antimicrobial and parasiticidal screening ppt

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INTRODUCTION Chemotherapy - term originally used to describe the use of drugs that are ‘ selectively toxic ’ to invading micro-organisms while having minimal effects on the host. All living organisms including humans are prey to infection. susceptible to diseases caused by viruses, bacteria, protozoa, fungi &helminthes. Word microbe - bacteria ,viruses ,fungi and word parasite -protozoa and helminths . The feasibility of selective toxicity depends on the ability to exploit the biochemical differences as may exist between the infecting organism & the host. 3


GENERAL METHODS FOR ANTIMICROBIAL SCREENING divided into types based on the principle applied in each system. They include : Diffusion methods. Dilution Methods. Dilution and Diffusion. 4

Disc diffusion methods :

Disc diffusion methods i.Kirby -Bauer method ii.Stokes Method 5

KB testing or disk diffusion antibiotic sensitivity testing:

KB testing or disk diffusion antibiotic sensitivity testing 1.Inoculation of test plates A defined inoculum (compared to McFarland 0.5 OD standard)is streaked as a lawn onto a large Mueller-Hinton agar or Blood agar plate in 3 directions to ensure confluence. This procedure is repeated – to ensure an even distribution of inoculum . 6

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Blood agar plate Mueller-Hinton agar 7

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2.Application of Discs to Inoculated Agar Plates antibiotic-impregnated disks are placed onto the agar surface. Each disc must be pressed down to ensure complete contact with the agar surface. They must be distributed evenly so that they are no closer than 24 mm from center to center. 8

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The plates are inverted and placed in an incubator set to 35  C within 15 minutes after the discs are applied. 3. Reading Plates and Interpreting Result After 16 to 18 hours of incubation, each plate is examined. Zones of inhibition - uniformly circular and there will be a confluent lawn of growth. The diameters of the zones of complete inhibition are measured, including the diameter of the disc. 9

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Measuring of zones -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 black, nonreflecting background and illuminated with reflected light. In blood agar media - the zones are measured from the upper surface of the agar illuminated with reflected light, with the cover removed. 10

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Read the plates in transmitted light 11

Stokes’ Method:

Stokes’ Method In original Stoke’s method, a control organism is inoculated on part of a plate and the test organism is plated on the remainder. Disks are placed at the interface and the zones of inhibition are compared . 12

Dilution Methods:

Dilution Methods Agar and Broth dilution methods for Minimum Inhibitory Concentration determination. Minimum Inhibitory Concentration (MIC)- To confirm resistance of micro organisms to an antimicrobial agent To monitor the activity of new antimicrobial agent s. 13

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14 Broth dilution method The Antibiotics are diluted to various dilution to test the minimum inhibitory concentration.

The Agar dilution Method :

The Agar dilution Method Agar dilutions are most often prepared in petri dishes. The dilutions are made in a small volume of water and added to agar which has been melted and cooled to not more than 60 o C. Antimicrobial agents and range of dilutions(mg/ml) Standard panel Pn G 0.008 - 64.0 Tetracycline 0.06 - 64.0 Cefixime 0 .002 - 2.0 Ciprofloxacin 0.001 – 16.0 15

Dilution and Diffusion:

Dilution and Diffusion E Test( epsilometer test ) quantitative method applies both the dilution of antibiotic and diffusion of antibiotic into the medium. 16

The strips are impregnated with various concentration of Antibiotics:

The strips are impregnated with various concentration of Antibiotics

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When this E test strip is applied onto an inoculated agar plate, there is an immediate release of the drug. Following incubation , a symmetrical inhibition ellipse is produced. The intersection of the inhibitory zone edge and the calibrated carrier strip indicates the MIC value over a wide concentration range (>10 dilutions). 18

Antibacterial Screening methods:

Antibacterial Screening methods Conditions for the Culture of Bacteria Temperature : Most bacteria cultured in the laboratory are classified as mesophiles - prefer temperatures between 20 and 40°C. pH: Most bacteria grow optimally in media with a pH between 6 and 8 . Very few bacteria can grow in acidic conditions. Nutrients: Bacteria need a source of carbon, nitrogen and mineral salts as raw ingredients for cellular growth. 19

