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'LOVELY PROFESSIONAL UNIVERSITY coping stone PROJECT REPORT TOPIC- germicide act OF DIFFERENT TYPES OF aftersen. PROJECT GUIDE- SUBMITTED BY- DR. AKSHAY GARG MOHIT KUMAR DEPT. OF BIOTECH nary(prenominal)OGY REG. NO. †10800037 ROLL NO- RB1R07B02 B. TECH BIOTECH. (8th sem. ) DATED- . 17-05-2012 CERTIFICATECertified that this project authorise â€Å"anti smallbial operation of contrary geeks of beloved ” submitted by MOHIT KUMAR , students of biotechnology Department, agree fitted Professioal University, Phagwara Punjab in the partial fulfillment of the need for the destine of Bachelors of technology (biotechnology) Degree of LPU, is a record of student’s hold claim carried under my supervision & axerophthol; guidance. This composition has non been submitted to t bulge ensemble an early(a)(prenominal) university or institution for the award of whatever full stop.Date: 17/5/2012 Name of find Guide Dr. Akshay Garg DECLARATION I, MOHIT KUMAR, s tudent of B. Tech ergonomics under Department of Biotechnology of Lovely paid Univerersity, Punjab, hereby affirm that whole the information furnished in this dissertation / capstone project report is based on my own in cardinalse research and is genuine. This dissertation / report does not, to the top hat of my knowledge, brook part of my buy the farm which has been submitted for the award of my degree either of this niversity or any another(prenominal)(a) university with come to the fore proper citation. Date †17/5/2012 Investigator- MOHIT KUMAR Regd. No. 10800037 Acknowledgement any(prenominal) attempt at any engage aim flowerpott be satisfactorily completed without the bear and guidance of learned people in my capstone projectâ€Å"Anti littlebial drill of assorted types of sexual love”.I would uniform to prove my immense gratitude to my guide Dr. Akshay Garg for his constant set apart up and motivation that has encouraged me to come up with this project. I in like manner would like to give thanks my group member who dish uped me in my project. MOHIT KUMAR rob lovemaking is a traditional contental pr all(prenominal)ing for infected wounds. It can be prep arive on antibiotic-resistant strains of bacterium. some(a)(prenominal)(prenominal)(prenominal) local anaesthetic anaesthetic brands of de best(Dabur) self-possessed from divers(prenominal) sources is employ in this take up. excessively the sweeten is collect from versatile apiaries from Pathankot(Punjab) and Saharanpur (Up).The bacteriuml culture were taken from the science lab of the Lovely professional University and was revived in alimentary blood line media and then sub-cultured in alimentary solid food nutritious agar media. The system utilize to test this antibiotic sensitivity of dear was done with the help of Kirby Bauer method . mark variations were find in the bactericide bodily function of these edulcorate patterns. bac terial species utilize were type B subtilis ,E. coli. , S. aureus and Burkholderia spp. The geographical regularize of stifling produced against assorted bacterium employ lovemaking of variant assiduity atomic number 18 as fol ridiculouss: E. oli (pathankot taste): blow% = 2. 6cm, 75%= 1. 9cm, 50%= 1. 7cm,and 25%= 1. 2cm; E. coli (dabur prototype): ascorbic acid% = 2. 2cm, 75%= 1. 6cm, 50%= 1. 3cm, and 25%= 1. 1cm; E. coli (saharanpur experiment): light speed% = 2. 8cm, 75%= 2. 4cm, 50%= 2. 2cm, and 25%= 1. 7cm; B. subtilis (pathankot): one hundred% = 3. 0cm, 75%= 2. 7cm, 50%= 2. 7cm, and 25%= 2. 5cm; B. subtilis (dabur): c% = 2. 7cm, 75%= 2. 5cm, 50%= 2. 4cm, and 25%= 2. 2cm; B. subtilis (saharanpur): coulomb% = 3. 2cm, 75%= 2. 7cm, 50%= 2. 3cm, and 25%= 1. 5cm; Burkholderia spp. (pathankot): vitamin C% = 2. 1cm, 75%= 1. 9cm, 50%= 1. 6cm, and 25%= 1. 4cm; Burkholderia spp. dabur): 100% = 2. 4cm, 75%= 1. 7cm, 50%= 1. 5cm, and 25%=1. 2cm; Burkholderia spp. (saha ranpur):100% = 2. 5cm, 75%= 1. 9cm, 50%= 1. 7cm, and 25%=1. 4cm; S. aureus (pathankot):100%=2. 1cm, 75%= 1. 7cm, 50%= 1. 4cm, and 25%=1. 2cm; S. aureus (dabur):100% =3. 0cm, 75%= 2. 7cm, 50%= 2. 2cm, and 25%=1. 8cm; S. aureus (saharanpur): 100% = 2. 9cm, 75%= 2. 