Biology Project (autorecovered).docx

2016 -2017

BIOLOGY PROJECT PRAGYA JAIN CLASS – XII-A ROLL NO. 14 ADMISSION NO. 74523

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Aim To study the effect of antibiotics on the microbes present in the soil sample.

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Contents S. No.

Topic

Page No.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Acknowledgement Certificate Definitions Some examples of bacteria found in soil About Antibiotics Types of Antibiotics How Do They Work? Uses and Limitations of Antibiotics Some Clinically Important Antibiotics Experiment Apparatus Procedure Observations Conclusion Bibliography

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Acknowledgement I would like to express my wholehearted gratitude towards my teachers- Dr. Rita sharma, and Ms. Akshita bej, for their constant support and guidance and for all facilities that they provided me for this project work. I am highly obliged to my family and my friends for their endless encouragement and continuous support during my project work.

Pragya Jain

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Certificate This is to certify that Pragya Jain of class XII A, Chowgule Public School, Delhi has completed this project in the stipulated time period as given by CBSE Board in the year 2016-17 under my supervision.

Dr. Rita Sharma Biology Teacher

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Definitions I. Flora It refers to the population of commensal bacteria normally present in the intensive, body openings, and on the skin.

II. Food borne Pathogens They are infectious organisms associated with livestock that can cause diseases in humans through intake of contaminated food. They include Salmonella, Campylobacter, and Listeria.

III. Gene  It is a segment of DNA that carries the direction for the structure of a given protein.  Antibiotics resistance genes direct the synthesis of antibiotic resistance proteins.

IV. Incidence It is the frequency of new occurrences of a disease within a defined time interval. Incidence Rate =

Number of new cases of a disease Total population over a period time (Usually 1 year)

V. Micro-organisms They are minute, microscopic or sub-microscopic living organisms including bacteria, fungi, and protozoa.

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Viruses are often included in this category, although, they are incapable of growth and reproduction outside the host cells.

VI. Multiple Drug Resistance It is a condition where some bacteria are resistant to more than one type of antibiotics.

VII. Narrow Spectrum Antibiotic It is an antibiotic which is effective against a limited number of bacterial species.

VIII. Broad Spectrum Antibiotic It is an antibiotic which is effective against a large number of bacterial species.

IX. Antibacterial It refers to a drug that kills or inhibits the growth of bacteria.

X. Antibiotics  They are a class of substances that kill or inhibit the growth of bacteria.  Example- Penicillin, Tetracycline, Fluoroquinolones.  Originally, antibiotics were derived from natural sources like Penicillin was derived from moulds but many currently used antibiotics are semi-synthetic and modified with additions of synthetic chemical components.

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 Some scientists reserve the term “antibiotics” for naturally produced substances and use antimicrobial to encompass both synthetic and natural forms.

Examples of Some Bacteria Found in Soil 1. Decomposers They serve as decomposers, digesting organic materials and breaking them down into soil and compost. They typically take simple compounds like roots and plant litter and convert it into forms that plants can use. Some decomposers can break down pesticides or herbicides, and are very good at retaining nutrients within their cells, preventing essential nutrient loss in the soil. E.g. Bacillus subtilis, Pseudomonas fluorescens, etc.

2. Nitrogen Fixers They extract nitrogen gas from the air and converting it into mineral form that the plant can take up. These bacteria often live in root nodules of plants like alfalfa, clover and legumes. E.g. Rhizobium, etc.

3. Actinomycetes These bacteria are decomposers that specialize in decomposing tough materials like cellulose and chitin. They are responsible for giving the freshly turned earth its recognizable “earthy” scent.

4. Aerobic Bacteria

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They are most common in well drained sols where there is no standing water. These bacteria make up the majority of the species found in most soils, and also play a vital role in nitrification. These bacteria turn ammonium into nitrates which are then used by the plants to turn into proteins.

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About Antibiotics What are they? The standard definition states that an antibiotic is a substance produced by micro-organisms that kill or inhibit other microorganisms. That is, antibiotics are strong medicines that can cure many bacterial illnesses and infections. Chemically antibiotics are complex organic compounds usually aromatic in nature and nowadays, minerals, metals and other compounds (e.g.- HCl, Sulphur, etc.) are added to the pure forms to increase their utility.

