefollowingcritical namely:

Immunodiagnositic Tests : Complement-fixation(trichinosis); precipitin test (schistosomiasis);

bentoniteflocculation(ascariasis); hemagglutination(echinococcosis); latexagglutination(trichinosis)

; cholesterol flocculation (schistosomiasis) ;fluorescentantibody (malaria) ; and methylene-bluedyetest (toxoplasmosis).

Methods Used for Standardization of Antibiotics

Official compendia invariably make use of the terminologythat essentially designates acontaining an appreciable quantum of a

chemicalentity which is causedto produce naturally by amicroorganismor by a semi-synthetic route artificially, andthat possesses the inherent ability to either destroy(bactericidal effect) or inhibit (bacteriostatic effect)microorganismsin relatively dilute solution.

Followingaresomeof the standardizationcertification of variousin a chronologicalorder :

YearEvent

1938 : FederalFood,Drugand CosmeticAct Introduction in stagesof thecertificationof meantfor humanor verternaryapplications.

1945: Penicillin

1948: Streptomycin

1949: Aureomycin,BacitracinandChloramphenicol.

1962 : Kefauver-HarrisAmendments as part of these amendmentsit wasmandatoryfor the of all antibiotics intended for humanuse.

1982 : Federal Drug Authority (FDA)-USAissued regulations which totallyexemptedthe from the batch certification requirementsso longas the articlescompliedwith standards; however,section507(i.e., related tocertification of Antibiotics) remainsintact andhenceapplicable.

Federal Register (USA): It essentially incorporates the Standards ofPotencyand Purity for Antibiotics asestablishedand determinedby theFDAin the form of regulationspublishedfrom time to time.

Thefollowing three important pointsmaybe takeninto considerationwithregardto the i.e., standardizationof Antibiotics,suchas:

(1) FDA-regulationsgoverningall aspectsof antibioticstestingare extremelydetailedandaresubjectto periodicamendment,

(2) FDA-regulationsneed to be referred to with regard to thefor the assayof individual antibiotics and their preparations,

and

(3) While evaluatingthe potencyof the actual andapparent measuredeffect is the of of the growth of asuitable strain of microorganismsi.e., the ultimate prevention of themultiplicationof the .

The proceduresusuallyemployedin the assayofmaybe categorizedunder two heads,namely: (a)

Cylinder-PlateMethod ; and (b) TurbidimetricMethod, whichshallnowbe treatedbriefly asunder:

1. Cylinder-PlateMethod

Thecylinder-plate method of assayof antibiotics potency issolelybasedupon the measurementof the specificdiameterof of microbial growth immediatelysurroundingcylinderscontainingvariousdilutionsof the

i.e., the substanceunder investigation, that arecarefully placed on the surface of a solid nutrient agarmedium previously inoculated with the of anappropriate organism. In actual practice the zone ofinhibition caused by the test compound is meticulouslycomparedwith that producedby a known concentrationof apure .

2. Turbidimetric MethodTheturbidimetric method of assayofantibiotics potency is exclusivelybased upon the inhibition ofmicrobialgrowth as indicatedby thecorrespondingmeasurementof theturbidity (i.e., transmittance) ofsuspensions of an appropriateorganism in a fluid medium intowhichthe gradedamounts

of the have beenaddedduly.

Consequently, the changes in thetransmittance caused by the

are duly comparedwiththose resulted by knownconcentrations of the ReferenceStandard.

SCREENING OF SOIL FOR ORGANISMS PRODUCINGANTIOBIOTICS

Soil is the best availablesourcefrom which one may obtain ultimately abroadspectrumof viable .

comprisesof variouskindsof microorganismsamongwhichmanyofthem exhibit the biosyntheticabilitieswhich areof genuineinterest.

why is soil invariablyregardedto be the idealsourcefrom whichto obtaindiversetypesof microorganisms

1. A sizablequantum,of the of the finds its normal passageeither onto or into the soil ; andultimately getsadequatelydecomposedby onemicroorganismor the other.

2. may be thought of as being of a specified kind of naturalfermentation wherein a plethora of organismsare activelyengagednot only in the actualdecompositionand resynthesisof simpleto complexorganic materials, but also in carrying out effectively the processofoxidation, reduction and other chemicalchangespertaining to inorganicmaterials.

3. It hasbeenduly demonstratedandestablishedthat more than one type, andoftenmany types,of soil microorganismsare invariablycapableof performing eachoftheseindividualchemicalor biochemicaltransformation.

