BIOTECH LAB MANUAL-10TH SEM

2012

Experiment No#1 To evaluate Anti-bacterial activity of a selected Bacterial specie Introduction: The process of placement of culture cell to reproduce it in a specific environment to make it viable (that can reproduce).the item is inoculated is known as inoculum. Viable: Those species which have ability to reproduce are called viable while which dont reproduce are non-viable like dust particle. Incubation: A process of growth /reproduction of culture at most favorable temperature in an incubator. Strain: Strain it is the lowest taxonomic rank, strain is actually the subtype of specie varies during replication. Strain is identified on the basis of genetic studies. Mutation: Genetic variation is known as mutation which may be physical or chemical change or due to stress. Stock solution: It is a solution (concentration) which must be diluted prior to use. Antibiotics: Organic compounds produced by a group of micro-organisms or a single micro-organism and are used to kill other micro-organisms. Classification of Antibiotics: On the basis of their Action Bacteriostatic: Bactericidal: Agents which inhibit further growth of micro- They kill the microorganisms as the organism e.g. sulphonamides . bacteriostatic inhibits the growth while bactericidal are effective when microbes are replicating, so these drugs are not given in combination Narrow antibiotics They are effective against limited number of microorganism e.g. benzyl penicillin, benzathene penicillin.Amino-glycosides are active only against Gram positive species. On the basis of spectrum of activity spectrum Broad spectrum antibiotics Extended spectrum They are effective against wider range of microorganism, cephalosporin amoxicillin, erythromycin, clarithromycin are active against both gram positive and gram negative species. These are blood spectrum antibiotics even effective against Gram negative resistant species. E.g. Pseudomonas aeruginosa. It is not treated by some broad spectrum agents due to its resistance and genetic make up

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Classification of Bacteria: Psychrophiles They grow below 10 C or 15 c .they are found on glaciers. Mesophiles They survive between 25c to 45c and found in plains of Pakistan.

2012

Thermophiles They survive at temperature higher than 40 c but less than 80c i.e. in deserts and higher areas of Pakistan. Extreme Thermophiles They can grow above 80c and they can even grow on hot boiling water e.g. geysers.

BIOTECH LAB MANUAL-10TH SEM

2012

Experiment No # 2 To evaluate the antibacterial activity of selective bacterial species screening method Methods Cross streak method Stabbing method Inoculation method Streaking is a method of purification of colonies. Requirements Producer strains Indicator strains Producer strains Following are the strains which are evaluated for their capacity of producing antibiotics. Bacillus subtilis (gram +ve) E.coli (gram-ve) They are reported to produce bacitracin and subtilin (peptide antibiotic) which inhibit cell wall synthesis. E.coli is not able to produce antibacterial activity. Indicator strain Following are the strains against which antibiotic producing strains will be tested Staphylococcus aureus Micrococcus luteus E .coli Other Requirements Wire loop Inoculation loop Cotton swab Nutrient agar plate Chloroform Filter paper Borer 6-8 mm in diameter. Procedure 1. Prepare nutrient agar plate and incubate at for 24 hours at 37c.(sterility check) 2. Refresh cultures of both the indicator and the producer strain. 3. Take the nutrient agar plate and draw a straight line of indicator strain in the center of plate with the help of wire loop. 4. With a gap of 1-2 mm from central line draw a line of the indicator strain. 5. Incubate the plate at 37c for 24 hours. 6. Then check the results. 3

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Result

2012

Positive results: A zone of inhibition will be observed near the junction of producer an indicator strain Negative Results: No zones of inhibition are observed Advantage The method is useful to evaluate more than one test strain against single indicator strain. We can also perform this test reversely with one test strain and more than one indicator strain. A. Stabbing method 1. Prepare nutrient agar plate. 2. Incubate it for 24 hours at 37c to check sterility. 3. Refresh cultures of both procedure and indicator strain by streaking method. 4. Incubate culture for 24 hours at 37c. 5. Take nutrient broth in test tube. 6. Now take test strain by the help of inoculating needle and place it within depth of nutrient agar plate. Incubate nutrient agar for 24 hours at 37c. Next day growth will be observed in this plate. Cut the filter paper of size of agar plate lid and tip in the chloroform. 7. Place the filter paper in the cover of nutrient agar and place over the plate. 8. Place this plate under Laminar Flow Hood for 30 minutes. 9. Now dilute inoculums of indicator strain with normal saline and compare it with McFarland solution. Now soak cotton bud in these inoculums and make lawn on nutrient agar plate. Ensure completion of formation of bed by horizontal, vertical swabbing. 10. Incubate at 37c for 24 hours. 11. Check for results next day.