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Gaseous environment: Aerobic bacteria Obligate anaerobes -( e.g.Clostridium ) Facultative anaerobes All bacterial cultures benefit from a low concentration of carbon dioxide. 20

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Bacterial Strain Incubation period Incubation temperature Disease Produced Corynebacterium diptheriae 1-8 days 36-37°C Diptheria Bordetella pertussis 7-10 days 35  C Pertussis Clostridium tetani 4-10 days 37  C Tetanus Clostridium botulinum 4-14 days 24-27  C Botulism Escherichia coli 3-8 days 37  C gastroenteritis Klebsiella pneumoniae 1-3 days 37°C Klebsiella pneumonia

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Staphylococcus aureus 1-6 hours 36-38 ° C Meningitis Osteomyelitis Endocarditis Streptococcus pyogens 1-3 days 37° C Scarlet fever Impetigo or cellulitis Bacillus anthracis 1-7 days 35° C Anthrax Haemophilus influenza 2-4days 37 ° C Bacteremia pnenomonia Pseudomonas aerugionasa 1-3 days 35 ° C Malignant external otitis Folliculitis Micrococcus luteus 24 hour 30°C Meningitis 22

In vitro screening methods:

In vitro screening methods 23

In vivo assay:

In vivo assay Fifteen male Balb /c mice (15–17 g) aged between 5 and 6 weeks were used for the experiments. Kept in a temperature-controlled room under a 12 h light 12 h dark cycle. Mice were divided into the following groups: Control, Salmonella-infected (SI) and Salmonella-infected + EtOH extract( SIEt ) Each treatment group contained five mice. S.typhimurium was grown overnight in Luria– Bertani broth , centrifuged, washed in phosphate-buffered saline (PBS) & diluted. 24

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The SI and SIEt groups- exclusively inoculated using gavage needle orally with approximately 105 CFU of S. typhimurium in a 0.1 mL volume. One hour after infection, animals in the SIEt group were orally administered 5 mg of the PGEt daily, whereas control and SI animals were not. Fecal samples were then collected 0, 1, 2, 3, 4, 5 and 6 days after the bacterial suspensions were administered and the numbers of the bacteria per gram of feces were determined. 25

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Aliquots (100 μl ) of fecal suspensions were serially diluted in PBS and then plated on Salmonella- Shigella agar plates , which were subsequently incubated overnight at 37°C. Typical colonies were then counted on plates that contained between 30 and 300 colonies . At Day 4 post-infection, the mice were sacrificed, and tissue specimens of the kidney, liver, intestine and spleen organs were transferred to 10% buffered neutral formalin for histopathologic examinations. 26


Salmonella on SS agar give a black color as a result H2S production 27

Antimycobacterial screening:

Antimycobacterial screening Human tuberculosis (TB) is a contagious-infectious disease mainly caused by M. tuberculosis. an aerobic pathogenic bacterium that establishes its infection usually in the lungs . 28

Invitro screening methods:

Invitro screening methods 1.Broth dilution method. 2.Agar diffusion method and dilution method. 3.Other methods. 1.Broth dilution method organism M. tuberculosis strain H37Rv were grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80 and 10% ADC at 37°C under biosafety level 3 conditions. Bacterial culture medium was prepared with endotoxin -free materials. 29

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viability of the bacilli - determined by colony assay on Middlebrook 7H11 agar plates. Method 100 µl of the drug solution twofold serially diluted with Middlebrook 7H9 broth medium supplemented with 0.05% Tween 80 and 10% ADC were prepared in a 96-well plate. 100 µl of the broth medium containing 1 × 10 4 to 2 ×10 4 organisms was added. . 30

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The plate was incubated at 37°C for 2 weeks. MIC - the lowest concentration of agents that inhibit visible growth of the bacteria. The partial reduction in turbidity was scored as negative. Control : M.tuberculosis strain H37Rv grown in Middlebrook 7H9 medium with out test drug. 31

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MIC of certain antimycobacterial agents as determined by BDT . Antibiotics MIC INH 0.107 STR 0.227 RIF 0.026 EMB 1.733 32

