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BI0217-BASIC BIOTECHNOLOGY LAB MANUAL Offered to II YEAR B.TECH BIOINFORMATICS DEPARTMENT OF BIOINFORMATICS SCHOOL OF BIOENGINEERING SRM UNIVERSITY KATTANGULATHUR S. PAGE DATE OF N NAME OF THE EXPERIMENT SIGN NO EXPERIM O ENT LABORATORY SAFETY-GENERAL RULES 1 AND REGULATIONS LABORATORY PROTOCOL 2 STERILIZATION TECHNIQUES 3 PREPARATION OF MEDIA ISOLATION, ENUMERATION AND 4 PURIFICATION OF MICROBES FROM A GIVEN SAMPLE 5 USE OF MICROSCOPE 6 SIMPLE STAINING 7 GRAM STAINING SPORE STAINING (SCHAEFFER-FULTON 8 METHOD) 9 HANGING DROUP PREPARATION OF BUFFERS AND 10 MEASUREMENT OF pH 11 ESTIMATION OF SUGARS ESTIMATION OF PROTEINS BY LOWRY’S 12 METHOD / BIURET METHOD ESTIMATION OF CHOLESTEROL BY ZAK’S 13 METHOD SEPARATION OF AMINO ACIDS - THIN 14 LAYER CHROMATOGRAPHY SEPARATION OF SUGARS - PAPER 15 CHROMATOGRAPHY BIOCHEMICAL ESTIMATION OF DNA /RNA 16 USING SPECTROPHOTOMETER INSTRUCTIONS: ALL DIAGRAMS IN THE LEFT HAND SIDE OF THE PAGE 2LABORATORY SAFETY - GENERAL RULES AND REGULATIONS A rewarding laboratory experience demands strict adherence to prescribed rules for personal and environmental safety. The former reflects concern for your personal safety in terms of avoiding laboratory setting to prevent contamination of experimental procedures by microorganisms from exogenous sources. Because most microbiological laboratory procedures require the use of living organisms, an integral part of all laboratory session is the use of aseptic techniques. Although the virulence of microorganisms used in the academic laboratory environment has been greatly diminished because of their long-term maintenance on artificial media, all microorganisms should be treated as potential pathogens (organisms capable of producing disease). Thus, microbiology students must develop aseptic techniques (free of pathogenic organisms) in preparation for industrial and clinical marketplaces where manipulation of infectious organisms may be the norm rather than the exception. The following basic steps should be observed at all times to reduce the ever-present microbial flora of the laboratory environment. 1. Upon entering the laboratory, place coast, books, and other paraphernalia in specified locations-never on bench tops. 2. Keep doors and windows closed during the laboratory session to prevent contamination from air currents. 3. At the beginning and termination of each laboratory session, wipe bench tops with a disinfectant solution provided by the instructor. 4. Do not place contaminated instruments, such as inoculating loops, needles, and pipettes, on bench tops. Loops and needles should be sterilized by incineration, and pipettes should be disposed of in designated receptacles. 5. On completion of the laboratory session, place all cultures and materials in the disposal area as designated by the instructor. 6. Rapid and efficient manipulation of fungal cultures and materials in the disposal area as designated by the instructor. 7. Rapid and efficient manipulation of fungal cultures is required to prevent the dissemination of their reproductive spores in the laboratory environment. To prevent accidental injury and infection of yourself and others, observe the following regulations at all times: 1. Wash your hands with liquid detergent and dry them with paper towels upon entering and prior to leaving the laboratory. 2. Wear a paper cap or tie back long hair to minimize its exposure to open flames 3. Wear a lab coat or apron while working in the laboratory to protect clothing from contamination or accidental discoloration by staining solutions. 4. Closed shoes should be worn at all times in the laboratory setting. 5. Never apply cosmetics or insert contact lenses in the laboratory. 6. Do not smoke, eat, or drink in the laboratory. These activities are absolutely prohibited. 7. Carry cultures in a test - tube rack when moving around the laboratory. Likewise, keep cultures in a test-tube rack on the bench tops when not in use. This serves a dual purpose to prevent accidents and to avoid contamination of yourself and the environment. 8. Never remove media, equipment, or especially, bacterial cultures from the laboratory. Doing so is absolutely prohibited. 39. Immediately cover spilled cultures or broken cultures tubes with paper towels and then saturate them with disinfectant solution. After 15 minutes of reaction time, remove the towels and dispose of them in a manner indicated by the instructor. 10. Report accidental cuts or burns to the instructor immediately. 