4. Thougha largevolume of different types of microorganismsdo occur in the soil ;however,it is not yet so clearand evident that actuallyupto what extent of theseorganisms,as on date, been pinned down and isolated in the form of purestlaboratory culture.

5. Theavailabilityof nutrients in soil is invariably found to be relatively at low ebb ;and, therefore, the prevailingmicrobial competition for these nutrients is quiteprevalent. In case,a highlydesiredand specificnutrient is timely incorporatedtothe and the treated soil is duly incubatedthen a relatively muchappreciable larger growth response takes place amongst the ensuing soilmicroorganismsthat are capable of attacking this specific nutrient therebyrendering the isolation of these particular organisms much simpler andconvenient. In other words, one may accomplishjudiciouslythe in

for specificmicroorganismsof our interest.

(7). In the same vein, the resulting soil may beadequately incubated in a particular liquidlaboratory culture media so as to causeenrichmentfor specific organisms before an isolation iscommenced.

ScreeningScreeningmay be defined as application ofhighlyselective,specificand sophisticatedsequentialproceduresto make the detection and isolation ofonly such microorganisms that are of genuineinterestout of a largemicrobial .

Variousunderlyingconceptsof screeningessentiallyinclude:

1. Segregationof ViableMicroorganisms: It shouldbe highlyeffectivein thesensethat either a few stepsor a singlestep would be able to discardamajor portion of the relatively not-so-useful microorganisms; whereas,simultaneously allowing the rapid and fast detection of the smallpercentageof viableandusefulmicroorganismswhichare usuallypresentin the population.

Example: In industrial researchprogrammesan attempt ismade from a natural microbial sourcee.g., soil is diluted toobtain a in such a fashion that whenaliquots spread, sprayed, or applied onto the surfacesofsterilizedagarplates, in an ascepticcondition, shall give riseto countable. Colonies not essentially touching theneighboringcolonies.

Detectionof Microorganismsby ColourChange: Thevarioustypes of microorganismsyielding organic acids (attributingacidic characteristics)or amines (attributing basic features)generatedfrom variouscarbonsubstratesquite often maybedetectedconvenientlyby the incorporationof a pH indicatingdye, for instance: bromothymol blue or neutral red, into aslightlybufferedagarnutrient medium.

In actualpractice,the productionof theseaforesaidis invariablyindicativeby exhibitinga definitechange

in colourof the previouslyincorporatedindicatingdye in theperipheryof the ensuingcolonyto a showingeitheran alkalineor an acidicreaction.

However, the usefulnessof this specific maybe augmentedappreciablyif a media having much higherbuffer capacityare utilized so that only suchmicroorganismswhich are responsible solely for producing significantquantum of either the or the can effectivelyinducecharacteristicchangesin the colourof the dye.

Toremember:

Agar infusedwith either acidicor basicindicator microbialinoculum applied they are then selectedbasedupon thethe chemical entity they produce eg. Acidic producecolor if the agar is infusedwith acidicindicator.

Drawback of this technique(a) It fails to give a definite indication about which amine or

organicacidhasbeenproducedactually. Hence,it shouldbeimmediately followed by further testing with the help ofcertain well-known analytical procedures e.g., paperchromatography, electrophoresisso as to determine andestablishwhether the acidicor basicproduct really is one ofinterest.

(b) Importantly, in such an event where colonies ofmicroorganismsby virtue of this initial screeningprocedure,seemto possess fermentative mustimmediately be subjected to purification ; and, therefore,subculturedsubsequentlyonto slantsof an appropriateagarmedium to be maintained adequately asduringfurther testingdevices.

(c) Sometimes, it is indeed quite discouraging todiscover a specific organism exhibitingfermentative only to observe that, viaerroneous technique, either contamination or forother reasons,the culture in questionhasbeen lostultimately.

(3) Microorganismsfor ProducingAntibiotics : In therecent past, the has beenexploited both extensively and intensively in themeticulous search for viable and specificmicroorganisms that are exclusively capable ofproducingantibiotics of interest to combat dreadlyhumandiseases.

Crowded-plate Technique :It is one of the simplest screening techniques invariablyemployedby the .

Many of the most useful antibiotics are derived fromcompounds originally isolated from microorganisms.Penicillin,as is well known,wasfirst discoveredin mold, andvariousother antibiotics were isolated from soil bacteria inthe 1950sand1960s.

