Microbiological Screening

7/31/2019 Microbiological Screening

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MICROBIOLOGICAL

SCREENING OFHERBAL EXTRACT


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INTRODUCTION

Antimicrobial activity of plants can be detected byobserving the growth response of various microorganismsto those plant tissues or extracts, which are placed incontract with them.

Many methods for detecting such activity are available, but

since they are not equally sensitive or even based on the,

by the method selected and the microorganisms used forthe test.

Biological evaluation in general can be carried out muchmore efficiently on water soluble, nice crystallinesubstances than on mixtures like plant extracts.


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In order to detect antimicrobial act ivity in plant

extracts, three conditions must be fulfilled:

1. The plant extract must be brought into contract with thecell wall of the microorganisms that have been selected forthe test.

2. Conditions must be adjusted so that the microorganisms

are able to grow when no antimicrobial agents are present.3. There must be some means of ud in the amount of

growth, if any, made by the test organism during the periodof time chosen for the test.

The currently available methods for antimicrobial screening fall intothree groups i.e. diffusion, dilution and bioautographic methods.

These methods are influenced by several factors such as extractionmethod, inocula volume, culture medium composition, PH andincubation temperature.


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PREPARATION OF PLANT SAMPLES

FOR ANTIMICROBIAL SCREENING: Plant extracts are usually prepared by maceration or

percolation of fresh green plants or dried powered plantmaterial with water or organic solvents.

Sometimes, a fractionation of the total extract is carried out

prior to testing in order to separate polar from non-polarcompounds and acid and neutral from basic substances.

In order to detect antimicrobial substances present in verysmall quantities in the plant extracts, testing is carried outon the extracts in the form in which they are prepared or onconcentrated extracts, obtained by evaporation of the

solvent in vacuo. This often results in the precipitation orco-precipitation of possibly active substances during thetesting procedure.


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It is advisable to extract the plants and to evaporate the

extracts at low temperature in order not to destroy anythermolabile antimicrobial agents present in the extracts.

Therefore, sterilization of the extracts by autoclaving or

other sterilization methods should be avoided as well . In

addition, sterilization by membrane filteration has its

disadvantages since many antimicrobial agents can bea sor e on e er ma er a , ren er ng e ex rac s

inactive.

The water insolubility of lipophilic samples, such as

essential oils or non polars extracts, makes it necessary

to use other solvents than water or to make aqueous

dispersions or emulsions with a surface active agents./


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Several solvents including alcohol, acetone,chloroform,

dimethylsulphoxide,dioxane, glycerol and other differentemulsifiers such as macrogol ethers, sorbitan and cellulose

derivatives etc. have been used.

In any case, solvents other than water should always be

tested simultaneously with the extracts to make sure that

they have no antibacterial properties in the test system.

It should also be pointed out that homogeneous

dispersions in water are principally only required in the

liquid dilution techniques, but not in the diffusion and agar

dilution methods.


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Nevertheless, diffusion methods are not the best choice fortesting non-polar or other samples, which are difficult todiffuse in the media, since there is no relation betweendiffusion rate and antimicrobial activity.

Also, aqueous dispersions containing high molecular weightsolubilisers ( mol. Wt. >100000) should be avoided indiffusion methods, since they cannot diffuse into 1% agar

media. Besides the polarity, the PH of the samples should also be

checked before testing , since microorganisms may not beable to grow in media which have been rendered too acidic ortoo alkaline by the sample.

In practice extracts are best adjusted to neutrality ( between

PH 6.0 & 8.0) or dissolved in buffer solutions such asphysiological tris buffer or others.


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General methods for antimicrobial screening:

The commonly used test methods can be classified by whetheror not they require sterile samples.

Sterilization by membrane filtration is excluded for aqueousdispersions or emulsions and can result in the loss ofantimicrobial activity from aqueous solutions.

Sterilization by gamma-irradiation is a very effective,inexpensive but rather time- consuming method

goo a erna ve s o prepare a samp es n an asep c waysterilized tubes and flasks, laminar flow, etc) and to useaqueous ethanol as extraction solvent for the plant material.

