Iycee Charles de Gaulle Summary Cup Plate Method Cultivation Of Microorganism Biology Essay

Cup Plate Method Cultivation Of Microorganism Biology Essay

An Adduct formed by stirring ( 0.01 mole ) of aromatic aldehyde with the 40 % of NaHSO3. O-phenylenediamine ( 0.01 mole ) was dissolved in 50 milliliter of warm Ethanol 80. The NaHSO3 adduct of the aldehyde is added easy with changeless stirring in the warm solution of O-phenylenediamine stirred for 20-30 min still solid merchandise obtained, so added 100 milliliter of Distilled H2O and filtered. Now the merchandise was recrystallised by utilizing Ethanol.

Measure 2: Nicotinoyl Cloride

0.1 mole of Nicotinic Acid was refluxed for 6 hour with the 20 milliliter of Thionyl Chloride. After this the surplus of Thionyl Chloride was distilled off and separated from the merchandise and dried it.

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Measure 3:

0.01 mole of 2-phenylbenzimidazole solution in 100 milliliters Pyridine stirred for 8 hours invariably with the 0.01 mole of Nicotinoyl Chloride, so the H2O added 50 milliliter to acquire a solid merchandise. The merchandise was filtered, dried and recrystallised utilizing Ethanol.

Scheme:

AIM AND OBJECTIVES

Molecular alteration of a promising lead compound is still a major line of attack for the find of new drug. Molecular alteration involves replacing, riddance, or adding new medieties to a parent lead compound, there by doing gradual alterations in the physico-chemical belongingss of the parent compound and therefore biological activity of the compound.

It is clear from the literature reappraisal that a figure of Benzimidazole derived functions are known for the, antibacterial, fungicidal and ant-inflammatory activities belongingss.

The present surveies were performed with the following aims:

Synthesis of new series of 1,2-substituted benzimidazole derived functions.

Word picture of freshly synthesized compounds by spectra methods viz.infrared spectra ( IR spectra ) , Nuclear magnetic resonance spectra ( ?H NMR spectra ) and ( Mass spectra ) .

Screening of the antibacterial and Antifungal of the freshly synthesized compounds utilizing assorted strains of bacteriums and Fungis by finding their MIC.

Screening of anti-inflammatory action of Benzimidazole derived functions.

Scope and Plan of work:

Literature study revealed that Benzimidazole karyon is a portion legion category of reported molecules exhibiting diverse scope of biological activities like antibacterial, fungicidal, antiviral, anticancer, analgetic, anti-inflammatory activity, antihyperlipidemic, antihistaminic, antiulcer, anti-arrhythmic, HIV-RT inhibitor. Sing the reported informations about Benzimidazole karyon we have tried to synthesise some Nicotinoyl derived functions of Benzimidazole. The Benzimidazole derived functions of all above mentioned activities are largely of 2-substituted type.The synthesis of 2- ( substituted phenyl ) -benzimidazolyl-1-pyridinyl-3-methanone was carried out and screened for antibacterial, fungicidal, and anti-inflammatory activity.

The present work was divided in to three subdivisions:

Synthesis of 1,2-substituted derived functions of Benzimidazole.

Chemical Characterisation of the synthesized compounds.

Biological rating of synthesized compounds.

Pharmacological showing of the synthesized compounds.

ANTIMICROBIAL Screening

An antibiotic is a chemical compound that in high dilution hinders the growing and the endurance of one or more species of microorganism.A drug is considered to hold bacteriostatic or fungistatic activity when it inhibits the growing of bacteriums or fungi severally and bactericidal or fungicidal activity when it kills the bacterium or Fungi. In vitro trials are used as screening process for new agents and for proving the susceptibleness of single isolates from infection to find which of the available drug might be utile therapeutically.

Important factors for antimicrobic activity are size of the inoculants, metabolic province of micro-organism, pH, temperature, and continuance of interaction, concentration of the inhibitor and presence of interfering substance.

ANTIBACTERIAL ACTIVITY STUDIES

Literature study reveals that the synthesis and rating of antibacterial activity of assorted 2-substituted benzimidazole derived functions. The development of resistant among assorted infective micro-organisms towards the antibiotics has increased the drift for look intoing new antimicrobic agent. When a compound are synthesized in the hope that one of them would be more effectual than the bing 1. The antimicrobic effectivity of a compound can be evaluated by consecutive dilution method and cup home base method. Dilution susceptibleness trials are used to find the Minimum Inhibitory Concentration ( MIC ) .

MIC is the lowest concentration of a drug that inhibits the growing of a peculiar being under specific status. The sensitiveness of a compound against a peculiar being can be studied by cup home base method.Initially the zone of suppression method was carried out to measure the sensitiveness of the being were selected for finding of MIC.

CUP PLATE METHOD:

Cultivation of Microorganism:

The undermentioned micro-organisms were used to analyze the antibacterial activity.

Bacillus subtilis – Gram positive bacteriums

Staphylococcus aureous – Gram positive bacteriums

Escherichia coli – Gram negative bacteriums

Salmonella typhi – Gram negative bacteriums

Standard: Streptomycin ( 1000mcg )

Solvent: DMF

All the trial compounds were tested at 250 µg, 500 µg, and 1000 µg.