History of Antibiotics Sir Alexander Fleming, who observed the inhibition of Staphylococci on a plate contaminated by a Penicillin mould, discovered the first antibiotic, Penicillin in 1929. By the mid-1940s, antibiotics were available for treatment against many bacterial infections including strep throat, pneumonia, skin infections, wound infections, scarlet fever, toxic shock syndrome, and other bacterial infections. By the mid-1950s, the discovery and introduction of streptomycin, tetracycline, and other antibiotics led to effective treatment of a vast array of formerly life-threatening infections, illnesses and diseases.

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Types of Antibiotics Even though many antibiotics are known to us today, each antibiotic doesn’t have an effect against all bacteria rather than antibiotics are selective. Most bacteria can be classified as Gram-Positive Bacteria (G+) and Gram-Negative Bacteria (G-) depending on their response to the gram stain. HANS GRAM, bacteriologist of the 1800s, developed this classification method. According to Gram’s system, many bacterial infections are either G+ or G- depending on the type of bacteria that causes them.

LIMITED SPECTRUM BROAD SPECTRUM ANTIBIOTICS ANTIBIOTICS i. When an antibiotic is i. These antibiotics are effective against some effective against a large micro-organisms, i.e., a range of micro-organisms specific spectrum or range (G+ and G-). and generally not effective against other bacteria which are not present in that spectrum, then such antibiotics are called limited spectrum antibiotics. ii.

Examples - Clindamycin, Eythromycin, Gentomycin, and Golistin

ii.

Examples- Tetracycline, and Chloramphenicol

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How do Antibiotics Work? Antibiotics fight pathogenic micro-organisms and cancer cells by interfering with their normal cell process. This occurs in three main ways: 1. Prevention of Cell Wall Formation 2. Disruption of Cell Membrane 3. Disruption of Chemical Processes

Prevention of Cell Wall Formation  Bacterial cells are enclosed in a membrane that is surrounded by a rigid wall that prevents the cells from splitting open.  Penicillin and some other antibiotics destroy pathogenic microbes by hindering the formation of this wall.  Peptidoglycan is one of the major wall targets because it is found only in bacteria.  Human cells don’t have nor need rigid cell walls, and so are not damaged by these antibiotics.

Disruption of Cell Membrane  Some antibiotics including amphotericin B and nystatin, disrupt the cell membrane of certain microbes.  This membrane controls the movement of materials in and out of the cell. If the cell membrane is disrupted, vital nutrients may escape from the cell or poisonous substances may enter the cell, and kill the cell.

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 However, the membranes of human cells are not affected because these antibiotics disrupt cell membranes that contain elements found only in the cells of microbes.

Disruption of Chemical processes  All cells produce proteins. Some of the antibiotics target bacterial protein synthesis, because bacterial ribosomes (70S) are different from the ribosomes (80S) of humans, and other eukaryotes.  Similarly, griseofulvin (antifungal agent) binds specifically to the tubulin proteins that make up the microtubules of fungal cells, these tubulins are somewhat different from the tubulins of humans.

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Uses and Limitations of Antibiotics Uses 1. Therapeutic Drugs They are used to fight bacterial infections and thus, are prescribed widely by doctors.

2. Treatment of Cancer A small number of antibiotics were developed to attack human cells; they are selectively toxic as they mostly damage cells that undergo unlimited mitosis. These types of antibiotics interact with DNA.

3. Treatment and prevention of diseases in animals Antibiotics are added to livestock feed to stimulate the animal’s growth and prevent the occurrence of diseases. If the animal has already been infected with a bacterial disease, antibiotics are then used by veterinarians to cure the animal.

4. Prevention of damage to fruits and grains Certain antibiotics are sprayed on the standing crops to prevent them from being spoiling.

5. Food Preservation Small quantities of antibiotics are added to packaged foodstuff to prevent it from spoiling by bacterial action.

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Limitations Many antibiotics are among the safest drugs when properly used but they can also cause unpleasant side effects at times. Thus, one must realize that these are only effective against bacterial diseases and also some fungi and protozoa. But they are not effective against viruses and hence, can’t be used to treat chickenpox, measles, cold, and other viral infections. The effectiveness of antibiotics is sometimes limited because of pathogenic microorganisms and become resistant to them. There are 4 main problems that might arise while using antibiotics:

1. Allergic Reactions In most cases allergic reactions are mild and produce only a rash or a mild fever. But a severe reaction to the drug can even result in death. Although, all antibiotics can produce allergic reactions, such reactions occur most frequently with penicillin.