Since, one is never sure of having eliminated all microbes, themethods that require sterile samples ( e.g. the liquid dilution

method ) should not be carried out on plant extract, whichhave not been sterilized by filtration or irradiation.

Among the methods to be considered as valuable for thetesting of plant extracts, which cannot be sterilized byfiltration, are the hole- plate diffusion and the agar dilutiontechniques.


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Diffusion method:

In the diffusion technique a reservoir containing the plant extractto be tested is brought into contract with an inoculated medium (eg. Agar) and after incubation, the diameter of the zone around thereservoir (inhibition diameter) is measured.

In order to lower the detection limit, the inoculated system is keptat a low temperature during several hours before incubation, whichfavors diffusion over microbial growth and thus increases the

inhibition diameter This method was originally designed to moniter the amounts of

an o c su s ances n ermen a on cu ures an as a so eenused for obtaining biograms and for testing essential oils

The different types of reservoirs have been employed, includingfilter paper discs, porcelain or stainless steel cylinders placed onthe surface and holes punched in the medium.

It is not necessary to sterilize the test samples since any bacteriapresent will be confined to the reservoirs and will not therefore beable to spread and ruin the place.


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The hole plate methods, however, is the only suitablediffusion technique for testing aqueous suspensions of

plant extracts. In this method, the presence of suspended particulate

matter in the sample being tested is much less likely tointerfere with the diffusion of the antimicrobial substanceinto the agar than in the filter paper disc and the cylinderplate methods.

Precipitation of water in soluble substances in the

antimicrobial substances into the agar.

Nevertheless, in order to limit precipitation as much aspossible in the hole plate method, pre-incubation shouldbe carried out at room temperature (250C) rather than at40C.

Advantages of the diffusion methods are the small size ofthe sample used in the screening and the possibility oftesting five or six compounds per plate against a singlemicroorganism.


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In most studies inhibition zones are comparedwith those obtained for antibiotics. This is useful

in establishing the sensitivity of the testorganism, but a comparison of the antimicrobialpotency of the samples and antibiotics cannot bedrawn from this, Since a large inhibition zone maybe caused by a highly active substance present inquite small amount or by a substance ofcomparatively low activity but present in highconcentration in the plant extract.

On the other hand, the filter paper disc method isa satisfactory and acceptable method for assaying

water soluble antibiotics. The zone diameter of inhibition is indeed inversely

related to the minimum inhibitory concentration (MIC), where as an appropriate MIC can beextrapolated from the zone diameter.


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Dilution method: In the dilution methods, samples being tested are mixed with a

suitable medium that has previously been inoculated with the testorganism.

After incubation, growth of the microorganism may be determinedby direct visual or turbidimetric comparison of the test culturewith a control cultures.

Usually a series of dilutions of the original sample in the culturemedium is made and then inoculated with the test organism.

After incubation, the end point of the test is taken as the highestdilution which will just prevent perceptible growth of the testorgan sm va ue .

These methods the best for assaying water soluble or lipophilicpure compounds and to determine their MIC values, which can becarried out in liquid as well as solid media. Also growth curves of amicroorganism can be recorded using this method.

Only the solid or agar dilution method, is suitable for testing non-sterile plant extracts, because aerobic organisms do not developwell under the solidified agar. The occasional contaminatingculture, which develops on the surface of the agar, is no problemsince it can be easily recognized.


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Moreover, non-polar extracts, essential oils, suspensionsof solids or emulsions and antimicrobial substances,

which do not diffuse through agar media, can be testeddirectly by incorporating them with the agar media as ifthey were aqueous solutions.

Unlike the diffusion methods, no concentration gradientoccurs during the testing procedure

In contrast with the dilution in a liquid medium however,

emulsions of, for example an essential oil in the medium,.

Several different test microorganisms may be testedsimultaneously on the same dilution, which makes theagar dilution method very quick and time saving

Taking into account all properties mentioned, the agardilution method seems to be the most convenient methodfor routine testing of complex samples such as plantextracts.

The method is also very useful to guide the isolation ofantimicrobially active components from plant extracts.


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Preparation of plant extract:

Fresh green plants (50.0g) or dried plant

material (5.0g) Macerated with 80% ethanol & filtered

Marc

Exhaustively percolated with the same solvent

80% ethanol ra e + perco a e com ne

Evaporated at 400C

Thick residue (extract).