In fact, this techniquehasan addedadvantagefor exclusivelylooking for microorganismswhich produce onwithout any specific consideration whatsoever about thetypes of microorganisms that may be sensitive to theantibiotic.

MethodologyVariousstepsinvolvedin the crowdedplate technique:

1. First,a sampleof organismsfrom soil or someother sourceis diluted inwater, then spread onto Petri dishes containing agar gel rich in thenutrients the bacteriawill needto grow. Scientistsselectplatesthat havea large number of colonies, then look for microorganismsthat haveinhibited the growth of other microorganismsin their vicinity. Thesemicrobesare possiblysecretingsomekind of compoundthat is killing orinhibitingtheir neighbors.

2. It isa commonpracticeto subculturesucha colony(coloniesthat maybeproducing antibiotics) further in an identical medium, and purifiedsubsequentlybe streaking, just prior to making . The

thus obtained is now almost ready for testing toestablishpreciselythe typesof microorganismsthat are sensitiveto theantibiotic under investigation, by, means of the inhibitionconcentration or the inhibition spectrum .

It's entirely possible,of course,that the colonywasreally just alteringthepH of its environment or making some other changethat killed otherbacteria,rather than secretinganantibiotic,sofurther testsareneededtoconfirm that it is indeedan antibiotic-producingstrain. Nonetheless,thecrowded plate technique was sometimes helpful in identifyingmicroorganismsthat couldserveassourcesof newantibiotics.

Advantages

The crowded plate technique is fairly simple -- indeed, the simplestmethod to find antibiotic-producingmicroorganismsin soil samples. It'salso fairly rapid, taking only a couple of days to produce results.Introducing "test organisms"can help to determine whether a specifickind of microorganism(e.g., a disease-causinggerm) is susceptibleto theantibiotic compound. If it doesindeedprove useful for this purpose,thecompoundcanbe isolatedfor further study.

DrawbacksThe crowded plate techniqueonly detects microorganismsthat producecompoundsto kill bacteriafound in their immediateenvironment. Thesecompoundscouldpotentially be toxic to humans,and they maybe lethalonly to certain types of bacteria (e.g., soil bacteria),as opposedto thebacteria that actuallycausediseasein humans. Moreover, they will onlydetect microorganismsthat start to produceantibiotic compoundswithina coupleof daysof beingculturedand incubated,so they might well missother compoundsthat couldpotentiallybeof interest.

Isolation of Pure Cultures From Mixed CulturesSeveral different methods for getting a pure culture from a mixed culture are available. These include:

Pour plate

Streak plate

Isolation using streak plate method

Steps for streak plate method1. Sterilize the wire loop in a flame.

2. Cool the hot loop by touching the loop wire into the sterilized agar.

3. Dip the loop into the sample (inoculum) and spread it froth and back across the agar surface.

4.by sterilizing (introducing) the loop in the flame.

5. After cooling the sterilized loop, the loop is dragged through the previous path,

plate.

6. After sterilizing and cooling the loop, the process is repeated once again. With each new path, the loop picks up a smaller number of bacteria, and therefore can spread them farther and farther apart.

7.found in the last streak represent the isolated strains.

Pour plate method

Secondary screeningPrimaryscreening(or preliminaryscreening)solelyenablesnot only the

but also the of such viable microorganismsthatessentially possesspotentially interesting and commercially feasibleapplications. Nevertheless,this screening is invariably followed by asecondaryscreeningso as to ascertainmore useful information abouttheseorganisms,besidestheir actualinherentcapabilities.

Primaryscreeningestablishesexclusivelythe capabilityof microorganismsthat are responsiblefor producing a compound without giving enoughidea either with respect to the yield or production potential for theorganisms.

Onthe contrary,the secondaryscreeningcategoricallyenablesthe furtherof thosespecificmicroorganismsthat essentiallypossessthe

inherent for feasible and gainful industrial processes; anddistinctlyeliminatingthosedevoidof sucha potential.

Thevariousstepsinvolvedareasfollows:

(1) Secondaryscreeningis usuallycarriedout onagarplatesaseptically.

(2) It may also be conductedin flasksor smallfermentors containing liquid media, or as acombinationof suchavailableprocedures.

(3) However,one may use as analternative to in the

method. Followingare someof theimportant merits and demerits of these twotechniquesstatedbriefly :