5cm, 50%= 2. 0cm, and 25%=1. 6cm; regularizes of proscription of antithetic treatment groups were measured by agar- intimately- public exposure undertake and compared with support. The comparison of sexual love archetype with distilled urine control had proved it significant.CONTENTS PAGE 1. opening 7 1. 1) love as bacteriumcide drug drug agent 7 1. neutral 9 2. literary works REVIEWS 10 3. 1 TYPES OF love 10 3. 2 COMPONENTS OF erotic love 11 3. MATERIALS AND METHODS 15 4. MATERIAL 15 4. 2 METHOD 16 4. RESULTS AND DISCUSSION 19 5. REFERENCES 32 1. ) INTRODUCTION edulcorate is a sweet food choose by bees using bitterweed from f baseers. The dearest is a sweet, viscous fluid produced by bees from the battle array of beebread, in the beginning from f depressive disorderers. It is considered to be a lifelike syrup. The Nectar is gathered by the bees and is soft transformed into lovemaking, through a keen-sighted rocess involving the addition of enzymes and the gradual reduction of moisture. love life is a rich source of carbohydrates primarily Fructose and Glucose. The chemical composition of love varies depending on the plant source, season and take methods. Therefore the Colour, Concentration and Compounds take off depending on the floral sources. separate compounds which can be fix in dulcorate al low gear Proteins and loony toonss much(prenominal) as Gluconic Acid (C6H11O7, too cognize as 2,3,4,5,6- pentahydroxyhexanoic Acid), Minerals and Anti-Oxidants such as enthalpy Peroxide (H202) and Vitamins (B6 and B12), (BD.Yates et. al. 1996). lovemaking has a low pH and a low moisture meat, which is usually on reasonable about 17 percent. The Gluconic Acid in beloved is produced when bees secrete Glucose Oxidase, speckle treat the nectar, this give sexual love a low pH. There are some(prenominal) varieties of beloved from around the world which come in three master(prenominal)(prenominal) types which are liquid, whipped and comb. some(prenominal) local brands of passion(Dabur) collected from diametric sources is utilise in this study. Also the honey is collected from heterogeneous apiaries from Pathankot(Punjab) and Saharanpur (Up) .Each of these honey’s were chosen because they are organic and readily easy in regainth food stores 1. 1) The bacteri virulente Effects of Honey The honeys are shown to throw bacteriacide properties, in particular Manuka honey. Manuka Honey has had all-inclusive research done on it. It has been shown in many studies that Manuka Honey has bring backthful raises (Barret J. , et. al, 2005; Coumbes A. L. , et. al. 2004; Mundo, M A. 2004). The Mechanism of antibacterial Properties Hone y has many natural properties which modify it to inhibit bacteria.These properties include, a low pH which is in the range of pH 3. 2 to 4. 5, approximately 3. 9 which is repayable to its content of vinegarishic compounds mainly Gluconic acid as stated above. A low pH is inhibitory to about bacteria. Since most bacteria live in milieus around pH 7, the pH of honey could inhibit the bacteria (Barrett, J et. al. al 2005). This is because pH affects the panache large proteins such as enzymes work. Which causes the settle of enzymes to change, which then alters the overall charge, this causes the protein to de reputation.Honey contains small tallys of Hydrogen bl severally (H202) this varies depending on the honey, it is produced as a event of the enzyme glucose oxidase body plow in producing Gluconic acid (Mundo, MA. et. al. 2004). C6H12O6 + H2O + O2 > C6H12O7 + H2O2 (glucose oxidase reaction) When honey is utilize locally (as, for example, a wound dressing), henry bl a piece is produced by dilution of the honey with body fluids. As a result, heat content hydrogen hydrogen bleach is released slowly and acts as an antiseptic. Hydrogen peroxide is a powerful oxidising agent (Free Radical) which has the ability to modify cells.In an aqueous dispelr hydrogen peroxide acts like an acid and can oxidise a variety of compounds, by evaluate free unpaired electrons. This allows the formation of other free radicals, which then causes a exhibitor effect. Therefore altering biological structures and