2. Destruction of helping organisms Certain areas of the body commonly harbour both harmless and pathogenic microorganisms. These two types microbes compete for food and so the harmless microbes help in restricting the growth of the pathogenic microbe. Many antibiotics especially broad-spectrum drugs do not always distinguish between harmless and dangerous microbes. If a drug destroys too many harmless microbes, the pathogenic ones will have a greater chance to multiply. This situation often leads to the development of a new infection called supra infection. Doctors usually prescribe a secondary drug to combat supra infection.

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3. Damage to organs and tissues It is rare in people using antibiotics that act only against the calls of pathogenic microbes. However, extensive use of some of these antibiotics may damage tissues and organs.

4. Resistance in bacteria Overuse or improper use of antibiotic causes bacteria to become resistant to the drug and hence, new drugs need in such cases.

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Some Clinically Important Antibiotics S. Antibiotic No.

Producer Organism

Type

Site or Mode of Action

1.

Penicillin

Penicillin chrysogenum

Gram Positive

Wall synthesis

2.

Cephalosporin

3.

Griseofulvin

Cephallosporium acremonium Penicillin griseofulvum

Broad Spectrum Dermatophytic

Wall synthesis Microtubules

4.

Bacitracin

Bacills subtilis

Gram Positive

Wall synthesis

5.

Polymyxin B

Gram Negative

6.

AmphotericinB

Bacillus polymyxa Streptomyces nodosus

Cell membrane Cell membrane

7.

Erythromycin

Streptomyces Erythreus

Gram Positive

Protein synthesis

8.

Neomycin

9.

Streptomycin

Streptomyces fradiae Streptomyces griseus

Broad Spectrum Gram Negative

Protein synthesis Protein synthesis

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Vancomycin

Streptomyces orientalis

Gram Positive

Protein synthesis

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Gentamycin

Micromonospora Broad purpurea Spectrum

Protein synthesis

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Tetracycline

Streptomyces rimosus

Broad Spectrum

Protein synthesis

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Rifamycin

Streptomyces mediterranei

Tuberculosis treating

Protein synthesis

Fungi

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Experiment Aim To show the effect of antibiotics on the microbes present in the soil sample.

Apparatus Laminar flow, spirit, pipettes, soil sample solution, eppendorfs, distilled water, pestle and mortar, cycle-mixer, spreader, burner, cork borer, petri plates, marker, camera, scale, screw gauge, and autoclave.

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Procedure This experiment involves six steps which are: A. Pouring the plates B. Preparation of the antibiotic solutions C. Spreading of the bacteria D. Formation of wells E. Addition of antibiotic solutions F. Recording observations

Before the experiment: Preparation of Luria and Bertani (LB) Medium Composition: 1. 2. 3. 4.

Yeast Extract(0.5g/100ml) Tryptone/Peptone(1.0mg/100ml) NaCl(0.5g/100ml) Agar(1.5g/100ml)

Preparation: 1. The materials needed for the medium are weighed accurately on a butter paper using a digital weighing machine. 2. The weighed substances are added to a clean conical flask. 3. Distilled water is added to this mixture to form a 100ml solution. 4. The conical flask is put on a magnetic stirrer to mix the contents properly. 5. A cotton plug is put onto the opening of the conical flask, and the solution is autoclaved to melt agar which transforms

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into a jelly like substance, and also to kill any unwanted microbes that might contaminate the medium.

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Prosecution of the steps: A.Pouring of Plates 1. After autoclaving, procured around 25ml of LB medium in a petri dish. 2. The medium is now allowed to solidify in the plate.

