VPT 607 Veterinary Chemotherapy Lab Manual

8/3/2019 VPT 607 Veterinary Chemotherapy Lab Manual

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Ex No: 1

GENERAL METHODS FOR ASSAY OF CHEMOTEHRAPEUTICAGENTS

Unlike tests for the evaluation of disinfectants wheredetermination of cidal activity is of paramount importance, tests

involving potential chemotherapeutic agents (antibiotics) invariably

have as their main focus determination of MIC.

Tests for bacteriostatic activity

Disc tests

These are modifications of the earlier cup or ditch-plate procedures

where filter-paper discsimpregnated with the antimicrobial replace the

antimicrobial-filled cups or wells. For disc tests, standard suspensions

(e.g. 0.5 McFarland standard) of log phase growth cells are prepared

and inoculated onto the surface of appropriate agar plates to form a

lawn. Commercially available ilter-paper discs containing known

concentrations of antimicrobial agent (it is possible to prepare your

own discs for use with novel drugs) are then placed on the dried lawn

and the plates are incubated aerobically at 35C for 18 hours.

Disc tests are basically qualitative, although it is possible to get

some information on the degree of activity depending on the zone

size . Although there are subtle variations of the disc test used in somecountries, the basic principles behind the tests remain similar and are

based on the original work of Bauer and colleagues. Some techniques

employ a control bacterial isolate on each plate so that comparisons

between zone sizesaround the test and control bacterium can be

ascertained (i.e. a disc potency control).

Control strains of bacteria are available which should have

inhibition zones of a given diameter with stipulated antimicrobial discs.

Use of such controls endorses the suitability of the methods (e.g.

medium, inoculum density, incubation conditions) employed. For slow-

growing microorganisms, the incubation period can be extended.

Dilution tests

These usually employ liquid media but can be modified to involve

solid media. Doubling dilutions usually in the range 0.12256mg/L

of the antimicrobial under test are prepared in a suitable broth

medium, and a volume of log phase cells is added to each dilution to

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result in a final cell density of around 5 \ 105 CFU/ml. After incubation

at 35C for 18 hours, the concentration of antimicrobial contained in

the first clear tube is read as the MIC..

Endpoints with dilution tests are usually sharp and easily defined,

although skipped wells (inhibition in a well with growth either side)

and trailing (a gradual reduction in growth over a series of wells) may

be encountered. The latter is especially evident with antifungal tests

Dilution tests can also be carried out using a series of agar plates

containing known antimicrobial concentrations. Appropriate bacterial

suspensions are inoculated onto each plate and the presence or

absence of growth is recorded after suitable incubation.

E-tests

The most convenient and presently accepted method of determining

bacterial MICs, however, is the E (Epsilometer)-test.Basically this is performed in a similar manner to the disc test

except that nylon strips that have a linear gradient of antimicrobial

lyophilized on one side are used instead of the filter-paper

impregnated antimicrobial discs.

On the other side of the nylon strip are a seriesof lines and

figures denoting MIC values (the nylon strips are placed antimicrobial

side down on the freshly prepared bacterial lawn and, after incubation,

the MIC is determined by noting where the ellipsoid (pear-shaped)

inhibition zone crosses the strip . For most microorganisms, there

appears to be excellent correlation between dilution and E-test MIC

results.

Tests for bactericidal activity

The MBC is the lowest concentration (in mg/L) of antimicrobial that

results in 99.9% killing of the bacterium under test. The 99.9% cutoff

is an arbitrary in vitro value with 95% confidence limits that has

uncertain clinical relevance.

MBCs are determined by spreading 0.1-ml(100-ml) volumes of all clear

(no growth) tubes from a dilution MIC test onto separate agar plates(residual antimicrobial in the 0.1-ml sample is diluted out over the

plate).

After incubation at 35C overnight (or longer for slow-growing

bacteria), the numbers of colonies growing on each plate are

recorded. The first concentration of drug that produces


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with MICs, the initial bacterial inoculum should result in about 5 \ 105

CFU/ml. Inhibition, but not killing of this inoculum, should therefore

result in the growth of 50 000 bacteria from the 0.1-ml sample. A

99.9% (3-log) kill would result in no more than 50 colonies on the

subculture plate. With most modern antibacterial drugs, the

concentration that inhibits growth is very close to the concentration

that produces death, e.g. within one or two dilutions. In general, only

MICs are determined for such drugs.

Ex No: 2

ANTIMICROBIAL SUSCEPTIBILITY TESTING

PrincipleThe principles of determining the effectivity of a noxious agent to a

bacterium were well enumerated by Rideal ,Walker and others at theturn of the century, the discovery of antibiotics made these tests(or

their modification)too cumbersome for the large numbers of tests

necessary to be put up as a routine. The ditch plate method of agar

diffusion used by Alexander Fleming was the forerunner of a variety of

agar diffusion methods devised by workers in this field .The Oxford

group used these methods initially to assay the antibiotic contained in

blood by allowing the antibiotics to diffuse out of reservoirs in the

medium in containers placed on the surface.

With the introduction of a variety of antimicrobials it became

necessary to perform the antimicrobial susceptibility test as a routine.

For this, the antimicrobial contained in a reservoir was allowed to

diffuse out into the medium and interact in a plate freshly seeded with

the test organisms. Even now a variety of antimicrobial containing

reservoirs are used but the antimicrobial impregnated absorbent paper

disc is by far the commonest type used. The disc diffusion method of

AST is the most practical method and is still the method of choice for

the average laboratory. Automation may force the method out of the

diagnostic laboratory but in this country as well as in the smaller

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laboratories of even advanced countries, it will certainly be the most

commonly carried out microbiological test for many years to come. It

is, therefore, imperative that microbiologists understand the principles

of the test well and keep updating the information as and when

necessary. All techniques involve either diffusion of antimicrobial agent

in agar or dilution of antibiotic in agar or broth.

