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Cup Plate Method | Cultivation Of Microorganism

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 ml of warm Ethanol 80. The NaHSO3 adduct of the aldehyde is added slowly with constant stirring in the warm solution of O-phenylenediamine stirred for 20-30 min still solid product obtained, then added 100 ml of Distilled water and filtered . Now the product was recrystallised by using Ethanol.
Step 2: Nicotinoyl Cloride
0.1 mole of Nicotinic Acid was refluxed for 6 hrs with the 20 ml of Thionyl Chloride. After this the excess of Thionyl Chloride was distilled off and separated from the product and dried it.
Step 3:
0.01 mole of 2-phenylbenzimidazole solution in 100 ml Pyridine stirred for 8 hrs constantly with the 0.01 mole of Nicotinoyl Chloride ,then the water added 50 ml to get a solid product. The product was filtered, dried and recrystallised using Ethanol.
Scheme:
AIM AND OBJECTIVES Molecular modification of a promising lead compound is still a major line of approach for the discovery of new drug. Molecular modification involves substituting, elimination, or adding new moieties to a parent lead compound, there by making gradual changes in the physico-chemical properties of the parent compound and thus biological activity of the compound.
It is clear from the literature review that a number of Benzimidazole derivatives are known for the, antibacterial, antifungal and ant-inflammatory activities properties.
The present studies were performed with the following objectives:
Synthesis of new series of 1,2-substituted benzimidazole derivatives.
Characterization of newly synthesized compounds by spectra methods viz.infrared spectra (IR spectra), Nuclear magnetic resonance spectra (¹H NMR spectra) and (Mass spectra).
Screening of the antibacterial and Antifungal of the newly synthesized compounds using various strains of bacteria and fungi by determining their MIC.
Screening of anti-inflammatory action of Benzimidazole derivatives.
Scope and Plan of work: Literature survey revealed that Benzimidazole nucleus is a part numerous class of reported molecules exhibiting diverse range of biological activities like antibacterial, antifungal, antiviral, anticancer, analgesic ,anti-inflammatory activity, antihyperlipidemic, antihistaminic, antiulcer, anti-arrhythmic , HIV-RT inhibitor. Considering the reported data about Benzimidazole nucleus we have tried to synthesize some Nicotinoyl derivatives of Benzimidazole. The Benzimidazole derivatives of all above mentioned activities are mostly of 2-substituted type .The synthesis of 2-(substituted phenyl)-benzimidazolyl-1-pyridinyl-3-methanone was carried out and screened for antibacterial, antifungal, and anti-inflammatory activity.
The present work was divided in to three sections:
Synthesis of 1,2-substituted derivatives of Benzimidazole .
Chemical Characterisation of the synthesized compounds.
Biological evaluation of synthesized compounds.
Pharmacological screening of the synthesized compounds.
ANTIMICROBIAL SCREENING An antibiotic is a chemical compound that in high dilution hinders the growth and the survival of one or more species of microorganism.A drug is considered to have bacteriostatic or fungistatic activity when it inhibits the growth of bacteria or fungi respectively and bactericidal or fungicidal activity when it kills the bacteria or fungi. In vitro tests are used as screening procedure for new agents and for testing the susceptibility of individual isolates from infection to determine which of the available drug might be useful therapeutically.
Important factors for antimicrobial activity are size of the inoculums, metabolic state of microorganism, pH, temperature, and duration of interaction, concentration of the inhibitor and presence of interfering substance.
ANTIBACTERIAL ACTIVITY STUDIES Literature survey reveals that the synthesis and evaluation of antibacterial activity of various 2-substituted benzimidazole derivatives. The development of resistant among various pathogenic microorganisms towards the antibiotics has increased the impetus for investigating new antimicrobial agent. When a compound are synthesized in the hope that one of them would be more effective than the existing one. The antimicrobial effectiveness of a compound can be evaluated by serial dilution method and cup plate method. Dilution susceptibility tests are used to determine the Minimum Inhibitory Concentration (MIC).
MIC is the lowest concentration of a drug that inhibits the growth of a particular organism under specific condition. The sensitivity of a compound against a particular organism can be studied by cup plate method.Initially the zone of inhibition method was carried out to evaluate the sensitivity of the organism were selected for determination of MIC.
CUP PLATE METHOD: Cultivation of Microorganism:
The following microorganisms were used to study the antibacterial activity.
Bacillus subtilis – Gram positive bacteria
Staphylococcus aureous – Gram positive bacteria
Escherichia coli – Gram negative bacteria
Salmonella typhi – Gram negative bacteria
Standard: Streptomycin (1000mcg)
Solvent: DMF
All the test compounds were tested at 250 µg, 500 µg , and 1000 µg.
Preparation of the medium:
Composition of nutrient 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 dissolving the specified quantity of the dehydrated medium in purified water by heating on a water bath and were dispensed in 100 ml volume conical flasks. The conical flasks were closed with cotton plugs and were sterilized by autoclaving at 121°C (15 lb psig) for 15 minutes.
The contents of the conical flasks were poured aseptically into sterile Petridishes are allowed to solidify. These sterilized Medias were used to subculture the bacterial culture.
PROCEDURE: Each Petridish was filled to a depth of 4-5 mm with a nutrient agar medium that was previously inoculated with suitable inoculums of suitable test organism, and then allowed to solidify. The petridish were specially selected with flat bottom and were placed on level surface so as to ensure that the layer of medium is in uniform thickness. The petridishes were sterilized at 160-170°C in hot air oven for 30 mins before use. Small sterile borer of uniform size was placed approximately at 10 cm height, having an internal diameter of approximately 6-8 mm and made of aluminium (or) stainless steel. Each plate was divided in to four equal portions along the diameter. To each portion one cylindrical cavity was made in medium with the help of sterile borer. Three cavities for test compounds and one cavity for the standard. The petridishes were incubated at 37°C for 18 hours. Diameter of the zone of inhibition was measured and the average diameter for each sample was calculated. The diameter obtained by the test sample was compared with that produced by standard Streptomycin.