B. Preparation of Antibiotic Solution of Different Concentration 1. One antibiotic is taken at a time, say first Terramycin- one 250mg tablet is grinded into a powder by a pestle and mortar. 2. This powder is carefully added to an eppendorf. Now, 1ml of distilled water is added to the eppendorf by a pipette. 3. The solution formed is naturally shaken vigorously to make the solute dissolve in the solvent; if it doesn’t dissolve, use a cyclo-mixer. 4. Once the solute dissolves, we get a solution of conc. 250mg. This is solution A. 5. Now, 500ul of the solution A is pipette out, and added to an eppendorf. To this 500ul of distilled water is added. 6. The solute is made to completely dissolve in the solvent, and the solution formed is of concentration 125mg. this is solution B. 7. Now, 500ul of the solution B is pipette out and added to an eppendorf to this 500ul of distilled water is added. 8. The solute is made to completely dissolve in the solvent, and the solution formed is of the concentration 62.5mg. This is solution C.

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9. The same method is used to prepare solutions of the rest of the antibiotic i.e., Amoxycillin.

C. Spreading of soil solution The cover of the Petri dish is lifted, and 50ul of the soil sample solution is pipette in the solid medium and spread over the LB agar plate.

D. Formation of Wells (Apparatus: Cork borer, spirit, Laminar flow, Petri plate prepared in the previous step) 1. The cork borer is made sterilized by dipping in spirit and heating over the burner for a few minutes. 2. The cover of the Petri plate is marked at different positions with a marker to ascertain the positions of the wells along with the concentration of antibiotic solution to be added later. 3. The cover of the Petri plate is removed and using the cork borer and the needle, wells are made into the solid medium at the inclined spots. 4. The spreader is sterilized using spirit and flame. 5. This spreader is used to spread Soil sample evenly over the surface of the medium. 6. The cover of the Petri plate is placed on top of it again.

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E. Addition of Antibiotic Solutions (Apparatus: 10ul pipette, Spirit, Laminar Flow, and Marker) 1. The three plates prepared are marked as control, Amoxycillin, and Terramycin. 2. A 10ul pipette is used to pipette put 10ul of one concentration of the antibiotic into its respective spot in the particular antibiotic Petri plate. 3. The same is done with different concentrations of the same antibiotic till its plate has 3 wells filled. 4. The plates are now ready, and he process is repeated for other antibiotic. 5. To prepare the control instead of the antibiotic solutions, 10ul of distilled water is added to the wells. 6. Once the Petri plates are ready, they are put into the incubator set at 37˚C for 24 hours.

F. Recording of Observations (Apparatus: Camera, scale, screw gauge, and autoclave) 1. After the incubation period is over, the Petri plate are taken out of the incubator, and the diameter of the inhibiton zones is noted with the help of a scale. Diameter is taken at 3 different points to ensure accurate results. 2. The diameter of the cork is noted with the help of a screw gauge. 3. Photograph of the petri plates are taken. 4. After the experiment is over, the Petri plates are autoclaved.

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Agar

Photo of performing experiment

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Observations A. Once the antibiotic solutions have been added to the wells, and incubation has been done, a zone of inhibition is seen very close to the wells, this inhibition zone is circular in shape, and has no whitish cream colour indicating the absence of the bacterial growth. B. From this, it can be inferred that the antibiotic solutions have diffused out form the wells into the medium, and have stopped the bacteria from growing. C. The diameter of the inhibition zone is more in the case of higher concentration of the same antibiotics from which it can be concluded that higher concentrations of antibiotics are more effective in killing bacteria. D. In the control plate, no inhibition zone is found, and the whole plate is covered with the whitish cream layer of bacterial growth. This can be explained as in the control plate only distilled water is added to the wells and since no substance that restricts the growth of bacteria is present in this control plate, bacteria grow unchecked.

Lawn of Bacteria in Control

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Antibiotic activity of Amoxycillin on soil bacteria

Antibiotic activity of Terramycin on soil bacteria

Observation Table Name

Concentration 250 mg Amoxycillin 125 mg 62.5 mg 250 mg Terramycin 125 mg 62.5 mg Control water -

Zone of Inhibition 1.8 cm 1.5 cm 1.5 cm 1.3 cm 1.25 cm 1.15 cm -

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Sample loaded in each well=50µL

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Conclusion The results are seen as the bacteria do not grow in the regions around the wells; the area far away from the wells is observed to have the whitish cream of bacterial growth. In contrast to this, in the control plate, a lawn of bacteria is observed- TNTC. Amoxycillin is more effective than Terramycin as the zone of inhibition in the case of Amoxycillin is wider as compared to Terramycin.

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Bibliography    

NCERT Biology Class XII www.google.com www.wikipedia.com www.wikihow.com