2.Agar diffusion method :

2.Agar diffusion method PROCEDURE Strains of M. tuberculosis used: Three strains : the rifampicin -sensitive 28-25271, rifampicin resistant TMC-331 strain and the H37Rv strain. Culture and preparation of M. tuberculosis inoculum : Stored strains were cultured using the Middle brook 7H11 agar supplemented with oleic acid albumin- catalase (OADC). No adjustments for pH were made. 33

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Method The stock solution (50mg/ml of test drug)was sterilized using 0.2 μm single use filters. Sterile distilled water was used for further dilution. A concentration of 50.0 μg per well was used for the general susceptibility tests. A stock solution of rifampicin , 3.0 mg/ml was prepared from a preserved frozen stock of a concentration of 20.0 mg/ml. A concentration of 1.0mg/ml was used for susceptibility testing in the case of rifampicin . 34

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The medium for incubation was sterile Middle brook 7H11 agar in 90-mm diameter Petri dishes with quadrants. In each quadrant of the Petri dish was put 5.0 ml of the medium. The solidified medium in the quadrants was inoculated using the flooding method so that a uniform surface distribution of inoculum was obtained. Wells of 5.0 mm diameter and 2.5 mm depth were then bored in the dry inoculate medium using a sterile cork borer. 35

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50 μl of the test extract, were dispensed into the well of the first quadrant in each Petri dish giving an extract concentration of 50.0 μg per well. A volume of 50 μl of the 1.0 mg/ml solution of rifampicin and an equal volume of each of the plant extracts were dispensed into the well of the second quadrant giving a drug concentration of 1.0 μg per well. The well in the third quadrant was left empty as a control, while the well in the fourth quadrant was filled with the solvent used to dissolve the extract, also as a control. 36

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The Petri dishes were then left in the hood overnight to allow diffusion of the extracts and drug . then sealed with a carbon dioxide-permeable tape . incubated at 37°C in a carbon dioxide incubator for up to four weeks. Activity of extracts and rifampicin was determined from the zone of inhibition surrounding the well. 37

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The sensitivity of M. tuberculosis to the test extracts and the drug was determined by measuring the zones of inhibition surrounding the well using a millimetre scale. The actual diameter of zone of inhibition was got after subtracting the diameter of the well. Three replicates of each test were done for each MTB strain. A rough Minimum Inhibitory Concentration (MIC ) was determined. 38

Agar dilution method:

Agar dilution method The agar dilution method was used to get the definitive MIC value. The test extract concentrations were dispensed in the medium at intervals of 5.0 mg/ml to determine the actual MIC. The MIC - concentration of the drug or extract that inhibited growth of M. tuberculosis by 99 percent or greater, in comparison to the positive growth control. 39

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Fresh cultures (up to 3 weeks old) were used as source of M. tuberculosis using a standardized inoculum prepared basing on McFarland No.1 standard. Serial dilutions of the test extracts and rifampicin were made and each solution added to the autoclaved medium cooled up to 50 ₒ C. The concentrations were made at 10 ml per ml of medium . 5 ml of the medium at each of the concentrations was then poured into quadrants corresponding to the concentrations in the Petri dishes. 40

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One quadrant in each Petri dish contained the medium with no drug as the growth control. The quadrants were then inoculated with10 -4 dilution of inoculum at McFarland standard No.1. Activity was then determined from the number of colonies in each quadrant. After the period of incubation, the numbers of colonies on the drug containing quadrants were determined and expressed as percentages of those on the drug-free quadrant 41

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Minimum Bactericidal Concentration (MBC )Tests: the concentration of the drug or extract that prevented growth by 99 percent or greater, compared with the untreated controls. OTHER INVITRO METHODS Microplate Alamar Blue Assay and Mycobacterium Growth Indicator Tube, MB/ BacT , and EPS II. Radiometric methods (BACTEC 460 system). (BACTEC TB 460 system 42

Screening of Antifungal agents:

Screening of Antifungal agents Fungal infections ( mycoses ) - generally associated with the skin or mucous membranes. Superficial fungal infections-classified into dermatomycoses and candidiasis . Dermatophytoses -most commonly caused by Trichophyton , Microsporum or Epidermophyton , giving rise to various types of 'ringworm or tinea . 43