The residue is suspended or dissolved inphysiological Tris buffer ( PH 7.2) or in a mixtureof polyethylene glycol 400 (PEG 400) &physiological Tris buffer (4.6) (10.0ml).


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A per warmed ( 500C) solubilized plant extract ( 2.0ml) or

its four- fold dilutions ( eg. & 1/6) are mixed with anequal amount of liquid double concentrated agar mediumat 500C. This gives dilutions of , 1/8 & 1/32 respectively.

The holes of the lanes B-D or F-H of a microtitre plate arefilled with these dilutions (0.3 ml per hole) under an infrared lamp in order to prewarm the plate and to prevent

solidification during filling.

consisting of a mixture of solubilizing buffer ( 2.0 ml) andculture medium (2.0ml ) ( 0.3 ml per hole ).

After solidification at room temperature, all holes areinoculated with a 1:100 dilution (5 l) of over night

cultures of test bacteria ( +_ 103 bacteria ). After incubation for 24h at 370C, inhibition of growth of the

test organisms is judged by comparing it with the amountof growth in the control holes using a light microscope.


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In this way, a concentration range of 7500-0.5 g of

potential antimicrobial agents in fresh plants is covered,assuming that the fresh plant material contains 10% drymatter and 90% water and that the active compounds arepresent within a range of 1% to 0.001%.

A prominent antibacterial effect, worthy of furtherinvestigation, is obtained if not only the , but also the 1/8& 1/32 dilutions show inhibitory activities.

The finding of inhibition for the dilution only mostly

and is less promising for further investigation.

Sterile plant extracts, obtained by membrane filtration oftheir aqueous solutions can be tested by the liquid dilutionmethod. Thus, serial two- fold dilutions of the plant extracts

in standard nutrient broth ( eg. Up to 1/32 dilutions) aremade in small glass tubes or dispensed in the holes ofmicrotitre plates.

After inoculation and incubation of all dilutions, growth ofthe microbes is determined as previously mentioned.


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Aqueous suspensions and emulsion of plantextracts, made sterile by gama- irradiation, can

also be tested, provided that the stability of theemulsion remains constant and sedimentationof suspended particles of the extract in theextract in the liquid culture medium is avoidedby shaking during the entire assay.

Dilution in liquid medium is the mostlaborious but also the most accuratetechnique.

The method is very much appreciated for assaypurposes of pure samples, particularly when ahigh degree of sensitivity is required, butestimated by many researchers as being lesssuitable than diffusion methods for qualitativework eg, the rapid screening of a large numberof plant extracts.


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It is, however, the only method to determine whether an

antimicrobial agent is bactericidal or only bacteriostatic tothe test organism at various concentrations.

The minimum bactericidal concentration ( MBC) can be

determined by plating out samples of completely inhibited

dilution cultures on to solid or liquid media containing no

antibiotic. en e germ oes no grow, e samp e s ac er c a .

All the methods described for bacteria are equally suitable

for yeasts and for certain other unicellular organisms.


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Bioautographic methods: Bioautography, as a method to localize antibacterial activity on a

chromatogram, has found wide spread application in the searchfor new antibiotics from microorganisms.

Most published procedures are based on the agar diffusiontechnique, whereby the antimicrobial agent is transferred fromthin layer or paper chromatogram to an inoculated agar platethrough a diffusion process.

Zone of inhibition are then visualized by appropriate vital stains.

The problems due to the differential diffusion of compounds from

bioautographic detection on the chromatographic layer.

This method, however, requires more complex microbiologicalequipment and is, in contrast to the contact bioautographicmethodology, easily affected by possible contamination fromairborne bacteria.

Although bioautographic methods are suitable for testing highlyactive antibiotic (MIC values of at least 10g/ml ), they did notprove very promising for testing plant extracts, which oftencontain much less potent antimicrobial agents than the currentlyavailable antibiotics.


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Some phytoconstituents with antimicrobial activity:

Numerous investigation have been carried out in individual

plants and the respective antimicrobial agents have beenidentified in a gratifying number of cases.

Various studies, it is clear that the chemical structures of

these agents belong to the most commonly encountered

classes of higher plant secondary metabolites.