CUP PLATE METHOD: Cultivation of Microorganism
The following fungal strains were used to study the antibacterial activity.
1. C.raphigera
2. A.polytricha
Standard: Ketocanazole (1000mcg)
Solvent: DMF
All the test compounds were tested at 250 µg, 500 µg , and 1000 µg.
Preparation of the medium:
Composition of nutrient agar medium
Sabraoud Dextrose broth………..64gm
Distilled water……………………….1000ml
pH………………………………………..7.2± 0.2
The medium was prepared by dissolving the specified quantity of the dehydrated medium in purified water by heating on a water bath and were dispensed in 100 ml volume conical flasks. The conical flasks were closed with cotton plugs and were sterilized by autoclaving at 121°C (15 lb psig) for 15 minutes.
The contents of the conical flasks were poured aseptically into sterile Petridishes are allowed to solidify. These sterilized medias were used to subculture the fungal culture.
ROCEDURE: Each Petridish was filled to a depth of 4-5 mm with a nutrient agar medium that was previously inoculated with suitable inoculums of suitable test organism, and then allowed to solidify. The petridish were specially selected with flat bottom and were placed on level surface so as to ensure that the layer of medium is in uniform thickness. The petridishes were sterilized at 160-170°C in hot air oven for 30 mins before use. Small sterile borer of uniform size was placed approximately at 10 cm height, having an internal diameter of approximately 6-8 mm and made of aluminium (or) stainless steel. Each plate was divided in to four equal portions along the diameter. To each portion one cylindrical cavity was made in medium with the help of sterile borer. Three cavities for test compounds and one cavity for the standard. The petridishes were incubated at 37°C for 18 hours. Diameter of the zone of inhibition was measured and the average diameter for each sample was calculated. The diameter obtained by the test sample was compared with that produced by standard Ketocanazole.