because damaging cells. Honey is primarily a saturated garland of both monosaccharide, with a low water system bodily function; most of the water molecules are associated with the gelts and few lodge available for microorganisms, so it is a abject environment for their growth.If water is mixed with honey, it loses its low water application, and therefore no semipermanent possesses this healthful spot. Honey consists of motley constituents such as water, carbohydrates, proteins, vitamins, amino acid, energy and minerals. alike the study ones, there must similarly be several minor constituents in honey, which whitethorn be playing a key role in find out the antimicrobial behaviour of honey. In the past, antimicrobial activeness of honey had been report encourage by using aqueous solution of honey.It is said that honey possesses antibacterial property hardly it is not separate whether it is the legal age honey or s wateryly fragment of it. The antibacterial properties of honey speed up the growth of naked as a jaybird tissue to heal the wound. The bactericidal effect of honey is reported to be dependent on assimilation of honey used and the nature of the bacteria Considering the fact that their might be some specific constituents which may be lend to the antimicrobial behaviour, it was decided to carryout the studies using contrasting solvents.The stick study therefore deals with the constituents in the differ ent solvents followed by evaluation of rend for their antimicrobial behaviour against certain species of bacteria. 2) OBJECTIVE Based on the above facts, the present study was defined with following documentary: 1) To check the antibacterial effect of different honeys on. a. positive staphylococci aureus, b. Gram- forbid Escherichia coli, c. Gram-positive group B subtilis, and d. Gram- negatively charged Burkholderia capicium 2) To check the effect of honey on bacterial strains using different-different dilutions. . 100% honey sample b. 75% honey sample c. 50% honey sample d. 25% honey sample 3) To compare the effect of honey and control (Distilled water) on a. Gram-positive Staphylococcus aureus, b. Gram-negative Escherichia coli, c. Gram-positive Bacillus subtilis, and d. Gram- negative Burkholderia capicium 3) LITERATURE REVIEWS Honey has been used with medicinal purposes since quaint times and there are reports of its topic use by Ayurvedic medicine, dating hold to 2500 b . c. Hippocrates prescribed the use of honey for several indications, including treatment of wounds and gastritis.Its healing properties are mentioned in the Koran and in Bible. Honey is a part of traditional medicine for centuries. The antimicrobial practise of honey is delinquent primarily to hydrogen peroxide produced enzymatically. However, in neat honey the acidity is also a significant antimicrobial factor. The pH, ranging from 3. 2 to 4. 5, is low enough to inhibit the development of pathogens, whose optimal pH range situates amid 7. 2 and 7. 4. When applied to affected areas of tissue, acidity, osmotic effect and phytochemical factors decrease with dilution; however, the exercise of hydrogen peroxide increases from 2. to 50 times. On these values, peroxide acts as an antiseptic, without causing cell damage. The composition of sugars in honeys, from different floral lines, inhibits the development of various intestinal bacteria. in all chemical and fleshly factors make the therapeutic properties of honey quaint: fast decline of infections and healing of wounds, dissipated inflammation recovery, minimizing of wounds, stimulation of angiogenesis, as wholesome as the development of epithelial and gritty tissues. Honey to be used with topical medicinal purpose, certain requirements are needed, like being free f herbicides, pesticides, dangerous metals and radioactive elements and sterilise to obstruct tri scarcelyary infections. 3. 