In many cases, investigation with modern methodology hascon rme o or c accoun s o e use o g er p an

preparations for the treatment of infections.

Up till now, however all antimicrobial substances from

higher plants have been found either to be toxic to animals

or not competitive therapeutically with the products of

microbial origin, due to their low potency and narrow

spectrum. Therefore, no antimicrobial compound from a

higher plant has yet come into significant clinical use.


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Research, however continues in the hope of

finding plant antimicrobials that are effective

for the systematic or tropical treatment of

human or agricultural infections.

Since the screening methodology for detection

of such agents, their isolation from the plants

and successive structure activity relation

studies are rather easy to perform, there are

grounds for cautions optimism to be successful

in the future.


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Antibacterial screening method: The antimicrobial activity of any drug of natural origin is

assayed separately using the agar diffusion method

employing 24 h culture of test bacterial and fungi strains.

For the antibacterial screening , bacterial strains used in

this study are Bacillus subtilis, Bacillus coagulans, Bacillus

megatorium, Pseudomonus cepacia, Staphyloccus aureus,

Escherichia coli etc.

The test organisms are seeded into sterile nutrient agar

media for bacterial screening. 1l of inoculum is mixed

uniformly with 20l of sterile melted nutrient agar and

cooled to 48-500

C in a sterile petridish.


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When the agar solidifies, holes of uniform diameter are

made using a sterile borer. 0.3l of each of the test solutionscontaining different extract solutions at varying

concentration as well as the standard drug solutions ( e.g.

tetracycline or other) and DMSO ( control- blank) are placed

in each hole separately under aseptic conditions.

The plates are then maintained at room temperature for 2hand allowed for diffusion of the solution into the s ecific

medium.

All the plates are then incubated at 250C for one week and

the zone of inhibitions is measured.

Usually for the screening of the antibacterial activity ofdifferent plant extracts of varying polarity like petroleum

ether, acetone, chloroform and methanol, extracts of plant

parts are screened for their activity by the above method.


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After preliminary screening, the effective extracts arefurther investigated for their activity at differentconcentrations and the optimum activity of the specific

extract is measured.

By using this model several plant species like Nelumbonucifera rhizome, leaf extract of Leucas lavendulaefolia ,Drymeria cordata, Cryptostegia gradiflora, Moringa oleiferaseveral species of the genus Hypericum- H. hookerianum,H. patulum and H. mysorense have been shown toproduce antimicrobial constituents.

The nature of the extracts of all the above species withreported potent antibacterial potential suggests the use ofthe plant extract as a source of active antimicrobialprinciples against infection caused by susceptibleorganisms.


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Occurrence of infrequent variation inconcentrations within species and relatedorganisms suggest that resistance to theindividual extracts, when it occurs, is due tothe intrinsic properties of the species involvedrather than acquired characters.

For this reason, it would be more effective and

much more useful if the plant extract or its

development of anti-microbialchemotherapeutic agents.

This can be considered in the line with the

current search for such substances to augmentor replace the antibiotics in current clinical use,which because of the spread of resistance areless useful than before.


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Assay for phytopharmaceuticalshaving antifungal activity

Phytoalexins appear to have a very broad range

of fungi for which they are quite toxic in the

10-100 g/ml concentration range. In fact,

their non-specificity could reduce their.


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Biological estimation procedures for screening/evaluation of antifungal agents:

Assays using sporelings in liquid mediaproduced the most reliable indication offungicidal activity. The results of other testswere less reliable and often, complete inhibitionof growth did not occur. For most fungi, spores

and 1-day-old sporelings were equally sensitive. However , the spores of some fungi were

markedly resistant to compounds that inhibitedmany other fungi.Similarly, 3-day-old sporelings

sometimes grew in relatively high levels of thefungicides tested.

The growth, which was produced afterprolonged incubation, arose from a few cellsthat survived the potentially fugicidal effects


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Mycelial growth in agar media containing phytoalexins

was quite variable and was rarely linear throughout theincubation period.

Skipp and Bailey (1977) divided the growth response of

fungi in their bioassays into three types:

a) Growth started immediately and was essentially linear.

b) Produce a growth curve with a lag period , but then

proceeded at a constant rate and

c)

The rate of growth increased progressively throughoutthe incubation period.