11. Never pipette by mouth any broth cultures or chemical reagents. Doing so is strictly prohibited. Pipetting is to be carried out with the aid of a mechanical pipetting device. 12. Do not lick labels. Use only self-stick labels for the identification of experimental cultures. 13. Speak quietly and avoid unnecessary movement around the laboratory to prevent distractions that may cause accidents. The specific precautions outlined below must be observed when handling body fluids of unknown origin due to the possible imminent transmission of the HIV and hepatitis B viruses in these test specimens. 1. Disposal gloves must be worn during the manipulation of these test materials. 2. Immediate hand washing is required if contact with any of these fluids occurs and also upon removal of the gloves. 3. Masks, safety goggles, and laboratory coast should be worn if an aerosol might be formed or splattering of these fluids is likely to occur. 4. Spilled body fluids should be decontaminated with a 1:10 dilution of household bleach, covered with paper toweling, and allowed to react for 10 minutes before removal. 5. Test specimens and supplies in contact with these fluids must be placed into a container of disinfectant prior to autoclaving. I have read the above laboratory safety rules and regulations and agree to abide by them. 4 LABORATORY PROTOCOL Student preparation for laboratory sessions The efficient performance of laboratory exercises mandates that you attend each session fully prepared to execute the required procedures. Read the assigned experimental protocols to effectively plan and organize the related activities. This will allow you to maximize use of laboratory time. PREPARATION OF EXPERIMENTAL MATERIALS Microscope Slides: Meticulously clean slides are essential for microscopic work. Commercially precleaned slides should be used for each microscopic slide preparation. However, wipe these slides with dry lens paper to remove dust and finger marks prior to their use. Labeling of culture vessels: Generally, microbiological experiments require the use of a number of different test organisms and a variety of culture media. To ensure the successful completion of experiments, organize all experimental cultures and sterile media at the start of each experiment. Label culture vessels with non- water- soluble glassware markers and / or self - stick labels prior to their inoculation. The labeling on each of the experimental vessels should include the name of the test organisms, the name of the medium, the dilution of sample, if any, your name or initials, and the date. Place labeling directly below the cap of the culture tube. When labeling Petri dish cultures, only the name of the organism(s) should be written on the bottom of the plate, close to its periphery, to prevent obscuring observation of the results. The additional information for the identification of the culture should be written on the cover of the Petri dish. INOCULATION PROCEDURES Aseptic techniques for the transfer or isolation of microorganisms, using the necessary transfer instruments is described fully in the experiments in Part I of the manual. Technical skill will be acquired through repetitive practice. Inoculating Loops and Needles: It is imperative that you incinerate the entire wire to ensure absolute sterilization. The shaft should also be briefly passed through the flame to remove any dust or possible contaminants. To avoid killing the cells and splattering the culture, cool the inoculating wire by tapping the inner surface of the culture tube or the Petri dish cover prior to obtaining the inoculum. When performing an aseptic transfer of microorganisms, a minute amount of inoculum is required. If an agar culture is used, touch only a single area of growth with the inoculating wire to obtain the inoculum. Never drag the loop or needle over the entire surface, and take care not to dig into the solid medium. If a broth medium is used, first tap the bottom of the tube against the palm of your hand to suspend the microorganisms. Caution: Do not tap the culture vigorously as this may cause spills or excessive foaming of the culture, Which may denature the proteins in the medium. Pipettes: Use only sterile, disposable pipettes or glass pipettes sterilized in a canister. The practice of pipetting by mouth has been discontinued to eliminate the possibility of auto- infection by the accidental imbibement of the culture or infectious body fluids. Instead, a mechanical pipetting device is to be used to obtain and deliver the material to be inoculated. INCUBATION PROCEDURE Microorganisms exhibit a wide temperature range for growth. However for most