Preparation of the medium:

Composition of alimentary agar medium

Beef extract………..10g

Peptone……………..10g

Sodium chloride……..5g

Agar………………….20g

Purified water………1000ml

pH 7.2± 0.2

The medium was prepared by fade outing the specified measure of the dehydrated medium in purified H2O by heating on a H2O bath and were dispensed in 100 milliliter volume conelike flasks. The conelike flasks were closed with cotton stoppers and were sterilized by autoclaving at 121 & A ; deg ; C ( 15 lb psig ) for 15 proceedingss.

The contents of the conelike flasks were poured aseptically into unfertile Petridishes are allowed to solidify. These sterilized Medias were used to subculture the bacterial civilization.

Procedure:

Each Petridish was filled to a deepness of 4-5 millimeter with a alimentary agar medium that was antecedently inoculated with suited inoculants of suited trial being, and so allowed to solidify. The petridish were specially selected with level underside and were placed on degree surface so as to guarantee that the bed of medium is in unvarying thickness. The petridishes were sterilized at 160-170 & A ; deg ; C in hot air oven for 30 mins before usage. Small unfertile bore bit of unvarying size was placed about at 10 centimeter tallness, holding an internal diameter of about 6-8 millimeter and made of aluminum ( or ) chromium steel steel. Each home base was divided in to four equal parts along the diameter. To each part one cylindrical pit was made in medium with the aid of unfertile bore bit. Three pits for trial compounds and one pit for the criterion. The petridishes were incubated at 37 & A ; deg ; C for 18 hours. Diameter of the zone of suppression was measured and the mean diameter for each sample was calculated. The diameter obtained by the trial sample was compared with that produced by standard Streptomycin.

Compound

Concentration

( µg )

Zone Of Inhibition ( millimeter )

S.subtilis

S.aureus

S.typhi

E.coli

A

250

500

1000

14

15

17

13

16

18

15

18

20

14

16

18

Bacillus

250

500

1000

8

9

10

7

8

9

8

9

10

8

9

10

C

250

500

1000

10

11

12

9

10

11

10

12

14

10

11

12

Calciferol

250

500

1000

10

11

12

9

10

10

10

11

12

10

11

12

Tocopherol

250

500

1000

9

9

10

8

9

10

10

11

11

9

10

11

F

250

50

1000

9

10

11

9

9

10

9

10

11

9

10

11

Gram

250

500

1000

10

10

11

9

10

11

10

11

12

10

11

12

Hydrogen

250

500

1000

10

11

12

10

10

11

9

10

11

9

10

11

Venereal disease

Streptomycin

1000

13

10

22

15

Fungicidal Activity:

CUP PLATE METHOD:

Cultivation of Microorganism

The undermentioned fungous strains were used to analyze the antibacterial activity.

1. C.raphigera

2. A.polytricha

Standard: Ketocanazole ( 1000mcg )

Solvent: DMF

All the trial compounds were tested at 250 µg, 500 µg, and 1000 µg.

Preparation of the medium:

Composition of alimentary agar medium

Sabraoud Dextrose stock… … … ..64gm

Distilled H2O… … … … … … … … … .1000ml

pH… … … … … … … … … … … … … … … ..7.2± 0.2

The medium was prepared by fade outing the specified measure of the dehydrated medium in purified H2O by heating on a H2O bath and were dispensed in 100 milliliter volume conelike flasks. The conelike flasks were closed with cotton stoppers and were sterilized by autoclaving at 121 & A ; deg ; C ( 15 lb psig ) for 15 proceedingss.

The contents of the conelike flasks were poured aseptically into unfertile Petridishes are allowed to solidify. These sterilized medias were used to subculture the fungous civilization.

ROCEDURE:

Each Petridish was filled to a deepness of 4-5 millimeter with a alimentary agar medium that was antecedently inoculated with suited inoculants of suited trial being, and so allowed to solidify. The petridish were specially selected with level underside and were placed on degree surface so as to guarantee that the bed of medium is in unvarying thickness. The petridishes were sterilized at 160-170 & A ; deg ; C in hot air oven for 30 mins before usage. Small unfertile bore bit of unvarying size was placed about at 10 centimeter tallness, holding an internal diameter of about 6-8 millimeter and made of aluminum ( or ) chromium steel steel. Each home base was divided in to four equal parts along the diameter. To each part one cylindrical pit was made in medium with the aid of unfertile bore bit. Three pits for trial compounds and one pit for the criterion. The petridishes were incubated at 37 & A ; deg ; C for 18 hours. Diameter of the zone of suppression was measured and the mean diameter for each sample was calculated. The diameter obtained by the trial sample was compared with that produced by standard Ketocanazole.

Table: Antifungal activity of Benzimidazole Derived functions

Compound

Concentration

( µg )

Zone of Inhibition

C.raphigera

A.polytricha

A

250

500

1000

23

24

25

24

25

26

Bacillus

250

500

1000

22

24

24

24

26

28

C

250

500

1000

24

26

28

19

22

27

Calciferol

250

500

1000

21

23

24

21

22

25

Tocopherol

250

500

1000

23

25

26

20

22

24

F

250

50

1000

23

24

26

25

26

27

Gram

250

500

1000

23

24

27

19

22

25

Hydrogen

250

500

1000

22

23

24

16

20

24

Venereal disease

Ketocanazole

1000

26

30