1) TYPES OF dulcorate †ON BASIS OF patterned SOURCE- Blended- Most commercially available honey is blended, meaning it is a mixture of twain or more(prenominal) honeys differing in floral source, color, flavor, density or geographic origin. Polyfloral †Polyfloral honey, also known as wildflower honey, is derived from the nectar of many types of flowers. The taste may vary from year to year, and the aroma and the flavor can be more or little(prenominal) intense, depending on which bloomings are pr evalent.Monofloral- Monofloral honey is made primarily from the nectar of one type of flower. Different monofloral honeys take in a characteristic flavor and color because of differences between their bargainer nectar sources. To produce monofloral honey, beekeepers keep beehives in an area where the bees let access to just one type of flower. winter melon vine honey- sooner of taking nectar, bees can take honeydew, the sweet secretions of aphids or other plant sap-sucking insects. Honeydew honey is very dark dark-brownness in color, with a rich posy of stewed fruit or image jam, and is not sweet like nectar honeys. . 2) COMPOSITION OF dear Carbohydrates Unsurprisingly, these comprise the major portion of honey †about 82%. The carbohydrates present are the monosaccharide levulose (38. 2%) and glucose (31%); and disaccharides (~9%) sucrose, maltose, isomaltose, maltulose, turanose and kojibiose. There are also some oligosaccharides present (4. 2%), including erlose, theanderose and panose, formed from incomplete breakdown of the high saccharides present in nectar and honeydew. Proteins and amino group Acids.Honey contains a number of enzymes, including turnase, which converts sucrose to glucose and levulose; amylase, which breaks starch down into smaller units; glucose oxidase, which converts glucose to gluconolactone, which in turn yields gluconic acid and hydrogen peroxide; catalase, which breaks down the peroxide formed by glucose oxidase to water and oxygen; and acid phosphorylase, which removes inorganic phosphate from organic phosphates. Honey also contains eighteen free amino acids, of which the most abundant is proline. Vitamins, Minerals and AntioxidantsHoney contains trace amounts of the B vitamins riboflavin, niacin, folic acid, pantothenic acid and vitamin B6. It also contains ascorbic acid (vitamin C), and the minerals calcium, iron, zinc, potassium, phosphorous, magnesium, selenium, chromium and manganese. The main group of an tioxidants in honey are the flavonoids, of which one, pinocembrin, is unique to honey and bee propolis. Ascorbic acid, catalase and selenium are also antioxidants. Generally speaking, the darker the honey, the greater its antioxidising properties. Other compoundsHoney also contains organic acids such as acetic, butanoic, formic, citric, succinic, lactic, malic, pyroglutamic and gluconic acids, and a number of aromatic acids. The main acid present is gluconic acid, formed in the breakdown of glucose by glucose oxidase. Honey also contains hydroxymethylfurfural, a natural product of the breakdown of simple sugars below pH 5. check to studies done by Patricia E. Lusby et al cardinal of the 13 bacteria were curb by all honeys used in this study with only Serratia marcescens and the yeast Candida albicans not inhibited by the honeys.Little or no antibacterial activity was seen at honey preoccupancys 1%, with minimal inhibition at 5%. No honey was able to produce complete inhibition o f bacterial growth. Although Medi honey and manuka had the overall best activity, the locally produced honeys had equivalent inhibitory activity for some, but not all, bacteria. He then postulated that honeys other than those commercially available as antibacterial honeys can bring equivalent antibacterial activity. These newly identified antibacterial honeys may prove to be a valuable source of future therapeutic honeys. gibe to Peter C.Molan (2001), honey may be the â€Å"natural cure” for most bacterial infections that could replace man-made antibiotics. lookers say that an enzyme in the honey turns into a tiny amount hydrogen peroxide when combined with embodied fluids killing nearby bacteria. Honey also causes an increase in lymphocyte and scavenger cell activity ( cooper et al. , 2011). The honeys are shown to have antibacterial properties, in particular Manuka honey. Manuka Honey has had extensive research done on it. It has been shown in many studies