used in 0 this manual, optimum growth occurs at 37 C over a period of 18 to 24 hours. Unless 5otherwise indicated in specific exercise, incubate all cultures under the conditions cited above. Place culture tubes in a rack for incubation. Petri dishes may be stacked; however, they must always be incubated in an inverted position (top down) to prevent water of condensation from dropping onto the surface of the culture medium. This resultant excess moisture may then serve as a vehicle for the spread of the micro-organisms on the surface of the culture medium, thereby producing confluent rather than discrete microbial growth. PROCEDURE FOR RECORDING OBSERVATIONS AND RESULTS The accurate accumulation of experimental data is essential for the critical interpretation of the observations upon which the final results will be based. To achieve this end, it is imperative that you complete all the preparatory readings that are necessary for your understanding of the basic principles underlying each experiment. Meticulously record all the observed data in the "Observations and Results" section of each experiment. In the exercises that require drawings to illustrate microbial morphology, it will be advantageous to depict shapes, arrangements, and cellular structures enlarged to 5 to 10 times their actual microscopic size, as illustrated below. For this purpose a number 2 pencil is preferable. Stippling may be used to depict different aspects of cell structure, e.g., endospores or differences in staining density. REVIEW QUESTIONS The review questions are designed to evaluate understanding of the principles and the interpretations of observations in each experiment. Completion of these questions will also serve to reinforce many of the concepts that are discussed in the lectures. The designated critical-thinking questions are designed to stimulate further refinement of cognitive skills. PROCEDURES FOR TERMINATION OF LABORATORY SESSION 1. All equipment, supplies and chemical reagents are to be returned to their original locations. 2. All capped test-tube cultures and closed Petri dishes are to be neatly placed in a designated collection area in the laboratory for subsequent autoclaving. 3. Contaminated materials, such as swabs, disposable pipettes, and paper towels, are to be placed in a biohazard receptacle prior to autoclaving. 4. Hazardous biochemicals, such as potential carcinogens, are to be carefully placed into a sealed container and stored in a fume hood prior to their disposal according to the institutional policy. CLEANING AND PREPARATION OF CLEANING SOLUTION THE NEED FOR CLEANING: i. To remove the stains in the glassware. ii. To remove the chemical residues. iii. To remove the microbes partially by using the cleaning solution. iv. To remove the impurities which were stick on the surfaces of the glass wares. v. To remove the greasy areas by using mild detergents. 6PREPARATION OF CLEANING SOLUTION AIM: To prepare the cleaning solution to clean the glass wares. REQUIREMENTS: Balance, erylyn Meyer flask (1 L), measuring cylinder, spatula, butter paper, potassium dichromate, conc sulphuric acid, metal distilled water etc. COMPOSITION FOR CLEANING SOLUTION: DILUTE SOLUTION CONC.SOLUTION Potassium dichromate 60 g 60 g. Water 1 litre 300 ml. Conc.sulphuric acid 60 ml 460 ml. PROCEDURE: About 800 ml of distilled water contained in a clean erylyn Meyer flask was dissolved with 60 gms of potassium dichromate and mixed with 60 gms of potassium dichromate and mixed with 200 ml of conc. H SO and made upto a volume of 1 litre with 2 4 distilled water. This solution is allowed to cool and used later. This cleaning solution which can be used to oxidise any organic matter and will clean the glassware like test tubes, petriplates, pipettes etc. The cleaning solution can be used until it terms into dark green colour solution. CLEANING: The glassware are soaked in the cleaning solution overnight and washed with soap water and rewashed in running tap water and then with distilled water. Glassware 0 were allowed to drain of water and dried in a hot air oven at 80 C for 2 hrs for further use. 7Experiment No: STERILIZATION TECHNIQUES Sterilization is the process of destroying all forms of microbial life-vegetative and sporulating. It is important that all equipment used in a microbiological experiment are sterilized in order that a particular organism of interest is grown, without contamination by the organisms present in the surrounding environment. Several methods of this sterilization are employed in the process of sterilization to sterilize the various equipments used in an experiment these methods are broadly classified as PHYSICAL and CHEMICAL METHODS OF STERILIZATION. I PHYSICAL AGENTS: The major physical agents used for the control of microorganisms are TEMPERATURE, RADIATION AND FILTRATION. 