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2012

B. Direct Inoculation Method: Prepare nutrient agar plate and incubate it. Prepare 100ml nutrient broth 250ml flask and autoclave it. Take fresh indicator and producer strain cultures. Inoculate producer / test strain in nutrient broth with the help of wire loop. Incubate it at 37c for 24 hours. Nutrient broth will become turbid next day, indicating that test strain produced. Take sample from nutrient agar flask. Transfer sample to test tube or centrifuge tubes previously sterilized. Centrifuge it for 25-30 minutes at 400 rpm or 2000 rpm for 15 minutes In this test supernatant clear solution is obtained and pellets are present in the bottom. Pellets are present in the bottom. Pellets are bacterial mass so we discard this and shift supernatant to another test tube. This supernatant is the antibiotic carrying solution. Now make nutrient broth in tube by placing 2ml solution in tube and then autoclave it. Transfer the indicator strain to nutrient broth tube. Incubate tube for 24 hours at 37c. Dilute it with 0.9% NaCl solution till turbidity match with macfarland solution. Dip cotton swab in it and form lawn on nutrient agar plate. Make wells in plate with the help of sterile bores incubate plate. Check for results. Results: If growth is inhibited and transparent area is obtained this indicator fine result. Negative result is indicating by overlapping due to growth. Precautions: Try to keep two solutions apart to avoid irregular zone.

BIOTECH LAB MANUAL-10TH SEM

2012

Experiment No #3 Enzymes Production Ability of Selected Bacterial Species Enzymes: These are the secondary metabolites of bacteria produced during the metabolic reaction. Enzymes are protein in nature and are used as catalyst to speed up the reaction Introduction: Enzymes have some impact on the reaction always but never consumed in the reaction. every cell has about 4000 enzymes in it known as biological enzymes they take put in metabolic reaction of cell every reaction has a specific enzyme , because every enzyme has specific sequence of amino acid. At least 62 amino acids constitute an enzyme , 2500 amino acid sequence enzymes are also reported known as complex enzymes Properties of enzymes: Each enzyme possesses certain specific properties for e.g.: 1. Specific in nature i.e. each enzyme catalyzes a specific reaction only. 2. Mostly increase the rate of reaction but they are also used for poisoning. 3. They are never consumed for take part in the reaction: but always affect the reaction. 4. They always the designed compounds without chemical contamination. 5. Enzymes can also be taken as nutrient supplement Enzyme production: Enzymes can be produced by two methods genetic engineering and fermentation. Bacteria produce specific enzymes so are screened for desired production Fermentation Method: Fermentation is biological process used to produced or generate a desirable products under specific condition Requirements: Substrate Casein media Petri plate

BIOTECH LAB MANUAL-10TH SEM

2012

Procedure Step 1: Preparation of nutrient agar plates Nutrient agar plates are prepared as per composition and number required. incubate for 24 hrs at 37c.Evaluate the sterility

Step2: Freshing of culture Culture of produce retain is freshed by streaking on nutrient agar plates a incubate. Step3: Preparation of casein agar plates Casein plates are prepared as per composition and number required and incubates. Step4: Transfer of culture Transfer the fresh culture of producer strain on casein-Agar plate using wire loop making any particular shape then incubate

BIOTECH LAB MANUAL-10TH SEM

2012

Experiment No # 4: Evaluation of enzyme production ability of selected bacterial species at fermentation level Fermentation is a biological process used to produced or generate a desirable product under specific conditions