that M anuka Honey has antimicrobial make (Barret J. , et. al, 2005; Coumbes A. L. , et. al. 2004; Mundo, MA. 2004).In this study the antibacterial activity of the vanadium honeys were compared, the Manuka honey was used as a positive control. The nonperoxide antibiotic activity is referable(p) to methylglyoxal (MGO) and an unidentified synergistic component. Most honeys contain very low takes of MGO, but manuka honey contains very high levels. The heraldic bearing of the synergist in manuka honey more than recurs MGO antibacterial activity. In vitro antibacterial activity of rude(a) and commercially available honey was tried against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Shigella spp. nd Salmonella spp. ). Both types of honey showed antibacterial activity against test organisms with the zone of inhibition ranging from 8. 13 to 30. 85 mm, era E. coli, S. aureus, and Shigella spp. showed sensibility towa rds both types of Honey. Both types of honey showed no make on Salmonella spp. The federal agency of honey at 100% engrossment was found to be higher than all other preoccupancys tested. However, no effect was notice at concentration of 6. 25% v/v honey in the miscue of both samples(Patricia E. Lusby 2004).To study the antimicrobial activity of honey, 60 samples of various botanical origin were evaluated antimicrobial activities against 16 clinical pathogens and their single reference strains. The bioassay applied for find out the antimicrobial effect employs the wholesome-agar airing method and the estimation of minimum active dilution which produces a 1 mm diam inhibition zone. Streptococcus pyogenes,Bacillus cereus and Bacillus subtilis were proven to be up to 60% more resistant than their equal reference strains thus emphasizing the variability in the antibacterial effect of honey and the need for further research (C.Voidarou 2010). Natural products, either as pure co mpounds or as govern plant extracts, provide unlimited opportunities for new drugs because of the ready availability of chemical salmagundi (Cos et al. , 2006). Honey and lemon-honey are traditional remedies in the Middle East and China and for many centuries and have been used in the treatment and prevention of the common cold and various upper respiratory tract infections (Molan, 1992; Zulma ; Lulat, 1989). bacteriacide activity of the honeys was assayed using standard intumesce diffusion methods. oncluded that the sandal wood from different sources and mixture of of †and -santanols were highly rough-and-ready against gram negative bacteria including Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae and as well as yeast Candida albicans. The present work gives strong evidence of antibacterial activities of honey, sandal oil and black pepper (Sheikh Ahmad 2002). In general, all types of honey have high sugar content as well as low water content and acidi ty, which prevent microbial growth.Osmotic effect, effect of pH and hydrogen peroxide are represented as an â€Å"inhibition” factor in honey (Postmes et al. , 1993). Most types of honey generate hydrogen peroxide when diluted because of the activation of the enzyme glucose oxidase, which oxidizes glucose to gluconic acid and hydrogen peroxide (Schepartz and Subers, 1964). Hydrogen peroxide is the major contributor to the antimicrobial activity of honey,and the different concentrations of this compound indifferent honeys result in their variable antimicrobial effects (Molan, 1992).Moreover, non peroxide factors also play lively role. The content of non peroxide factors are relate to the floral source and sometimes posting for the major part of the antibacterial activity in honey(Molan and Russell, 1988). Forty samples of different honey types (Acacia, Ziziphus , Brassica and Citrus) were collected from different areas of Pakistan and analyzed for moisture, pH, total acidit y, ash, electrical conductivity, hydroxyl group methyl furfural (HMF), sucrose, total sugars, invert sugar, protein, proline contents as well as macro and micro elements.The variation in composition of honey samples was spy due to different types of flora. Likewise, a significant level (P ; 0. 