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In superficial candidiasis -the yeast-like organism may infect the mucous membranes of the mouth or vagina (thrush), or the skin Commonest systemic fungal disease is candidiasis . Other more serious conditions are cryptococcal meningitis, endocarditis, pulmonary aspergillosis , and rhinocerebral mucormycosis . 44

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Organism Principal disease(s) Incubation period Incubation temperature 1.Yeasts Cryptococcus neoformans Meningitis 3-4days 30-35°C 2.Yeast-like fungus Candida albicans Thrush, systemic candidiasis Several days to several weeks 25-30 °C 3.Filamentous fungi Trichophyton spp. Epidermophyton floccosum Microsporum spp All these organisms cause skin and nail infections and are referred to as tinea or ‘ringworm’. 10-15 days 25 or 30 °C 45

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4.Dimorphic fungi Histoplasma capsulatum Histoplasmosis 2-4 weeks Below 30°C Coccidioides immitis Coccidiomycosis 7-21 days 30°C Blastomyces dermatides Blastomycosis 30-100 days 33°C 46

Skin mycosis:

Skin mycosis In vitro inhibitory activity The test organisms are : various strains of dermatophytes : Trichophytum mentagrophytes , T. rubrum, T. verrucosum, T. equinum, T. gallinum,Microsporum canis, Microsporum gypseum and yeasts : Candida albicans , Ca. tropicalis , Ca. pseudotropicalis , Ca. krusei , Ca. parapsilosis , Ca. lipolytica,Ca . brumpti , Ca. utilis , Torulopsis glabrata . 47

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PROCEDURE The studies are performed by means of conventional serial dilution procedures in test medium without and with addition of 4% bovine albumin. The test medium - Sabouraud dextrose broth containing 1% Neopeptone and 2% glucose. The basic medium is sterilized in an autoclave at 121 °C for 15 min. The pH is adjusted to 6.5 with 1 N NaOH . The medium containing albumin is sterilized by filtration through a membrane filter. 48

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preparation of the test series - the inhibiting substances are dissolved in methanol and then rapidly diluted with slightly warmed test medium. Each test tube contains 3 ml. The organisms are pre-cultured on a modified Grütz agar at 28 °C for periods of 1–4 weeks. The suspensions are adjusted by photometry that about 105 microconidia of dermatophytes & 104 yeast cells per ml are obtained in each inoculated test tube. The minimal inhibitory concentrations - measured after 14 days incubation at 28 °C. 49

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EVALUATION The percentage of strains of Trichophytum mentagrophytes , Trichophytum rubrum , and Candida albicans is plotted against dilution steps for each test compound with and without albumin and IC50 values are calculated. 50

In vivo activity in the guinea pig Trichophytosis model:

In vivo activity in the guinea pig Trichophytosis model PROCEDURE Male albino guinea pigs ( Pirbright White), bred mycosis- free, weighing 450–550 g are taken. On both sides of the back,areas of 5 × 12 cm are shorn to a fur length of 1 mm. Three areas with a diameter of 3 mm are inoculated with a pipette on either side. 51

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Per injection site-104 spores of Trichophyton mentagrophytes 2 114 in 0.05 ml suspension in physiological saline solution are inoculated. Three days after inoculation, infections with reddening and scale formations are observed. From days 3–7 after the infection, 1 ml of the test preparation or standard is applied onto the right animal sides and rubbed in once daily. The diameters (mm) of all alopecias are measured with a ruler 3.5 weeks after the infection. 52

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EVALUATION The values of alopecias , separated according to the treated group and animal side, are determined and statistically evaluated using Duncan’s new multiple range test. 53

Antiparasitic Screening:

Antiparasitic Screening Molluscicidal & Piscicidal Activity Evaluation Schistosomiasis - a parasitic disease A general strategy for schistosomiasis control is to remove a link of the vital cycle i.e. the snail Inorganic or organic molluscicides : -cause serious environmental hazards & possible buildup of snail resistance & toxicity in non target organisms. 54