Types of fermentation: Fermentation is usually carried out at 3 levels small size production pilot plant scale production large scale fermentation Small size production: It is also known as lab scale or flask scale in which 1 liter or 2 liter flask is used. Pilot plant scale production: It involves trial scale production of desired material Large scale fermentation: It is known as rector scale based or industrial based fermentation Enzymes: Enzymes are protein molecules composed of certain excipients of amino acids. Enzymes are composed of 60-2500 amino acid sequence. Requirements: Substrate (Casein media) Nutrient agar plates Centrifugation machine Synthetic media Wire loop Nutrient broth Producer strain Autoclave Incubator

BIOTECH LAB MANUAL-10TH SEM

2012

Procedure: Small scale production: Fresh the producer strain on nutrient agar plates. Prepare 100 ml of nutrient broth in flask. Prepare 100ml of synthetic media in flask. Autoclave both flask. Prepare casein media plates; incubate to evaluate its sterility. Incubate producer strain in 100 ml nutrient broth after introduction. Now introduce 10% inoculums i.e. 10 ml in 90 ml of synthetic media incubate it at 37 C for 24 hrs. There must be production of enzymes. Centrifuge by taking sample in centrifuge tube at 10000 rpm for 25-30 mins. Collect the supernatant at anti sterile plate and discard the pallets. Now evaluate supernatant in sterile plates. Result: Milky appearance which conform the presence of enzymes Transparent zone due to hydrolysis of proteins .i.e. complex proteins are dissolved.

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Experiment No # 5 Antibiotic production By Fermentation

2012

Fermentation: Biological process preceded by the decomposition of certain substances under suitable environmental conditions. It may be aerobic or anaerobic e.g. yogurt or cheese. Level of fermentation: Three production level for fermentation method 1. small scale production 2. Pilot batch trial based fermentation done by RND department from 2-10 l also called scale up/pilot plant production. 3. Large scale production, rector based on industrial production up to several 1000 L. Optimization of conditions: Max result of products (derived product)obtained from availability resources done on small scale also called standardization. Physical parameter Temperature Time of incubation Nutrient medium Aeration tube Carbon sucrose Glucose (0.5,1%.....not more tha 5%) increase conc lactic acid,conc acidic decrease bacterial growth Nitrogen source Glycin,glutamic acid,aspartate acid Primary media Natural media,growth media,con is unknown,consc of which bacterial growth facilitated e.g Agar (1.5-2.5) ptimize conc is not known Secondary media Synthetic media, production media concentration is known at which production is facilitated optimize concentration is find out at which production is max .e.g. carbohydrates, proteins; minerals concentration can be variated to get max product. Compounds are optimized to get maximum production of antibiotic so called as production media or synthetic media.

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BIOTECH LAB MANUAL-10TH SEM

2012

Example of fermentation Cheese production Yogurt production Antibiotics Hormones Immounu-modulater Requirements Agar plates Wire loops Synthetic media 0.9% Nacl solution Cotton swab Cork Flask Beakers Burner Graduated cylinder Procedure A. Preparation of nutrient agar plates Nutrient agar plates are prepared As per composition and number required these are inoculated at 37 C for 24 hour to evaluate sterility B. Freshening of culture Using a wire loop streak strains of producer and indicator Organism on freshly prepared nutrient agar C. Preparation of media Prepare 100ml of nutrient broth in a flask as nutrient media prepare 90ml of synthetic media in another flask .Prepare three test tubes containing 2ml of nutrient broth each. Incubate these solution at 37 C for 24 hours, to evaluate sterility D. Inoculation of producer stain Inoculate the producer strain in 100ml nutrient broth flask and incubate for 24 hours. The media becomes turbid due to bacterial growth and antibiotics E. Inoculation of indicator stains Indicator strain are inoculated in the test tube containing nutrients broth and inoculated for 24 hours F. Shifting of inoculam Shift 10ml of inoculum of producer stain to 90 m of synthetic media. Incubate it .it will turbid due to bacterial growth and antibiotic produced G. Centrifugation Centrifuge synthetic media with the antibiotics at 10,000 RPM for 15-20 min. Take supernatant in fresh sterile test tube and discard the plate H. Evaluation Evaluate the antibiotic method produced 11