Even automated techniques are variations of the above methods.

Methods of Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing methods are divided intotypes based on the principle applied in each system. They

include:

Diffusion DilutionDiffusion&Dilution

Stokes method Minimum Inhibitory Concentration

E-Test method

Kirby-Bauer method i) Broth dilution

ii)Agar Dilution

Quantification of antimicrobial concentration in plasma/tissues is thebasic prerequisite to determine the bioavailability / bioequivalence(for new commercial formulation), pharmacokinetic parameters andfixation of dose/dosing schedule. The different methods available forassay of antimicrobial agents are:

1. Microbiological assay ( disc-diffusion method)2. Colorimetric/ Spectrophotometric : eg. Sulphonamides3. Fluorimetric : Tetracycline/ Doxycycline4. HPLC: any antimicrobial at residue levels can be quantified by

using suitable detection electrode

DISK DIFFUSION

Reagents for the Disk Diffusion Test1. Meller-Hinton Agar MediumOf the many media available, Meller-Hinton agar is considered to bethe best for routine susceptibility testing of nonfastidious bacteria forthe following reasons:* It shows acceptable batch-to-batch reproducibility for susceptibility

testing.* It is low in sulphonamide, trimethoprim, and tetracycline inhibitors.* It gives satisfactory growth of most nonfastidious pathogens.

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* A large body of data and experience has been collectedconcerning susceptibility tests performed with this medium.

Although Meller-Hinton agar is reliable generally for susceptibilitytesting, results obtained with some batches may, on occasion, varysignificantly. If a batch of medium does not support adequate growth

of a test organism, zones obtained in a disk diffusion test will usuallybe larger than expected and may exceed the acceptable qualitycontrol limits. Only Meller-Hinton medium formulations that havebeen tested according to, and that meet the acceptance limitsdescribed in, NCCLS document M62-A7- Protocols for EvaluatingDehydrated Meller-Hinton Agar should be used.

Preparation of Meller-Hinton AgarMeller-Hinton agar preparation includes the following steps.1. Meller-Hinton agar should be prepared from a commercially

available dehydrated base according to the manufacturer's

instructions.2. Immediately after autoclaving, allow it to cool in a 45 to 50C water

bath.3. Pour the freshly prepared and cooled medium into glass or plastic,

flat-bottomed petri dishes on a level, horizontal surface to give auniform depth of approximately 4 mm. This corresponds to 60 to70 ml of medium for plates with diameters of 150 mm and 25 to30 ml for plates with a diameter of 100 mm.

4. The agar medium should be allowed to cool to room temperatureand, unless the plate is used the same day, stored in a refrigerator

(2 to 8C).

5. Plates should be used within seven days after preparation unless

adequate precautions, such as wrapping in plastic, have been

taken to minimize drying of the agar.

6. A representative sample of each batch of plates should be

examined for sterility by incubating at 30 to 35C for 24 hours orlonger.

Preparation of antibiotic stock solutions

Antibitiotics may be received as powders or tablets. It is recommended

to obtain pure antibiotics from commercial sources, and not use

injectable solutions. Powders must be accurately weighed and dissolved

in the appropriate diluents (Annexure III) to yield the required

concentration, using sterile glassware. Standard strains of stock cultures

should be used to evaluate the antibiotic stock solution. If satisfactory,

the stock can be aliquoted in 5 ml volumes and frozen at -20C or -60C.

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Stock solutions are prepared using the formula (1000/P) X V X C=W,

where P+potency of the anitbiotic base, V=volume in ml required,

C=final concentration of solution and W=weight of the antimicrobial to

be dissolved in V.

Preparation of dried filter paper discs

Whatman filter paper no. 1 is used to prepare discs approximately 6 mm

in diameter, which are placed in a Petri dish and sterilized in a hot air

oven.

The loop used for delivering the antibiotics is made of 20 gauge wire and

has a diameter of 2 mm. This delivers 0.005 ml of antibiotics to each

disc.

Storage of commercial antimicrobial discs

Cartridges containing commercially prepared paper disks specifically forsusceptibility testing are generally packaged to ensure appropriate

anhydrous conditions. Discs should be stored as follows:* Refrigerate the containers at 8C or below, or freeze at -14C orbelow, in a nonfrost-free freezer until needed. Sealed packages ofdisks that contain drugs from the -lactam class should be storedfrozen, except for a small working supply, which may berefrigerated for at most one week. Some labile agents (e.g.,imipenem, cefaclor, and clavulanic acid combinations) may retaingreater stability if stored frozen until the day of use.

* The unopened disc containers should be removed from therefrigerator or freezer one to two hours before use, so they mayequilibrate to room temperature before opening. This procedure

minimizes the amount of condensation that occurs when warm aircontacts cold disks.

* Once a cartridge of discs has been removed from its sealedpackage, it should be placed in a tightly sealed, desiccatedcontainer. When using a disc-dispensing apparatus, it should befitted with a tight cover and supplied with an adequate desiccant.The dispenser should be allowed to warm to room temperaturebefore opening. Excessive moisture should be avoided byreplacing the desiccant when the indicator changes color.

* When not in use, the dispensing apparatus containing the discsshould always be refrigerated.

* Only those discs that have not reached the manufacturer'sexpiration date stated on the label may be used. Discs should bediscarded on the expiration date.