1. TEMPERATURE: Microorganisms can grow over a range of temperatures, from very low temperatures characteristic of Psychrophiles, to very high temperatures characteristic of thermophiles. Temperatures above a maximum generally kill microbes. Such high temperatures can be produced by DRYHEAT or MOIST HEAT. DRY HEAT STERILIZATION: Can be used where it is either undesirable or unlikely that steam under pressure will make direct and complete contact. This is true of glassware such as Petriplates, erlyn meyer flasks, pipettes, test tubes. Such material can be sterilized 0 by placing in hot air, at a temperature of 160 C for 120 min. Alternatively, equipment like forceps, inoculation needle etc can be sterilized by direct heating on a flame till red hot. Thus it brings about destruction of unwanted organisms without changing the nature (flavour) of the material. This process involves heating at that 0 temperature for 15 min and then cooking it quickly to 0- 5 C. PRINCIPLE INVOLVED IN DRY HEAT STERILIZATION: Dry heat oxidise chemical components of organisms thus destroying them. MOIST HEAT STERILIZATION: High temperatures combined with high moisture is one of the most effective ways of sterilization. PRINCIPLES INVOLVED IN MOIST HEAT STERILIZATION: Moist heat coagulates microbial proteins, and is hence more rapid in killing microbes. Moist heat can be applied in the following ways in order to bring about sterility. STEM UNDER PRESSURE: Provides temperatures higher than those obtainable by any other method. 2t has advantages of rapid heating, penetration and moisture which facilitates coagulation of proteins. Autoclave is, a device used in the laboratory to sterilize media solution and to kill discarded cultures. It is operated at 15lbs/ sq inch pressure, which yields 0 a temperature of 121 C effective in bringing about sterility in 15 min FRACTIONAL STERILIZATION OR TYNDALLISATION: Some microbial 0 solutions cannot be heated over 100 C without being damaged. Such materials are 0 sterilized by Tyndallization, which involves heating at 100 C on three successive days with incubation periods in between. Resistant spores germinate during the period of incubation that is killed on heating on the subsequent day. PASTEURISATION: Milk, cream and other alcohol beverages are subjected to controlled heat treatment which kills microbes of a certain type alone. 2. RADIATION: When ionising radiation pass through cells, they create free hydrogen radicals, hydroxyl radicals and peroxides that cause intracellular damage, resulting in destruction of microbes. This method of sterilization is effective for sterilization heat labile material / also called (OLD STERILIZATION) 8U.V.light is the most effective region of the electromagnetic spectrum, and is employed in disinfecting of inoculation chamber and hospital operating rooms. UV light alters nucleic acids and results in a pyrimidine diamer, thus inhibiting DNA replication. 3. FILTRATION: This technique is used when the material to be used is heat labile and cannot be sterilized by heating for eg. Solutions of proteins, vitamins etc. filters of pore size 0.02μ -0.08μ are used to filters off microbes, thus rendering the filtrate sterile. Pore size, electric charge of the filter, charge carried by the organisms and nature of fluid being filtered affect efficiency of filtration e.g. Acity filters, Berkefeld filter, Berkefeld filter, membrane filter are all microbial filters. II CHEMICAL AGENTS Several groups of chemicals can be used as antimicrobial agents: S.No GROUP ACTION EXAMPLE 1. Alcohol Denature proteins and solubilize Ethyl 2. Aldehyde lipids Glutaraldehyde 3. Halogens Alkylate, reacts with – NH , SH- Co I Cl 2 2, 2 4. Heavy oH metals I inactivates proteins oxidise cells. Hg Cl 2 2 5. Gases Precipitates and inactivates proteins Ethylene dioxide 6. Detergents (used for surface sterilization) Alkylates organic compounds Disrupt cell membrane 7. Phenols Denature protein and disrupt cell membrane Ethyl alcohol (70%) finds indispensable use for disinfecting hands before and after a microbiological experiments, and also to disinfect the inoculation chamber or area where the experiment is conducted. CONCLUSION: Sterilization is the first indispensable step of any microbiological experiment. Clean and sterile equipment are pre-requisites for culture isolation and characterisation of any microorganism in a laboratory. Several methods can hence be employed to sterilize the various materials required for an experiment. 