05) of ash, electric conductivity, sucrose, total sugar as well as macro and micro elements was also found in these honey types. Different formulations of honey has significantly inhibited growth of pathogenic microorganisms, Staphylococcus aureus, Escherichia coli, Candida albicans and genus Aspergillus niger when compared to control group, which is an evidence that honey is a therapeutic agent being used since ancient time throughout the world. Feng et. al. 1994) The edition in the observed antibacterial activity can be due to several reasons. One possibility might be related to the differences in susceptibility of each species of microorganism to the antibacterial activity of honey used. Similar observations are reported by others (Nzeako and Hamdi 2000; Ceyhan and Ugur 2001; Taormina et al. 2001). As reported by others (Melissa et al. 2004) dilution of honey enhances hydrogen peroxide mediated antibacterial activity may explain some of the discrepancies of observed with the antibacterial activity of these honey.The presence of unstable putative agents and/or thermolabile antibacterial agent(s) could also be inactivated during the data-based process and thus may be considered as possible explanation of the observed insensitivity of some honey samples found in the preliminary study. 4. ) MATERIALS AND METHOD 4. 1) Materials Several local brands of honey and Dabur honey collected from different sources is used in this study. Also the honey is collected from various apiaries from- Pathankot(Punjab) and Saharanpur (Up). In the antibacterial study, several bacterial species known to be pathogenic to human such as E. oli. , S. aureus and Burkholderia capicium and Bacillu s subtilis was used. These strains were obtained from biotechnology lab. in accordance to given requirement. tripe wares used:- conelike flask (500 milliliter), Conical flask (100 milliliter), Petri cuticles, glass rod, test tubes spreader, glass beakers, Glass slides and cover slip, Plastic wares used:- Beakers, Conical flask (500 milliliter), Conical flask (100 milliliter), dropper, appendrof tubes (2ml), political machine pipette tips (10? l, 100? l, and 1000? l) Instruments used:- Hot air oven, Autoclave machine, bedded air flow hood, Incubator, Microwave ovenOther textile used:- Sprit lamp, cork borer,test tube stand, elevator car pipette (10? l, 100? l, and 1000? l), parafilm wax,,filter reputation Chemicals used:- Nutrient agar, nutrient broth, Mueller Hinton agar, 70% Methanol, 100% methanol, ethyl alcohol ,sodium chloride. 4. 2) Methods physicochemical study: show of different honeys was observed. pH is determined using conventional procedure like using ph scale. bactericide study: Antibacterial study was carried out in steps. In the first step, an in vitro screening bequeath be carried out using either disc diffusion or well diffusion method.Well diffusion was carried out using plate diffusion, which I preferred. Preparation of test materials: Test materials will be prepared by diluting each honey at different dilutions, 25 ? l/100 ? l, 50 ? l/100 ? l, 75 ? l/100 ? l and one with no dilution. Moreover, exculpate honey was also used as test material. All dilutions were carried out with double distilled and deionised sterilized water. 3. 2. 1) Source of bacterial strains The E. coli. , S. aureus, Burkholderia capicium and Bacillus subtilis were revived from the stock available in the various Biotechnology laboratories of Lovely Professional University, Phagwara (Punjab), India.Sub-culturing of bacterial strains in nutrient broth:- 100ml nutrient broth was prepared and ten test tubes were taken, autoclaved, and after this the bacterial str ains was inoculated in the nutrient broth in different test tubes and then incubated for 24 hours in incubator . Sub-culturing of the bacterial strains on good media:- For All Bacterial strains:- 5. 6g of Nutrient agar was added to the 200ml of purified water. Then solution was het up(p) and stewed for 1 minute to completely answer the powder. Then media was autoclaved.Then media(10-15 ml) was poured in Petri plates. Then Bacterial strains were added to media. Then Petri plate were kept in incubator for 18-48 hours at 37 degree C. Saline preparation : Take . 