Turbidity standard for inoculum preparationTo standardize the inoculum density for a susceptibility test, a BaSO4turbidity standard, equivalent to a 0.5 McFarland standard or its opticalequivalent (e.g., latex particle suspension), should be used. A BaSO4 0.5McFarland standard may be prepared as follows:

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1. A 0.5-ml aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2 . 2H2O) isadded to 99.5 ml of 0.18 mol/L H2SO4 (1% v/v) with constantstirring to maintain a suspension.

2. The correct density of the turbidity standard should be verified byusing a spectrophotometer with a 1-cm light path and matched

cuvette to determine the absorbance. The absorbance at 625 nmshould be 0.008 to 0.10 for the 0.5 McFarland standard.3. The Barium Sulfate suspension should be transferred in 4 to 6 ml

aliquots into screw-cap tubes of the same size as those used ingrowing or diluting the bacterial inoculum.

4. These tubes should be tightly sealed and stored in the dark atroom temperature.

5. The barium sulfate turbidity standard should be vigorouslyagitated on a mechanical vortex mixer before each use andinspected for a uniformly turbid appearance. If large particles

appear, the standard should be replaced. Latex particlesuspensions should be mixed by inverting gently, not on a vortexmixer

6. The barium sulfate standards should be replaced or their densitiesverified monthly.

DISC DIFFUSION METHODS The Kirby-Bauer and Stokes' methods are usually used forantimicrobial susceptibility testing, with the Kirby-Bauer method beingrecommended by the NCCLS. The accuracy and reproducibility of thistest are dependent on maintaining a standard set of procedures asdescribed here.

NCCLS is an international, interdisciplinary, non-profit, non-governmental organization composed of medical professionals,government, industry, healthcare providers, educators etc. It promotesaccurate antimicrobial susceptibility testing (AST) and appropriatereporting by developing standard reference methods, interpretativecriteria for the results of standard AST methods, establishing qualitycontrol parameters for standard test methods, provides testing andreporting strategies that are clinically relevant and cost-effectiveInterpretative criteria of NCCLS are developed based on internationalcollaborative studies and well correlated with MICs and the resultshave corroborated with clinical data. Based on study results NCCLS

interpretative criteria are revised frequently. NCCLS is approved byFDA-USA and recommended by WHO.Procedure for Performing the Disc Diffusion Test

Inoculum Preparation

Growth Method

1. At least three to five well-isolated colonies of the same

morphological type are selected from an agar plate culture. The

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top of each colony is touched with a loop, and the growth is

transferred into a tube containing 4 to 5 ml of a suitable broth

medium, such as tryptic soy broth.

2. The broth culture is incubated at 35C until it achieves or exceedsthe turbidity of the 0.5 McFarland standard (usually 2 to 6 hours)

3. The turbidity of the actively growing broth culture is adjusted with

sterile saline or broth to obtain a turbidity optically comparable to

that of the 0.5 McFarland standard. This results in a suspension

containing approximately 1 to 2 x 108 CFU/ml for E.coli ATCC

25922. To perform this step properly, either a photometric device

can be used or, if done visually, adequate light is needed to

visually compare the inoculum tube and the 0.5 McFarland

standard against a card with a white background and contrasting

black lines.

Direct Colony Suspension Method

1. As a convenient alternative to the growth method, the inoculum

can be prepared by making a direct broth or saline suspension of

isolated colonies selected from a 18- to 24-hour agar plate (a

nonselective medium, such as blood agar, should be used). The

suspension is adjusted to match the 0.5 McFarland turbidity

standard, using saline and a vortex mixer.

2. This approach is the recommended method for testing the

fastidious organisms, Haemophilus spp., N. gonorrhoeae, and

streptococci, and for testing staphylococci for potential methicillin

or oxacillin resistance.

Inoculation of Test Plates

1. Optimally, within 15 minutes after adjusting the turbidity of the

inoculum suspension, a sterile cotton swab is dipped into the

adjusted suspension. The swab should be rotated several times

and pressed firmly on the inside wall of the tube above the fluid

level. This will remove excess inoculum from the swab.

2. The dried surface of a Meller-Hinton agar plate is inoculated bystreaking the swab over the entire sterile agar surface. This

procedure is repeated by streaking two more times, rotating the

plate approximately 60 each time to ensure an even distributionof inoculum. As a final step, the rim of the agar is swabbed.

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3. The lid may be left ajar for 3 to 5 minutes, but no more than 15

minutes, to allow for any excess surface moisture to be absorbed

before applying the drug impregnated disks.

NOTE: Extremes in inoculum density must be avoided. Never use

undiluted overnight broth cultures or other unstandardized inocula for

streaking plates.

Application of Discs to Inoculated Agar Plates

1. The predetermined battery of antimicrobial discs is dispensed onto

the surface of the inoculated agar plate. Each disc must be

pressed down to ensure complete contact with the agar surface.

Whether the discs are placed individually or with a dispensing

apparatus, they must be distributed evenly so that they are no

closer than 24 mm from center to center. Ordinarily, no more than12 discs should be placed on one 150 mm plate or more than 5

discs on a 100 mm plate. Because some of the drug diffuses

almost instantaneously, a disc should not be relocated once it has

come into contact with the agar surface. Instead, place a new disc

in another location on the agar.

2. The plates are inverted and placed in an incubator set to 35Cwithin 15 minutes after the discs are applied. With the exception

ofHaemophilus spp., streptococci and

N. gonorrhoeae, the plates should not be incubated in an

increased CO2 atmosphere, because the interpretive standards

were developed by using ambient air incubation, and CO2 will

significantly alter the size of the inhibitory zones of some agents.