BIOTECH LAB MANUAL-10TH SEM

2012

I. Extraction Extract the supernatant of centrifuge using chromatography techniques J. Purification After during product is purified using chromatography techniques K Structure determination Structure of antibiotics is determined using different techniques e.g NMR, IR etc

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BIOTECH LAB MANUAL-10TH SEM

Experiment No # 6 ISOLATION OF DNA FROM MAMMALIAN CELLS. THEORY:

2012

DNA is a part of genetic material which carries specific sequence of nucleotides that code for a particular protein. The sequence of nucleotide remains same for generation to generation until a mutation occurs. DNA contains basic hereditary material called genes. DNA isolation is done for: Forensic reason, e-g, DNA finger printing. Genetic engineering & genetic recombination for research purposes. Genetic therapy, e-g to evaluate the missing/defective gene.

PURPOSE

REQUIREMENTS: Eppendorf tubes 1.5ml Falcon tubes Centrifuge machine DNA concentrator Micropipette Chemical reagents Incubator Solution CHEMICAL REQUIREMENTS: Solution A: Tris-EDTA (buffer at pH 7.5) Sucrose 0.32M (precipitation) Magnesium chloride 25m.meter (cell lysis, ppt) Triton X-100 .1% ( se permeability of cells) Solution B: Tris-EDTA NaCl 400mm (cell lysis) SDS (increase permeability) Protenase-K-Enzymes (hydrolyze cellular enzyme) Solution C: Phenol pH 7.8 buffer Solution D: Izoamyl Alcohol Chloroform Solution C & D are used for solvents extraction. Phenol = 1 Isoamyl alcohol = 1 Chloroform = 25 13

BIOTECH LAB MANUAL-10TH SEM

2012

Experiment No # 7 Determine the antifungal activity of different synthetic compounds Introductions: Fungus: A fungus is a member of a large group of eukaryotic organisms that includes microorganisms such as yeasts and molds (British English: moulds), as well as the more familiar mushrooms. These organisms are classified as a kingdom, Fungi, that is separate from plants, animals and bacteria. One major difference is that fungal cells have cell walls that contain chitin, unlike the cell walls of plants, which contain cellulose. These and other differences show that the fungi form a single group of related organisms, named the Eumycota (true fungi or Eumycetes), that share a common ancestor (a monophyletic group). This fungal group is distinct from the structurally similar myxomycetes (slime molds) and oomycetes (water molds). The discipline of biology devoted to the study of fungi is known as mycology, which is often regarded as a branch of botany, even though genetic studies have shown that fungi are more closely related to animals than to plants Drugs: Many species produce metabolites that are major sources of pharmacologically active drugs. Particularly important are the antibiotics, including the penicillins, a structurally related group of -lactam antibiotics that are synthesized from small peptides. naturally occurring penicillins such as penicillin G (produced by Penicillium chrysogenum) antibiotics produced by fungi include: ciclosporin, commonly used as an immunosuppressant during transplant surgery; and fusidic acid. Widespread use of these antibiotics for the treatment of bacterial diseases, such as tuberculosis, syphilis, leprosy. Bioremediation: Certain fungi, in particular "white rot" fungi, can degrade insecticides, herbicides, pentachlorophenol, creosote, coal tars, and heavy fuels and turn them into carbon dioxide, water, and basic elements.[203] Fungi have been shown to biomineralize uranium oxides, suggesting they may have application in the bioremediation of radioactively polluted sites Procedure: There are different methods to evaluate the antifungal activity of synthetic compounds. Pour plate technique Hyphical extension inhibitory method