58g of Nacl and evaporate it to 10ml of distled water and make saline in the lead 15 min of spreading and take the colonies from the nutrient agar plates and mix flop in the saline. Nutrient agar sensitive for antimicrobial testing: 7g of nutrient agar moderate was added to the 250 ml of distilled water and heated for 1 minute to complete dissolve medium then autoclaved .After that pouring the petriplates and leave it 20 minute for solidify medium and make swell , after solidify put the different Honeys in the wells and kept it to incubate for 18-48 hours at 37C. exam of antibacterial activity using agar well diffusion method: The bacterial strains were inoculated into 10 ml of sterile nutrient broth, and incubated at 37 °C for 18 h. Each culture was then spreaded on the surface of sterile nutrient agar plate and also pour-plated in nutrient agar media to perform the test in triplet, one with the control and the other two ith the test sample. Making the wells With the help of a cork borer make wells in the agar guardedly without tearing the gel. In two agar plate of all quadruple sets, four wells were prepared with the help of sterilized cork borer. Then with the hep of micropipette put 100 micro litre of honey sample into the wellIn the wells of two plates of each set, samples of following concentrations: (1) 100% sterile Honey(2) 75% sterile Honey(3) 50% sterile Honey and (4) 25% sterile Honey; were added by using micropipette. Also in the a well water was added as negative control.Then with the help of micropipette put 100 micro litre of honey sample into the well Replace the lid of the plate between putting sample into wells to calumniate exposure to air-borne contaminants. Cover the lid tightly with paraffin tape to avoid contamination. pensiveness of the plates . A temperature range of 35°C ± 2°C is required for 24-48 hours. Do not incubate plates in degree centigrade dioxide as this will decrease the pH of the agar and result in errors due to incorrect pH of the media. Measuring zone of inhibition 1.Following incubation, measure the zone sizes to the near millimeter using a ruler or caliper; include the diam of the well in the measurement . 2. All measurements were made with the unaided eye while viewing the back of the petri dish. Hold the plate a few inches above a black, nonreflecting surface illuminated with reflected light . 3. Rec ord the zone size on the recording sheet. Figure1: Testing of antibacterial activity using agar well diffusion method 4. )RESULTS Sub-culturing of bacterial strains in nutrient broth:- Figure2: Culture E. coli. , S. ureus and Burkholderia capicium and Bacillus subtilis in Nutrient broth media. Sub-culturing of the bacterial strains on solid media:- Fig. â€3 E. coli subcultured on nutrient agar Fig. †4 B. Subtilis subcultured on nutrient agar Testing of antibacterial activity using agar well diffusion method: Fig 5 : No antibacterial activity seen as no honey is poured (taken as negative control) in which E. coli is grown Fig 6 : No antibacterial activity seen as no honey is poured (taken as negative control) in which Bacillus subtilis is grown ANTIMICROBIAL military action OF divers(a) HONEY’S AGAINST E. coliFIG. 7 †ZONES OF quelling BY different DILUTIONS OF HONEY(pathankot sample) IN E. coli: 100% = 2. 6cm, 75%= 1. 9cm, 50%= 1. 7cm,and 25%= 1. 2cm FIG. 8â⠂¬ ZONES OF prohibition BY dissimilar DILUTIONS OF HONEY (dabur sample) IN E. coli : 100% = 2. 2cm, 75%= 1. 6cm, 50%= 1. 3cm, and 25%= 1. 1cm FIG. 9†ZONES OF ban BY VARIOUS DILUTIONS OF HONEY (saharanpur sample) IN E. coli: 100% = 2. 8cm, 75%= 2. 4cm, 50%= 2. 2cm, and 25%= 1. 7cm; ANTIMICROBIAL ACTIVITY OF VARIOUS HONEY’S AGAINST B. subtilis FIG. 10†ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY IN B. subtilis (pathankot sample): 100% = 3. cm, 75%= 2. 7cm, 50%= 2. 7cm, and 25%= 2. 5cm FIG. 11†ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY in B. subtilis (dabur sample): 100% = 2. 7cm, 75%= 2. 5cm, 50%= 2. 4cm, and 25%= 2. 2cm; FIG. 12†ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY in B. subtilis (saharanpur):100% = 3. 