Reading Plates and Interpreting Results

1. After 16 to 18 hours of incubation, each plate is examined. If the

plate was satisfactorily streaked, and the inoculum was correct,

the resulting zones of inhibition will be uniformly circular and there

will be a confluent lawn of growth. If individual colonies areapparent, the inoculum was too light and the test must be

repeated. The diameters of the zones of complete inhibition (as

judged by the unaided eye) are measured, including the diameter

of the disc. Zones are measured to the nearest whole millimeter,

using sliding calipers or a ruler, which is held on the back of the

inverted petri plate. The petri plate is held a few inches above a

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black, nonreflecting background and illuminated with reflected

light. If blood was added to the agar base (as with streptococci),

the zones are measured from the upper surface of the agar

illuminated with reflected light, with the cover removed. If the test

organism is a Staphylococcus or Enterococcus spp., 24 hours of

incubation are required for vancomycin and oxacillin, but other

agents can be read at 16 to 18 hours. Transmitted light (plate

held up to light) is used to examine the oxacillin and vancomycin

zones for light growth of methicillin- or vancomycin- resistant

colonies, respectively, within apparent zones of inhibition. Any

discernable growth within zone of inhibition is indicative of

methicillin or vancomycin resistance.

2. The zone margin should be taken as the area showing no obvious,

visible growth that can be detected with the unaided eye. Faintgrowth of tiny colonies, which can be detected only with a

magnifying lens at the edge of the zone of inhibited growth, is

ignored. However, discrete colonies growing within a clear zone of

inhibition should be subcultured, re-identified, and retested.

Strains ofProteus spp. may swarm into areas of inhibited

growth around certain antimicrobial agents. With Proteus spp.,

the thin veil of swarming growth in an otherwise obvious zone of

inhibition should be ignored. When using blood-supplemented

medium for testing streptococci, the zone of growth inhibition

should be measured, not the zone of inhibition of hemolysis. With

trimethoprim and the sulfonamides, antagonists in the medium

may allow some slight growth; therefore, disregard slight growth

(20% or less of the lawn of growth), and measure the more

obvious margin to determine the zone diameter.

3. The sizes of the zones of inhibition are interpreted by referring to

Tables 2A through 2I (Zone Diameter Interpretative Standards and

equivalent Minimum Inhibitory Concentration Breakpoints) of the

NCCLS M100-S12: Performance Standards for AntimicrobialSusceptibility Testing: Twelfth Informational Supplement, and the

organisms are reported as either susceptible, intermediate, or

resistant to the agents that have been tested. Some agents may

only be reported as susceptible, since only susceptible breakpoints

are given.

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DILUTION METHODS

Dilution susceptibility testing methods are used to determine theminimal concentration of antimicrobial to inhibit or kill themicroorganism. This can be achieved by dilution of antimicrobial ineither agar or broth media. Antimicrobials are tested in log2 serialdilutions (two fold).

Minimum Inhibitory Concentration (MIC)Diffusion tests widely used to determine the susceptibility of organismsisolated from clinical specimens have their limitations; when equivocalresults are obtained or in prolonged serious infection e.g. bacterial

endocarditis, the quantitation of antibiotic action vis-a-vis thepathogen needs to be more precise. Also the terms Susceptible andResistant can have a realistic interpretation. Thus when in doubt, theway to a precise assessment is to determine the MIC of the antibioticto the organisms concerned.There are two methods of testing for MIC:(a) Broth dilution method(b) Agar dilution method.

Broth Dilution Method The Broth Dilution method is a simpleprocedure for testing a small number of isolates, even single

isolate. It has the added advantage that the same tubes can betaken for MBC tests also:

MaterialsSterile graduated pipettes of 10ml, 5ml, 2ml and 1ml Sterile capped7.5 x 1.3 cm tubes / small screw-capped bottles, Pasteur pipettes,overnight broth culture of test and control organisms ( same as for discdiffusion tests), required antibiotic in powder form (either from themanufacturer or standard laboratory accompanied by a statement ofits activity in mg/unit or per ml. Clinical preparations should not beused for reference technique.), required solvent for the

antibiotic, sterile Distilled Water - 500ml and suitable nutrient brothmedium.Trimethoprim and sulphonamide testing requires thymidine free mediaor addition of 4% lysed horse blood to the mediaA suitable rack to hold 22 tubes in two rows i-e 11 tubes in each row.

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Stock solutionStock solution can be prepared using the formula

1000------- x V x C= WP

Where P=Potency given by the manufacturer in relation to the baseV= Volume in ml required; C=Final concentration of solution (multiplesof 1000)W= Weight of the antimicrobial to be dissolved in the volume VExample: For making 10 ml solution of the strength 10,000mg/l frompowder base whose potency is 980 mg per gram,the quantities of theantimicrobials required is

W = 1000------- x 10 x 10=102.04mg

980

Note:the stock solutions are made in higher concentrations to maintaintheir keeping qualities and stored in suitable aliquots at -20oC .Oncetaken out,they should not be refrozen or reused.MethodPrepare stock dilutions of the antibiotic of concentrations 1000 and100 g/L as required from original stock solution (10,000mg/L).Arrange two rows of 12 sterile 7.5 x1.3 cm capped tubes in the rack. Ina sterile 30ml (universal) screw capped bottle, prepare 8ml of brothcontaining the concentration of antibiotic required for the first tube ineach row from the appropriate stock solution already made. Mix thecontents of the universal bottle using a pipette and transfer 2ml to the

first tube in each row. Using a fresh pipette ,add 4 ml of broth to theremaining 4 ml in the universal bottle mix and transfer 2ml to thesecond tube in each row. Continue preparing dilutions in this way butwhere as many as 10 or more are required the series should be startedagain half the way down. Place 2ml of antibiotic free broth to the lasttube in each row. Inoculate one row with one drop of an overnightbroth culture of the test organism diluted approximately to 1 in 1000 ina suitable broth and the second row with the control organism ofknown sensitivity similarly diluted. The result of the test is significantlyaffected by the size of the inoculum.The test mixture should contain106 organism/ml.If the broth culture used has grown poorly,it may be

necessary to use this undiluted. Incubate tubes for 18 hours at 37oC.Inoculate a tube containing 2ml broth with the organism and keep at+4oC in a refrigerator overnight to be used as standard for thedetermination of complete inhibition.Calculations for the preparation of the original dilution.