Receptors in Intestinal Muscles

The experiment was conducted in order to gain better understanding of the function of the receptors in the guinea pig ileum. For this reason, various agonists and antagonists were used and the muscle reaction was monitored. The results of our experiment are summarized in the following table.
As we can see acetylcholine and hexamethonium both have a triethylamine at one end and a straight chain of carbons. The basic difference is that hexamethonium has two tertiary amines, one on each end of the chain, whereas acetylcholine has the group -O-C(=O)-CH3 on one end. According to the SAR theory (Structure Activity Relationship) similar molecules in structure tend to have similar biological activity. As we know, both acetylcholine and hexamethonium bind to the nicotinic receptor, the first one to trigger a response and the second one to prevent acetylcholine from binding. Hexamethonium, having two active groups, can probably bind more easily to the receptor, effectively blocking the acetylcholine action.
b)
Histamine and mepyramine have less similarities in structure. Both of them have three nitrogen and an aromatic ring. Histamine has the two nitrogen inside the aromatic ring whereas mepyramine has only one nitrogen bound in the ring. Both compounds bind to the H1-Histamine receptor, to trigger different reactions. The difference in structure can be explained by the different action of the two compounds. Histamine causes contraction of the muscle and mepyramine causes its relaxation.
The drugs tested were classified as agonists and antagonists.
Acetylcholine: Acts as neurotransmitter. It binds on the muscarinic and nicotinic receptors and causes muscle contraction.
Histamine: Is also a neurotransmitter. It binds on the H1-Histamine receptor and causes smooth muscle contraction.
Nicotine: It acts on the nicotinic cholinergic receptors and mimics the neural transmission. It stimulates the muscle, then blocks stimulation.
Isoprenaline: Although isoprenaline was apparently an antagonist, it is actually a selective agonist for the β- adrenergic receptors that causes muscle relaxation. It is a sympathomimetic drug that mimics the effect of stimulating the postganglionic adrenergic sympathetic nerves.
Hexamethonium: It is a nicotinic antagonist and a ganglionic blocker. It binds to the nicotinic cholinergic receptors and blocks the actions of acetylcholine or cholinergic agonists. It has no effects on muscarinic (mAch) receptors.
Mepyramine: It is a histamine H1 antagonist and targets the H1- Receptor. Although it was believed to be an antagonist merely to block the actions of endogenous histamine without activating the receptors, it has recently been classified as an inverse agonist decreasing the spontaneous activity of gp-H1r. It also inhibits histamine induced inositol phosphate (InsP) production and intracellular calcium mobilization. It causes a marked decrease in the maximal response to histamine at high concentrations.
Atropine: It is a competitive antagonist for the muscarinic cholinergic receptor (mAch). It binds to the receptor without activating it, thus blocking the actions of endogenous acetylcholine or exogenous agonists.
a) The drug in this experiment were acting on three receptors. H1-Histamine receptors, muscarinic (mAch) receptors and nicotinic (nAch) receptors. Each agonist was acting on a different receptor and that is apparent from our results. When using an antagonist that blocked a specific receptor it only inhibited the action of the drug acting on that particular receptor, and had no effect on the rest of the drugs.
b) The receptors were obviously located on the surface of the muscle, so that the access of the drugs would be possible.
The first apparent antagonist which turned out to be an agonist was isoprenaline. It acts on the β- adrenergic receptors causing muscle relaxation and antagonized all the three agonists who acted on different receptors. This type of antagonism is called a physiological antagonist and describes the interaction of two drugs who cause opposing actions in the body and tend to cancel each other. In this case, the isoprenaline acts on the β- adrenergic receptors and causes relaxation of the muscle, whereas the agonist act on the histaminic, nicotinic and muscarinic receptors and cause contraction of the muscle.
The second apparent antagonist was mepyramine, which acts on the histamine receptor and blocks the action of histamine. It has recently been classified as an inverse agonist, causing muscle relaxation. This type of agonists show selectivity to the resting state of the receptor.
Atropine acts on the muscarinic receptors and blocks their action. Thus it prevents acetylcholine from binding to the receptor and stimulating it. Nicotine though activates the nicotinic receptor that apparently has nothing to do with atropine. The reversal of nicotine action indicates the presence of inhibitory postganglionic (terminal) neurones, which respond to stimulation of their ganglion-cells by inducing relaxation of the gut. It is also suggested by other experiments [Phillis

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