9Experiment No: PREPARATION OF MEDIA PURPOSE: To prepare the nutrient agar medium for bacterial culture. COMPOSITION OF NUTRIENT AGAR MEDIUM: pH (7.2) Beef extract 20g 1L Peptone 20g 1L Nacl 10 g 1L Agar 20g 1L Distilled water 1L COMPOSITION OF ROSE BENGAL AGAR MEDIUM pH: 5.6 ± 0.2 at 25 °C. (g/litre) Peptone 5.0; glucose 10.0; potassium dihydrogen phosphate 1.0; dichloran 0.002; magnesium sulfate 0.5; Rose Bengal 0.025; agar-agar 15.0. Distilled water 1L PROCEDURE: A clean erlynmeyer flask was taken with 200 ml of distilled water. The chemicals were weighed accurately and dissolved one by one taking care to add the next chemical only after the dissolution of the first one, expect agar and streptomycin in the case of rose bengal medium agar was melted separately and added to other ingredients. The pH was adjusted to 7.2 for nutrient agar medium and 6.5 for rose bengal agar medium. The total volume of the medium was made upto 1 litre. The medium was then distributed into 250 ml. Erlynmeyer flasks of 100 ml each and were plugged tightly with paper (from) and tied with thread. 0 Then the flasks were sterilized at 121 C for 15 minutes with 15lbs/in2 in an autoclave. Streptomycin was added only after the sterilization of the rose bengal agar medium. PREPARATION OF PLATES, SLANTS AND DEEPS: The prepared media can be poured into plates, slants and deeps for cultures and pure- cultures. PLATES: 0 The sterilized medium is cooled to 45 –50 C and poured into sterile petriplates. About 20 ml of the medium is poured into each of the sterile petriplates and allowed to solidify. Incubate in an incubator overnight before use. SLANTS: The molten medium after adjusting the pH is poured into text tubes, 5 ml of each and the test tubes are placed in a slanted position so that they with solidify with maximum surface area. DEEPS: Deeps are prepared by pouring the molten medium in sterile glass text tubes and then placed in a vertical position. The tubes are then cooled in cold water and are used to maintain cultures for long periods of incubation. RESULT: Thus nutrient agar and rose bengal agar media were prepared and used to prepared and used to prepare plates, slants and deeps for inoculation. 10Experiment No: ISOLATION, ENUMERATION AND PURIFICATION OF MICROBES FROM A GIVEN SAMPLE PURPOSE: To isolate, enumerate and purify various microbes from the given sample. PRINCIPLE The techniques commonly used for isolation of discrete colonies initially require that the number of organisms in the inoculum be reduced. The resulting diminution of the population size ensures that, following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to effect a separation of the different species present. The serial dilution is used to accomplish this. There are three techniques to do isolation of pure cultures. REQUIREMENTS: Sterile blanks (9ml), 12 sterile test tubes for slants, 1 sterile blank (10 ml), samples, 18-20 Petriplates(20 ml), 10 sterile pipettes (1 ml), bacterial growth medium- NUTRIENT AGAR (5 X 100 Ml), inoculation loop and wire, burner, marker pen, sterile chamber. PROCEDURE: 1. SERIAL DILUTION: Exactly 1 ml of the given sample was added to 9ml of blank,. -1 This solution was labeled as 10 . From this test tube, 1 ml of solution was taken and added -2 to a 9 ml blank, mixed evenly and labeled as dilution 10 for the same manner, the -5 procedure was separated during sterile pipettes for each transfer, until dilutions upto 10 -3 -4 are obtained. For the given soil sample, dilutions upto 10 and 10 were taken for fungal -4 -4 -5 isolation and dilutions 10 were taken for fungal isolation and dilutions 10 and 10 were -4 -5 fungal isolation and dilutions 10 and 10 were taken for bacterial isolation. 2. ISOLATION OF MICROBES : POUR PLATE METHOD. Pour plating is a technique useful for isolation and enumeration of microbes 1 ml of the selected dilutions were pipetted into sterile petriplates in close proximity to the flame. Molten agar (nutrient agar in case of bacterial culture and Rose bengal agar in case of 11fungal culture) was poured over the inoculum and the plates were swirled to evenly distribute the inoculum. Plates of a particular dilution were prepared in duplicates. The agar 0 was allowed to solidify and the plates were incubated at a temperature of 35 C in an inverted fraction for a period of 24-72 hours. SPREAD PLATE METHOD The spread-plate technique requires that a previously diluted mixture of microorganisms be