This often presents problems to those unaccustomed to

performing these tests. The following method advocated by Pamela M

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Waterworth is presented. Calculate the total volume required for the

first dilution. Two sets of dilution are being prepared (one for the test

and one for the control), each in 2ml volumes i-e a total of 4 ml for

each concentration as 4ml is required to make the second dilution, the

total requirement is 8ml. Now calculate the total amount of the

antibiotic required for 8ml. For 64 g/l concentration, 8x64mg/l =512g

in 8 ml. Place a decimal point after the first figure (5.12) and take this

volume in ml (i.e 5.12 ml) of the dilution below 512mg/l and make upto

8ml with broth. In this example given above, the series has to be

started again mid way at 2 mg/l which would be obtained in the same

way:

8ml of 2mg/l=16g in 8ml.1.6 ml of 10 mg/ l + 6.4 ml of broth.

READING OF RESULTMIC is expressed as the lowest dilution, which inhibited growth judgedby lack of turbidity in the tube.Because very faint turbidity may begiven by the inoculum itself, the inoculated tube kept in therefrigerator overnight may be used as the standard for thedetermination of complete inhibition.Standard strain of known MICvalue run with the test is used as the control to check the reagents andconditions.

Micro-broth dilution test This test uses double-strength Meller-Hinton broth, 4X strengthantibiotic solutions prepared as serial two-fold dilutions and the test

organism at a concentration of 2x106/ml. In a 96 well plate, 100 l ofdouble-strength MHB, 50 l each of the antibiotic dilutions and theorganism suspension are mixed and incubated at 35C for 18-24 hours.The lowest concentration showing inhibition of growth will be consideredthe MIC of the organism.Reading of resultMIC is expressed as the highest dilution which inhibited growth judgedby lack of turbidity in the tube. Because very faint turbidity may begiven by the inoculum itself,the inoculated tube kept in the refrigeratorovernight may be used as the standard for the determination of

complete inhibition. Standard strain of known MIC, run with the test isused as the control to check the reagents and conditions.

The Agar dilution MethodAgar dilutions are most often prepared in petri dishes and haveadvantage that it is possible to test several organisms on each plate.If only one organism is to be tested e.g M.tuberculosis,the dilutionscan be prepared in agar slopes but it will then be necessary to preparea second identical set to be inoculated with the control organism.The

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dilutions are made in a small volume of water and added to agar whichhas been melted and cooled to not more than 60oC.Blood may beadded and if chocolate agar is required,the medium must be heatedbefore the antibiotic is added.It would be convenient to use 90 mm diameter petri dishes and add

one ml of desired drug dilutions to 19 ml of broth.The factor of agardilution must be allowed for in the first calculation as follows.final volume of medium in plate = 20 ml Top antibiotic concentrations = 64mg/l Total amount of drug = 1280g to beadded to 1 ml ofwater2ml of 1280 g /ml will be required to start the dilution = 2560gin 2 ml

= 1.28ml of 2000g /ml

0.72 ml of water.1 ml of this will be added to 19 ml agar.

(Note stock dilution of 2000g /ml is required for this range of MIC)

Reading of results: The antibiotic concentration of the first plate

showing 99% inhibition is taken as the MIC for the organism.

DILUTION AND DIFFUSION

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E test also known as the epsilometer test is an exponential gradienttesting methodology where E in E test refers to the Greek

symbol epsilon ( ).The E test(AB Biodisk) which is a quantitativemethod for antimicrobial susceptibility testing applies both the

dilution of antibiotic and diffusion of antibiotic into the medium.. Apredefined stable antimicrobial gradient is present on a thin inertcarrier strip. When this E test strip is applied onto an inoculatedagar plate, there is an immediate release of the drug. Followingincubation , a symmetrical inhibition ellipse is produced. Theintersection of the inhibitory zone edge and the calibrated carrierstrip indicates the MIC value over a wide concentration range(>10 dilutions) with inherent precision and accuracy .

E test can be used to determine MIC for fastidious organismslike S. pneumoniae,

-hemolytic streptococci, N.gonorrhoeae, Haemophilus sp. andanaerobes. It can also be used for Nonfermenting Gram Negativebacilli (NFGNB) for eg-Pseudomonas sp. and Burkholderiapseudomallei.

Resistance of major consequence may be detected for e.g., the test isvery useful in detecting glycopeptide resistant Enterococci (GRE) andglycopeptide intermediate S.aureus (GISA) and slow growingpathogens such as Mycobacterium tuberculosis. Further it can be usedfor detection of extended spectrum beta lactamases (ESBL). Inconclusion E test is a simple, accurate and reliable method to

determine the MIC for a wide spectrum of infectious agents.

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Ex No: 3

ESTIMATION OF SULPHONAMIDES IN BIOLOGICAL FLUIDS

Aim: To estimate sulphonamide concentration in plasma

Principle: Sulphonamides react with nitrite in acid medium to formdiazonium salts. The excess nitrite is destroyed by ammoniumsulphamate and the diazonium salts couples with N-(1-naphthyl)ethylene diamine to form a stable dye (purple colour) and thecorresponding concentration can be quantifiedspectrophotometrically(545 nm).Requirements:Drugs/chemicals:Trichloroaceticacid(TCA,15%sol.),Sodium nitrite (0.05%),Ammonium sulphamate(0.5%),N-1-naphthyl-ehhylene-diamine dihydrochloride(0.5% sol.), Hydrochloric acid (4.0N),Sulphonamide(10% sol.)