Bioassay Molluscicidal tests - made with Biomphalaria glabrata , Snails reared in aquaria with a continuous circulation of water through a filter system,at a temp. of about 24°C. Aquatic snails employed for the tests are young-mature & relatively uniform in age & size(avg. diameter of shell 8mm). The snails are placed in de-chlorinated tap water at 26º-28°C under laboratory lighting conditions with normal diurnal light changes. Exposures of 24h to potential molluscicides are performed & death is established by examination (discoloration ,heart rate, activity of muscle.) 55

Evaluation of Acute Toxicity by Rapid Screening Procedure:

Evaluation of Acute Toxicity by Rapid Screening Procedure The test drug is dissolved or suspended in water at a conc.of 1000 ppm . Two containers at this concentration with 2 snails each are used,with the volume per snail not less than 40ml. Control- one container with two snails each is included. In the event of both snails dead ,a series of dilutions(eg:500,250,100,50 ppm ),are tested. For these assays ,2 containers at each concentration with 10 snails are used with the volume per snail not less than 40ml. 56

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As a control, 1 or 2 containers with 10 snails each are included. to confirm the mortality of the snails- placed in a beaker containing distilled water alone after 24h their condition is re-checked. The minimal lethal concentration required to kill both snails after 24h is thus established . 57

Antiprotozoal Screening:

Antiprotozoal Screening Giardiasis is a common g.i infection caused by Giardia lambia . Evaluation of Antigiardial Effect In vitro Activity A 0.1ml inoculum of exponentially growing(24h old)culture of Giardia lamblia containisng approx. 2000 trophozoites is dispensed into a cavity slide. The test sample in the desired conc. of 0.1ml TYI-S-33 medium is added in the cavity mounted with a glass coverslip & the edges of the cavity are sealed with paraffin wax. 58

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The slides are kept in a moist chamber at 37°C & incubated for 24h. The no: of dead & live trophozoites is recorded to determine the antigiardial activity of the sample. Invivo Screening Swis mice(18-20g) free from Giardia muris infection are used . An axenic strain of Giardia lamblia , grown in TYI-S-33 medium for 48hr, is harvested by centrifugation. The supernatent is discarded & the pellet is dispersed in fresh medium. 59

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An aliquot of 0.25ml,containing 0.5-1.0×10 6 trophozoites , is injected intrajejunally in mice. After 48 h, the animals are divided into two groups of 6 each. One group was given an oral administration of varying doses of drug daily for 5 consecutive days while the other set without drug served as the control. 60

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Animals are then killed & jejunum is removed and fleshed with PBS (pH=7.2). The fleshing in 1ml aliquot is examined microscopically for live trophozoites . Recovery in the experimental set (E: infected + drug treated) is compared with control set(C: infected without drug treatment). % Recovery= C-E ×100 C 61

Antimalarial Screening:

Antimalarial Screening In vitro screening tests The test is carried in a 96 flat-bottomed wells in microtitre plates. Extract is dissolved or micronised in ethanol & a series of 6 conc. prepared by 10 fold dilutions in RPMI 1640 medium . The ethanol conc. of dilutions for testing is kept below 0.1%. Aliquots(50µl) of culture medium is then added to each well. Human blood(50µl) containing 1% parasitaemia is added to each well. 62

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Two series of controls are set up 1. with parasitised blood without addition of extract 2. with uninfected red blood cells. After incubation in 3% O 2 ,4% CO 2 & 93% N 2 gas phase for 18h at 37°C,50µl of G-[ 3 H] hypoxanthine is added to each well. Incubation continued at 37°C for a further 18-24h. RBC’s are harvested with a Titertek Cell Harvester washing out the wells with normal saline & filtering through glass fibre membrane. 63

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RBC’s are harvested with a Titertek Cell Harvester washing out the wells with normal saline & filtering through glass fibre membrane. Membranes are dried , placed in scintillation fluid & incorporation of [ 3 H] hypoxanthine determined by scintillation counting. The % inhibition of incorporation & IC 50 values are determined. 64

Invivo Screening Tests :