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In general, assays using older mycelial inocula in liquidmedia, which may reflect the situation during lesionlimitation, gave results similar to those often with 1-day-

old sporelings . When prolonged incubation led toincreased MIC values, this was probably due to survival ofsome cells within the inoculum and subsequent mycelialgrowth. Assays measuring inhibition of mycelial growth onagar media are widely used.

The lag period in the growth of several fungi was probablycaused by the death of most cells within the inoculum.

row , w c even ua y occurs, ar ses rom yp ae esurvive for some reason. The end of the lag period consistswith alteration in the sensitivity to the compound byadaptation and/or detoxification.

Chemicals may not be equally active against closely relatedfungi or may be equally active against unrelated fungi.Pathogens differ appreciably in their sensitivity toparticular chemicals and each disease reveals a number ofchemicals that are not active against any of the otherpathogens.


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Media used:

Solid agar and liquid broth culture media C and D as a

specified in pharmacopoeia of India (1985) may be used for

sensitivity, turbidity, spore germination and to determine

the zone of inhibition.

Cultures:

Pure cultures of the organisims are to be procured from

the laboratory where the test is being performed . The

fungalstrains usally used for this study are Aspergilus

niger, Aspergillus flavus, Candida albicans, candida

tropicalis, Cryptococcus neoformans and Trichophytonlignorum etc.


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Sensitivity test :

This is performed by tube dilution technique.A series of test tubes (16x 125 mm).,

containing 9ml of sterile culture medium, C

and 1ml of various concentration of test drugs

ten tubes for each concentration are taken.

All tubes are inoculated with microorganisms

to be tested and than incubated at20-25 C

for 48 h. Turbidity produced is observed thereafter by determine the absorbance at 530 nm.


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Turbidity method :

This is performed as per the method describe inpharmacopoeia (Indian pharmacopoeia 1985 ).One ml of each concentration of the test drug tobe screened is placed .

To each tube 9ml of nutrient medium, Cpreviously seeded with the appropriate test

organism is added. One control containing thenocu a e cu ure me um an ano er an ,which is identical with the control broth treatedimmediately with 0.5ml of dilute formaldehydesolution is used.

All the tubes are incubated at the specifiedtemperature form 48 h. The growth of the testorganisms is measured by determining theabsorbance at 530nm in spectrophotometer.


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Spore germination methods:

Spore suspensions of 7 days old culture areprepared in the test compounds and standard

drug for comparison griseofulvin (in specific

concentration) . A control is prepared as

identical with this but without using the testcompoun s. rop o spore suspens on s

placed on a sterilized slide and incubated in

humid chamber for 12h and scored the

number of spores germinated to calculate thepercentage of spore germination .


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Disc diffusion method :

Filter paper disc agar diffusion method is used (Mukherjeeet al. 1995). 20ml of the sterilized medium D is taken ineach petridish. 2ml of 24h old broth culture of sub-culturedorganisms is distributed evenly over the surface of theplates.

Sterilized Whatman filter no.1 discs (6 mm diameter)

thoroughly moistened with different concentration of testand standard dru s are laced on the surface of the late.

Discs moistened with sterile water are used as control.

The test organisms are seeded into sterile SDA media forfungal screening.

1l of inoculum is mixed uniformly with 20l of sterile

melted nutrient agar and cooled to 48-500C in a sterilepetridish.


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When the agar solidifies, holes of uniform diameter are made

using a sterile borer.

0.3 l of each of the test solutions containing different extracts

solutions at varying concentrations as well as the standard drug

solutions ( e.g. griseofulvin or other ) and DMSO (control-blank)

are placed in each hole separately under aseptic condition.

The plates are then maintained at room temperature for 2h and

allowed for diffusion of the solution into the specific medium.

All the plates are then incubated at 250C for one week and the

zone of inhibition is measured.

By using these models several plant species like Nelumbo

nucifera rhizome, leaf extract of cassia tora, several species of the

genus Hypericum-H. hookerianum, H. patulum and H. mysorensehave been show to produce antifungal potentials.

Microbiological Screening

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