Instruments required: Spectrophotometer, Water bathProcedure: (Bratton & Marshal method, 1939)(i) Quantification of freesulphonamide:(a) Sample:1. Take one ml of sample (plasma/ urine) in a test tube and add 14 mlof distilled water and 5ml of TCA(15%). Mix the contents thoroughly.2. Keep the tube(s) at room temperature for 10min. for completedeproteinization. Filter the contents using Whatman filter paper # 1 toget the protein free filtrate.3. Take 5 ml of protein free filtrate in a test tube and add 0.5 ml ofsodium nitrite (0.05%). Thoroughly mix the contents and wait for 5

min.4. Add 0.5 ml of ammonium sulphamate(0.5%). Mix thoroughly andwait for 2 min.5. Add 0.5 ml of N-1-naphthyl-ehhylene-diamine dihydrochloride(0.5%sol.) and shake the tubes thoroughly and wait for 5 min.6. Measure the optical density (O.D) spectrophotometrically at 545nmagainst the blank within 30min of development of colour.(b) Blank:

Prepare blank as described above except that the sample isreplaced by 1ml of distilled water.(c) Standard: Prepare working standard solutions of sulphonamide

to be estimated from the stock solution (10%) to get aconcentration in the range of 2-50 g/ml. Take one ml of thesesolutions and add 14ml of distilled water and 5ml TCA (15%). Addother reagents as described above, but the filtration step may beomitted.

(ii) Quantification of total- sulphonamide: This includes free andacetylated sulphonamide present in the blood1. Make protein free filtrate as described above (step 1& 2)

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2. Take 5ml of protein free filtrate into a graduated glass tube and add0.5ml of 4NHCl

3. Cover the tube with a marble and place it in a water bath at 100 Cfor 1hour.4. Cool the tube at room temperature and make up the volume to 5ml

by adding dist.water5. Follow the same procedure as detailed in the case of freesulphonamide

(iii) acetyl-sulphonamide:he difference between the concentrationof free and total sulphonamide gives the concentration -acetylcompound.

Calculation: Plot the standard curve (sulphonamide concentrationv/s O.D) and the read the values of sulphonamide present in thesamples.

Note: Strictly follow the time schedule as mentioned in the procedure, The colour development is sensitive to sunlight, therefore wrap thetest tubes with black paper & switch off light

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Ex No: 4

ESTIMATION OF OXYTETRACYCLINE IN BIOLOGICAL FLUIDS

Aim: To estimate the concentration of Oxytetracycline in plasma

Requirements:Drugs/ chemicals:Trichloroaceticacid(TCA,15%sol.), Para-nitroaniline ,

Conc. HCl,Glacial acetic acid,Sodiun nitrite (15%) freshly preparedOxytetracycline standardNote: Para-nitroaniline colour reagent can be prepared by mixing 0.2gp-nitroaniline +

3ml con. HCl + 5ml glacial acetic acid + 2ml 15% sodium nitriteInstruments required: Spectrophotometer

Procedure:( Snell and Snell, 1971)(a) Sample:1. Take I ml of sample (plasma) and add 2ml of 15% TCA. Mixthoroughly and centrifuge at 4000rpm for 15 minutes.2. To 2ml of supernatant, add 0.2ml of p-nitroaniline colour reagent

and 2.0 ml of glacial acetic acid. Mix them and incubate at 65C for45min.3. Measure the resultant colour intensity at 435nm in thespectrophotometer against blank4. Derive the concentration of Oxytetracycline from the standardcurve.

(b) Blank: Prepare the blank in plasma and run along with the sample.

(c) Standard: Prepare the working standard solutions from the stocksolution of Oxytetracycline having concentrations in the range of 5-

200 g/ml in plasma and run along with the sample.Note: The limit of detection of Oxytetracycline is 0.6 g/ml of

plasma

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Ex No: 5

Introduction to High performance liquid chromatography

(HPLC)

High-performance liquid chromatography [HPLC] is now one of

the most powerful tools in analytical chemistry. It has the ability to

separate, identify, and quantitate the compounds that are present in

any sample that can be dissolved in a liquid. Today, compounds in

trace concentrations as low as parts per trillion [ppt] may easily be

identified. HPLC can be, and has been, applied to just about any

sample, such as pharmaceuticals, food, nutraceuticals, cosmetics,

environmental matrices, forensic samples, and industrial chemicals.

The components of a basic high-performance liquid chromatography

[HPLC] system are shown in the simple diagram in Figure E.

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High performance (pressure) liquid chromatography (HPLC) is an

efficient technique to separate, characterise and quantify organic

molecules in biological fluids or drug formulations. The essential

principle of HPLC is the partitioning (or distribution) of substances

between two liquid phases otherwise called partition chromatography.

In gas-liquid chromatography (GLC) high temperature are used to

volatilise the compounds so that separation can be done, however

elevated temperature may cause thermal degradation of some

compounds/drugs.

HPLC analysis of drugs is non-destructive in nature since: a.

there is no need for high temperature and b. the mobile phase is liquid

instead of gas (in GLC) and solubility is the limiting factor and not

volatility. Therefore, appropriate solvent (i.e. inert to the compound)

can be chosen so that no chemical changes will occur to the compound

/or its metabolite of our interest during separation.The major setback of the HPLC is sensitivity, but this limitation can beovercome by adopting good sample clean-up procedures. Thesensitivity of HPLC is generally in the nanogram range but in few casesit can measure picogram level

The various components of HPLC are: a. Pump b. Injection

system c. Column stationary phase) d. Mobile phase and e. Detector

Pump: it is used to deliver the solvent mobile phase through the

column. There are two types of column: a. Isocratic pump- means if

one or fixed solvent (mobile phase) at a set rate is used b. Gradient

pump- means two or more solvents in varying concentrations at adifferent flow rate are used (eg. in peptide purification).