Invivo Screening Tests 1.Peter’s Test Male mice( swiss albino) weighing 20±2 g are maintained at 22°C in batches of 5 & fed on std diet. Blood from donor mouse with rising parasitaemia is diluted in tissue culture medium so that each 0.2ml contains 10 7 infected red cells. Each mouse receives 0.2ml i.v via the tail on day zero. 65

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Extract is either suspended or dissolved by trituration or sonication after the addition of 0.2% solution of Tween 80 or 0.5 DMSO. Initial aq. conc. of doses ranging b/w 1 and 100 mg/kg are administered daily from initial day of infection for 4 successive days either by s.c or oral routes. On the 5 th day samples are taken from tail blood & stained with a suitable stain( eg.Giemsa ) Parasitised red cells are recorded as a percentage of total ED 50 values( i.e 50% suppression of parasites when compared with controls) 66

2.The Rane Test:

2.The Rane Test The basis of the test is to compare the effect of a std inoculum of P.berghei, which kills mice within 6 days with extension of survival time to 12 days by a single dose of test compound. An inoculum of 10 6 infected donor cells is given i.p on day 1 . Test compound is suspended in arachis oil are given s.c at an initial dose range of 640,320,160 & 80 mg/kg on day 4. The minimum effective dose(MED) is obtained & compared with the maximum tolerated dose(MTD) that produces no more than one in five toxic deaths. 67

Amoebicidal Screening:

Amoebicidal Screening Amoebiasis was defined by WHO as ‘a condition in which a patient is harbouring the organism Entamoeba histolytica in the bowel’. E.histolytica exist either as mobile trophozoites or as cysts. In vivo screening Many animals models have been used in the study of amoebiasis,the rat is used as the host for intestinal infections & the hamster for hepatic infections. E.histolytica is preferred to other Entamoeba species for chemotherapeutic evaluation. 68

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Amoebae are introduced directly into the caecum or liver by injection or through the abdominal wall. One successful technique for the production of hepatic amoebiasis utilizes small pieces of sponge impregnated with amoebae & inserted into the right liver lobe. Procedure The animals are treated 7 times by oral administration of test drug,the I st dose being given one day prior to challenge infection & the II nd 2h after infection. 69

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The remaining 5 doses are given on the next 5 consecutive days after infection. standard drugs - Metronidazole & emetine. Hepatic infections in golden hamsters are introduced by injection of trophozoites of axenic E.histolytica into liver lobes. The inoculum per hamster is approx. 1×10 5 .Six infected & untreated animals are used as controls. Autopsy of the hamsters is carried out on the 6 th day after infection & liver tissue smears from the date of infection examined microscopically for mobile trophozoites . 70

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Animals with no liver necrosis & free of amoebae are considered as cured. To study intestinal infection,a polyaxenic strain of E.histolytica is used to infect weaning rats by inoculation of 1×10 5 trophozoites in 0.5 ml into the caecal lumen. Ten infected & untreated rats are used as controls. The animals are killed on the 7 th day after infection & examined for amoebae & intestinal lesions. Animals without intestinal abscesses & free of amoebae are considered cured. 71

Invitro screening methods :

Invitro screening methods Sufficient test compound to give a serial dilution with the range of 0.01-100 µg /ml is dissolved in the appropriate volume of growth medium with the aid of 40µl of DMSO. The solution sterilized through a 0.22 µm filter. 0.1ml of amoebal suspension is added to each 12ml flat sided test tubes which is filled with medium to exclude air . Incubated at 37°C for 72h with the flat surface down. After incubation, the amoebae are examined through an inverted microscope. 72

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The amoebae are detached from the tube walls by cooling in ice water for 30 min & then centrifuged at 1000 rpm for 30 min. The supernatant is removed carefully from each tube & the pellet suspended in 250µl of medium. An equal volume of 1% Trypan Blue in normal saline is added & the amoebae counted on a haemocytometer. The no: of dead & alive amoebae are recorded. 73


REFERENCE PuloK . K.Mukherjee,Quality Control of Herbal Drugs, Second edition,615-625. H. Gerhard Vogel , Drug Discovery and Evaluation - Pharmacological Assays,second edition,1345-1346. Oto Zak et. Al, Hand book of animal model of infection: experiment model ,page 887. 74

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