used. During inoculation, the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent rod. The step-by-step procedure for this technique is as follows: Place the bent glass rod into the beaker and add a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion. a. With a sterile loop, place a loopful of culture in the center of the appropriately labeled nutrient agar plate that has been placed on the turntable. Replace the cover. b. Remove the glass rod from the beaker and pass it through the Bunsen burner flame, with the bent portion of the rod pointing downward to prevent the burning alcohol from running down your arm. Allow the alcohol to burn off the rod completely. Cool the rod for 10 to 15 seconds. c. Remove the Petri dish cover and spin the turntable. d. While the turntable is spinning, lightly touch the sterile bent rod to the surface of the agar and move it back and forth. This will spread the culture over the agar surface. e. When the turntable comes to a stop, replace the cover. Immerse the rod in alcohol and reflame. f. Keep the plate for incubation 3. ENUMERATION OF MICROBES OBSERVATIONS AND CALCULATIONS BACTERIAL CIULTURE The number of bacteria in a given sample is usually too great to be counted directly. However, if the sample is serially diluted and then plated out on an agar surface the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution. However, keep in mind that if the organism normally forms multiple cell arrangements, such as chains, the colony-forming unit may consist of a chain of bacteria rather than a single bacterium. In addition, some of the bacteria may be clumped together. Therefore, when doing the plate count technique, we generally say we are determining the number of Colony-Forming Units (CFUs) in that known dilution. By extrapolation, this number can in turn be used to calculate the number of CFUs in the original sample. Normally, the bacterial sample is diluted by factors of 10 and plated on agar. After incubation, the number of colonies on a dilution plate showing between 30 and 300 colonies is determined. A plate having 30-300 colonies is chosen because this range is considered statistically significant. If there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together. Generally, one wants to determine the number of CFUs per milliliter (ml) of sample. To find this, the number of colonies (on a plate having 30-300 colonies) is multiplied by the number of times the original ml of bacteria was diluted (the dilution factor of the plate 12counted). For example, if a plate containing a 1/1,000,000 dilution of the original ml of sample shows 150 colonies, then 150 represents 1/1,000,000 the number of CFUs present in the original ml. Therefore the number of CFUs per ml in the original sample is found by multiplying 150 x 1,000,000 as shown in the formula below: DILUTION P P AVG 1 2 -4 10 -5 10 No of bacterial C.F.U No of colonies Dilutions factor x amount of sample added = C.F.U / ml of sample. 4. STREAK PLATING: The streak-plate method is a rapid qualitative isolation method. It is essentially a dilution technique that involves spreading a loopful culture over the surface of an agar plate. Although many types of procedures are performed, the four-way, or quadrant, streak is described. Refer to figure, which schematically illustrates this procedure. Streaking cultures for isolated colonies allows you to: • separate mixed cultures • purify a single type of bacterium • propagate a clonal population of bacteria • help with the identification of a bacterium Streak plating is of two types: Quadrant streak: a. Place a loopful of culture on the agar surface in Area 1. Flame and cool the loop and drag it rapidly several times across the surface of Area 1. 0 b. Reflame and cool the loop and turn the petri dish 90 . Then touch the loop to a corner of the culture in Area 1 and drag it several times across the agar in Area 2. The loop should never enter Area 1 again. 0 c. Reflame and cool the loop and again turn the dish 90 .Streak Area 3 in the same manner as Area 2. 0 d. Without reflaming the loop, again turn the dish 90 and then drag the culture from a corner of Area 3 across Area 4, using a wider streak. Don't let the loop touch any of the previously streaked areas. The flaming of the loop at the points indicated is to effect dilution of the culture so that fewer organisms are streaked in each area, resulting in the final desired separation. 