Injector: it is to introduce sample into the column by syringe through

a spectrum of injection system (rheodune injector)

Column: it is the basic unit for stationary phase and it can be

described under three headings.

a. Column dimension or size- column are tubular structure and

usually made with stainless steel with internal diameter x length of:

4.6mm x 250mm. The size may vary if (600-1000mm in length) you

are performing a large scale preparative chromatography or shorter

(50 or 30mm length) if you want to do fast chromatography at higher

flow rate than normal speed.

b. Column particle and pore size- most column are packed with

inert hydrocarbon usually spherical shaped silica beads/particles of 3-

50m size, with 5m particles are most popular for peptides. Larger

particles will generate less pressure while smaller particles create high

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system pressure as well as higher separation efficiency. The pore size

of these silica particles is expressed as A and generally range

between 100-1000 A. A pore size of 100A is suitable for most of the

small molecules while 300A is ideal for proteins or peptides.

Stationery phase- it is generally made up of hydrophobic alkyl

chains (-CH2-CH2-CH2-CH3) that interacts with the analyte(drug

molecules) and there are three chain lengths. C8and C18(octadecyl

silane, ODS) is used for smaller hydrophilic peptides and drug

molecules respectively. C8 is used for normal phase while C18(non-

polar) is used for RPHPLC. C4 column is generally used for proteins

since larger protein molecules are likely to have more hydrophobic

moieties to interact with the column.

C18, 4.6 x 250mm, 5m, 300A

chain length column size particle size pore

size

Eg. Column specifications:

Mobile phase: The mobile phase is used to control retention and

selectivity. There are two types of HPLC assay.

A. Normal phase: Here the stationary phase is polar in

physiochemical terms and the retention of molecules is governed by

polar interaction between stationary and mobile base. In normal phase

HPLC the most popular mobile phase is either hexane or heptane to

which varying concentrations of more polar solvents like propanol or

ethyl acetate are added. The mobile phase containing proton donor

acceptors will interact with basic solutes. Conversely, mobile phases

containing proton donar acceptors will interact strongly with acidic

solvents. When excessive interaction occurs between solute and the

mobile phase peak tailing can result. Addition of basic modifier

(triethylamine) can overcome such strong interactions and thereby

improve peak shape. Normally increase in temperature results in

decrease in retention and this effect dependent on the specific polarmodifier in the solvent, for example ethanol increases retension.

B. Reverse phase (RPHPLC): Here mobile phase is more polar than

stationary phase (non-polar) and compounds are separated based on

their hydrophobic character. Normally, isocratic pump is the choice

when ever RPHPLC is employed.

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The reverse phase HPLC solvents are by convention installed in

the system channels A and B. The solvent A is the aqueous solvent

(HPLC grade water + 0.1% v/v acetic acid) while solvent B is the

organic solvent (HPLC grade acetonitrile, methanol or proponol + 0.1%

v/v acetic acid). The acid is added usually to improve the

chromatography peak shape and serve as a source protons in reverse

phase HPLC or LC/MS. The acids most commonly used are formic acid,

acetic acid or triflouroacetic acid. Usually, 0.1 %v/v solution is made by

adding 1ml/liter of solvent.

Quality of solvents: Solvents should be chosen to be compatible

with the column employed and the solubility and chemistry of the

analyte (drug). While formulating mixed solvents, they should be fully

miscible with each other; otherwise droplets of a second phase may be

trapped in the column and ruin separation. None of the mobile phase

constituents should interact with stationary phase and affect itadversely.Always one should use HPLC grade.

Sample cleanup: Biological samples may contain proteins, salts

and a host of organic compounds with widely differing polarities.

Pharmaceutical samples also contain a wide range of soluble and

insoluble excipients. The sample clean-up procedures can be designed

tentatively on the basis of extraction and polarity profiles.

Conventionally, solvent extraction is most commonly used to

extract organic molecules from biological fluids or tissues. If the

compound(s) are extracted in polar solvents then reverse phase HPLC

system would be the logical choice.

sample + Solvent to precipitate protein

Centrifuge and decant

supernatant

(Repeat twice /thrice for maximum

extraction)

Evaporate the solvent

Reconstitute with mobile phase

Inject 10/20l to HPLC

Fig. Steps involved in solvent

extraction

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Stationary Phase

The stationary phase in HPLC refers to the solid support contained

within the column over which the mobile phase continuously flows. The

sample solution is injected into the mobile phase of the assay through

the injector port. As the sample solution flows with the mobile phase

through the stationary phase, the components of that solution will

migrate according to the non-covalent interactions of the compounds

with the stationary phase. The chemical interactions of the stationary

phase and the sample with the mobile phase, determines the degree of

migration and separation of the components contained in the sample.

For example, those samples which have stronger interactions with the

stationary phase than with the mobile phase will elute from the column

less quickly, and thus have a longer retention time, while the reverse isalso true. Columns containing various types of stationary phases are

commercially available. Some of the more common stationary phases

include: Liquid-Liquid, Liquid-Solid (Adsorption), Size Exclusion, Normal

Phase, Reverse Phase, Ion Exchange, and Affinity.