2cm, 75%= 2. 7cm, 50%= 2. 3cm, and 25%= 1. 5cm ANTIMICROBIAL ACTIVITY OF VARIOUS HONEY’S AGAINST Burkholderia capacium FIG. 13†ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY in Burkholderia capacium (pathankot sample ): 100% = 2. 1cm, 75%= 1. 9cm, 50%= 1. 6cm, and 25%= 1. 4cm; FIG. 4†ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY Burkholderia capacium (dabur): 100% = 2. 4cm, 75%= 1. 7cm, 50%= 1. 5cm, and 25%=1. 2cm FIG. 15†ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY Burkholderia capacium (saharanpur):100% = 2. 5cm, 75%= 1. 9cm, 50%= 1. 7cm, and 25%=1. 4cm; ANTIMICROBIAL ACTIVITY OF VARIOUS HONEY’S AGAINST S. Aureus FIG. 16†ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY S. aureus (pathankot):100%=2. 1cm, 75%= 1. 7cm, 50%= 1. 4cm, and 25%=1. 2cm FIG. 17†ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY S. aureus (dabur):100% =3. 0cm, 75%= 2. 7cm, 50%= 2. 2cm, and 25%=1. cm FIG. 18†ZONES OF INHIBITION BY VARIOUS DILUTIONS OF HONEY IN S. aureus (saharanpur): 100% = 2. 9cm, 75%= 2. 5cm, 50%= 2. 0cm, and 25%=1. 6cm Appearance Appearance of each of honey brand was examined and it was observed that honey were brown to dark brown in color, whereas other honey w ere halcyon yellow in color. Dabur honey- golden twine,transparent Pathankot sample †golden colour with sufficient enhancer Saharanpur sample †dark yellow colour with zero transparency The transparency is due to processing of honey,while less transparent samples are unprocessed and taken instanter from bee hive.Table 1- diam of the district of proscription by different concentration of Pathankot honey sample on different Bacterial strains. Bacterial strains| Zone of Inhibition (in cm)| | Control(sterile DistilledWater)| 100%Honey sample| 75%Honey sample| 50%Honey sample| 25%Honey sample| E. coli. | 0. 0| 2. 6| 1. 9| 1. 7| 1. 2| Bacillus subtilis| 0. 0| 3. 0| 2. 7| 2. 7| 2. 5| S. aureus| 0. 0| 2. 1| 1. 7| 1. 4| 1. 2| Burkholderia capicium| 0. 0| 2. 1| 1. 9| 1. 5| 1. 4| Table2- Diameter of the Zone of Inhibition by different concentration of dabur honey sample on different Bacterial strains.Bacterial strains| Zone of Inhibition (in cm) | | Control(sterile DistilledWat er)| 100%Honey sample| 75%Honey sample| 50%Honey sample| 25%Honey sample| E. coli. | 0. 0| 2. 2| 1. 6| 1. 3| 1. 1| Bacillus subtilis| 0. 0| 2. 7| 2. 5| 2. 4| 2. 2| S. aureus| 0. 0| 3. 0| 2. 7| 2. 2| 1. 8| Burkholderia capicium| 0. 0| 2. 4| 1. 7| 1. 5| 1. 2| Table 1- Diameter of the Zone of Inhibition by different concentration of Saharanpur honey sample on different Bacterial strains. Bacterial strains| Zone of Inhibition (in cm)| | Control(sterile DistilledWater)| 100%Honey sample| 75%Honey sample| 50%Honey sample| 25%Honey sample| E. oli. | 0. 0| 2. 8| 2. 4| 2. 3| 1. 7| Bacillus subtilis| 0. 0| 3. 2| 2. 7| 2. 3| 1. 5| S. aureus| 0. 0| 2. 9| 2. 5| 2. 0| 1. 6| Burkholderia capicium| 0. 0| 2. 5| 1. 9| 1. 7| 1. 4| In the preliminary screening process was observed that some honey brands have more while some other have less antibacterial activity. nigh honey showed antibacterial effect against E. Coli and some showed strongest activity against Bacillus subtilis ,S. aureus and Burkholde ria capicium. Clear zones of inhibition were produced by strong without dilution.Through the analysis of total values of the diameters of each honey, it is possible to reason out a possible pattern in which commercial honeys, such as DABUR honey, have a greater average diameter while the homemade honeys from different regions, have a lower average diameter even at less concentrations, but the best antimicrobial activity was shown by Saharanpur honey sample with was collected directly from the apiary without any processing and it showed clear and large inhibition zones for all bacterial strains which were used for this test.Though after the dilutions were made, the low concentrated samples of Saharanpur honey resulted in rather poor antimicrobial activity. The wells in which water was loaded shoed absolutely no antimicrobial activity which was used as negative control. 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