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BIOTECH LAB MANUAL-10TH SEM

2012

Preparation of the crude extracts: Hundred grams of each of the air-dried and coarsely powdered plant material was exhaustively extracted for 2 hours with petroleum ether (60-80oC) in soxhlet apparatus. The petroleum ether extract was filtered and evaporated under reduced pressure using Rota-vapor. The extracted plant material was then air-dried, repacked in the soxhlet apparatus and exhaustively extracted with methanol (98.8%) for 2 hours. The methanol extract was filtered and evaporated under reduced pressure using Rota-vapor. The extracts were dissolved in dimethyl-sulphoxide to make the final concentrations which kept in refrigerator till used. Simultaneously, water extract was prepared by adding (10ml) of boiled distilled water to 5gm of coarsely powdered plants leaves in a beaker on water bath with occasional stirring for 4 hours. The aqueous extract was then filtered and rewashed with small volume of boiled distilled water and added to the filtrate, which were then adjusted to (5ml) volume and used immediately Preparation of the tested organisms: The fungal cultures (Aspergillus niger (ATCC 9763), Candida albicans (ATCC 7596) were maintained on sabouraud dextrose agar, incubated at 25C for 4days. The fungal growth was harvested and washed with sterile normal saline and finally suspended in (100ml) of sterile normal saline and the suspension was stored in refrigerator till used. Preparation of fungal culture: Sabouraud glucose-agar media. Following composition was used for this purpose: Pepton10g, glucose 20g, Agar 20g, distil water 1000 ml with 5.4 pH. All the contents were mixed and dissolved in distilled water. The solution was autoclaved at 1200C, 15 Lb/sq inch pressure for20 minutes. Pathogens were isolated with the help of sterilized forceps and plated on sterilized potato dextrose agar (PDA) medium (potato starch: 20 g, dextrose: 20 g, agar: 20 g and distilled water to make the volume 1 liter, which was sterilized in a gas operated autoclave at 15 pounds pressure per square inch (PSI) for 20 minutes. Plates were incubated at 25C and observed daily for emergence of colonies. Sub-culturing was done from single spore to obtain pure culture.

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In vitro testing of extracts for antimicrobial activity: Hyphal extension inhibitory method:

2012

Diffusates were added in Potato dextrose agar (PDA) @ 10, 50, 100 and 200 g L-1 and poured into Petri dishes. PDA medium added only with ethanol and water served as control. Each Petri dish was inoculated with 5 mm plug of pure Isolate taken from margins of actively growing culture of pathogen. Then Petri plates were incubated at 25 2oC. Mycelial growth was recorded when the growth of three selected pathogens were completed in the control treatment. Each treatment was repeated five times. Mean radial mycelial growth of each plant Diffusates was recorded and data were subjected to Statistical analysis. Radial mycelial growths on different Diffusates were transformed into inhibition percentage by using the following formula (Naz et al., 2006): Inhibition percentage =100 - Mycelial growth on Diffusates Mycelial growth on control X 100

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BIOTECH LAB MANUAL-10TH SEM

2012

Pour plate technique: The cup-plate agar diffusion method (pour plate) was adopted according to Kavanagh, (1972) to assess the antifungal activity of the prepared extracts. One full loop spore of fungus was thoroughly mixed with 60 Ml of SDA. 20 ml of the inoculated SDA were distributed into Sterile Petri dishes. The agar was left to set and in each of these plates 4 cups, 10 mm in Diameter, was cut using a sterile cork borer No. 4 and the agar discs were removed. Alternate cups were filled with 0.1ml of each extracts using micro titer-pipette and Allowed to diffuse at room temperature for two hours. The plates were then incubated in the upright position at 25C for 4 to 7 days. Two replicates were carried out for each extract against each of the test organism. Simultaneously addition of the respective solvents instead of extracts was carried out as controls. After incubation the diameters of the results and growth inhibition zones were measured, averaged and the mean values were tabulated

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Experiment No # 8 Antioxidant activity of different chemical compounds Antioxidant

2012

An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid or polyphenols.[1] Although oxidation reactions are crucial for life, they can also be damaging; hence, plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxidases. Low levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells. As oxidative stress might be an important part of many human diseases, the use of antioxidants in pharmacology is intensively studied, particularly as treatments for stroke and neurodegenerative diseases. However, it is unknown whether oxidative stress is the cause or the consequence of disease. Antioxidants are widely used as ingredients in dietary supplements in the hope of maintaining health and preventing diseases such as cancer, coronary heart disease and even altitude sickness. Although initial studies suggested that antioxidant supplements might promote health, later large clinical trials did not detect any benefit and suggested instead that excess supplementation may be harmful. [2][3] In addition to these uses of natural antioxidants in medicine, these compounds have many industrial uses, such as preservatives in food and cosmetics and preventing the degradation of rubber and gasoline

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