Liquid-Solid operates on the basis of polarity. Compounds that

possess functional groups cabable of strong hydrogen bonding will

adhere more tightly to the stationary phase than less polar

compoounds. Thus, less polar compounds will elute from the column

faster than compounds that are highly polar.

Liquid-Liquid operates on the same basis as liquid-solid. However,

this technique is better suited for samples of medium polarity that are

soluble in weakly polar to polar organic solvents. The separation of

non-electrolytes is achieved by matching the polarities of the sample

and the stationary phase and using a mobile phase which possesses a

markedly different polarity.

Normal Phase operates on the basis of hydrophilicity and lipophilicity

by using a polar stationary phase and a less polar mobile phase. Thus

hydrophobic compounds elute more quickly than do hydrophilic

compounds.

Reverse Phase operates on the basis of hydrophilicity and

lipophilicity. The stationary phase consists of silica based packings with

n-alkyl chains covalently bound. For example, C-8 signifies an octyl

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chain and C-18 an octadecyl ligand in the matrix. The more

hydrophobic the matrix on each ligand, the greater is the tendancy of

the column to retain hydrophobic moieties. Thus hydrophilic

compounds elute more quickly than do hydrophobic compounds.

HPLC PUMPSThee several types of pumps available for use with HPLC

analysis are: Reciprocating Piston Pumps, Syringe Type Pumps, and

Constant Pressure Pumps.

Reciprocating Piston Pumps consist of a small motor driven piston

which moves rapidly back and forth in a hydraulic chamber that may

vary from 35-400 L in volume. On the back stroke, the separation

column valve is closed, and the piston pulls in solvent from the mobile

phase reservoir. On the forward stroke, the pump pushes solvent out

to the column from the reservoir. A wide range of flow rates can beattained by altering the piston stroke volume during each cycle, or by

altering the stroke frequency. Dual and triple head pumps consist of

identical piston-chamber units which operate at 180 or 120 degrees

out of phase. This type of pump system is significantly smoother

because one pump is filling while the other is in the delivery cycle.

Syringe Type Pumps are most suitable for small bore columns

because this pump delivers only a finite volume of mobile phase before

it has to be refilled. These pumps have a volume between 250 to 500

mL. The pump operates by a motorized lead screw that delivers mobilephase to the column at a constant rate. The rate of solvent delivery is

controlled by changing the voltage on the motor.

Constant Pressure Pumps- the mobile phase is driven through the

column with the use of pressure from a gas cylinder. A low-pressure

gas source is needed to generate high liquid pressures. The valving

arrangement allows the rapid refill of the solvent chamber whose

capacity is about 70 mL. This provides continuous mobile phase flow

rates.

MOBILE PHASE:The mobile phase in HPLC refers to the solvent being

continuously applied to the column, or stationary phase. The mobile

phase acts as a carrier for the sample solution. A sample solution is

injected into the mobile phase of an assay through the injector port. As

a sample solution flows through a column with the mobile phase, the

components of that solution migrate according to the non-covalent

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interactions of the compound with the column. The chemical

interactions of the mobile phase and sample, with the column,

determine the degree of migration and separation of components

contained in the sample. For example, those samples which have

stronger interactions with the mobile phase than with the stationary

phase will elute from the column faster, and thus have a shorter

retention time, while the reverse is also true.

COLUMNS The are various columns that are secondary to the

separating column or stationary phase. are: Guard, Derivatizing,

Capillary, Fast, and Preparatory Columns.Guard Columns are placed

anterior to the separating column. This serves as a protective factor

THE DETECTOR

The detector for an HPLC is the component that emits a response dueto the eluting sample compound and subsequently signals a peak on

the chromatogram. It is positioned immediately posterior to the

stationary phase in order to detect the compounds as they elute from

the column. The bandwidth and height of the peaks may usually be

adjusted using the coarse and fine tuning controls, and the detection

and sensitivity parameters may also be controlled (in most cases).

There are many types of detectors that can be used with HPLC. Some

of the more common detectors include: Refractive Index (RI), Ultra-

Violet (UV), Fluorescent, Radiochemical, Electrochemical, Near-Infra

Red (Near-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance(NMR), and Light Scattering (LS).

Ultra-Violet (UV) detectors measure the ability of a sample to absorb

light. This can be accomplished at one or several wavelengths:

Fluorescent detectors measure the ability of a compound to absorb

then re-emit light at given wavelengths. Each compound has a

characteristic fluorescence.Radiochemical detection involves the use

of radiolabeled material, usually tritium (3H) or carbon-14 (14C). It

operates by detection of fluorescence associated with beta-particle

ionization, and it is most popular in metabolite research. Two detector

typesomogeneous- Where addition of scintillation fluid to column

effluent causes fluorescence; Heterogeneous- Where lithium silicate

and fluorescence caused by beta-particle emission interact with the

detector cell

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INJECTORS : Samples are injected into the HPLC via an injection

port. The injection port of an HPLC commonly consists of an injection

valve and the sample loop. The sample is typically dissolved in the

mobile phase before injection into the sample loop. The sample is then

drawn into a syringe and injected into the loop via the injection valve.

A rotation of the valve rotor closes the valve and opens the loop in

order to inject the sample into the stream of the mobile phase. Loop

volumes can range between 10 l to over 500 l. In modern HPLC

systems, the sample injection is typically automated.

Stopped-flow Injection is a method whereby the pump is turned off

allowing the injecion port to attain atmospheric pressure. The syringe

containing the sample is then injected into the valve in the usual

manner, and the pump is turned on. For syringe type and reciprocation

pumps, flow in the column can be brought to zero and rapidly resumedby diverting the mobile phase by means of a three-way valve placed in

front of the injector. This method can be used up to very high

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VPT 607 Veterinary Chemotherapy Lab Manual