13 SLANTS AND STABS: For long term storage of bacterial culture, suspended in a slow state of growth, slants and states can be used. Desired bacterial colonies were picked up with the sterile loop and streaked onto the surface of the slant (maximising the surface area) sterile inoculation were dipped into bacterial culture maintained in nutrient broth, and were stabbed into agar both slants and states were incubated. OBSERVATIONS: Purified and stored bacterial cultures were deserved and various types of colony morphology studied. SIZE: SHAPE/MARGIN: ELEVATION: PIGMENTATION: TEXTURE: RESULT: The given soil sample contained C.F.U of bacteria per ml of sample. Desired bacterial cultures more purified, their morphology studied and subcultured for storage. 14Experiment No: USE OF MICROSCOPE A microscope is a scientific instrument with one or more lenses that allow you to observe specimens so small it is not visible to the naked eye, e.g. microorganisms (bacteria) and microsopic materials placed on the stage General Procedures 1. Make sure all backpacks and junk are out of the aisles. 2. Plug your microscope in to the extension cords. Each row of desks uses the same cord. 3. Always start and end with the Scanning Objective. Do not remove slides with the high power objective into place - this will scratch the lens 4. Always wrap electric cords and cover microscopes before returning them to the cabinet. Microscopes should be stored with the Scanning Objective clicked into place. 5. Always carry microscopes by the arm and set them flat on your desk. Focusing Specimens 1. Always start with the scanning objective. Odds are, you will be able to see something on this setting. Use the Coarse Knob to focus, image may be small at this magnification, 15but you won't be able to find it on the higher powers without this first step. Do not use stage clips, try moving the slide around until you find something. 2. Once you've focused on Scanning, switch to Low Power. Use the Coase Knob to refocus. Again, if you haven't focused on this level, you will not be able to move to the next level. 3. Now switch to High Power. (If you have a thick slide, or a slide without a cover, do NOT use the high power objective). At this point, ONLY use the Fine Adjustment Knob to focus specimens. 4. If the specimen is too light or too dark, try adjusting the diaphragm. 5. If you see a line in your viewing field, try twisting the eyepiece, the line should move. That's because its a pointer, and is useful for pointing out things to your lab partner or teacher. Drawing Specimens 1. Use pencil - you can erase and shade areas 2. All drawings should include clear and proper labels (and be large enough to view details). Drawings should be labeled with the specimen name and magnification. 3. Labels should be written on the outside of the circle. The circle indicates the viewing field as seen through the eyepiece, specimens should be drawn to scale - ie..if your specimen takes up the whole viewing field, make sure your drawing reflects that. Cleanup 1. Store microscopes with the scanning objective in place. 2. Wrap cords and cover microscopes. 3. Wash slides in the sinks and dry them, placing them back in the slide boxes to be used later. 4. Throw coverslips away. 16Experiment No: SIMPLE STAINING PURPOSE To perform the simple staining procedure to compare morphological shapes and arrangements of bacterial cells. PRINCIPLE In simple staining, the bacterial smear is stained with a single reagent. Basic stains with a positively charged chromogen are preferred, because bacterial nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the cationic chromogen. The purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells. The most commonly used basic stains are methylene blue, crystal violet, and carbol fuchsin. MATERIALS Cultures 24-hour nutrient agar slant cultures of Escherichia coli and Bacillus cereus, and a 24-hour nutrient broth culture of Staphylococcus aureus. Reagents Methylene blue, crystal violet, and carbol fuchsin. Equipment Bunsen burner, inoculating loop, staining tray, microscope, lens paper, bibulous paper, and glass slides. PROCEDURE 1. Prepare separate bacterial smears of the organisms following the procedure described. Note: All smears must be heat fixed prior to staining. 2. Place a slide on the staining tray and flood the smear with one of the indicated stains, using the appropriate exposure time for each: Carbol fuchsin, 15 to 30 seconds; methylene blue, 1 to 2 minutes. 3. Wash the smear with tap water to remove excess stain. During this step, hold the slide parallel to the stream of water; in this way you can reduce the loss of organisms from the preparation. 4. Using bibulous paper, blot dry but do not wipe the slide. 5. Repeat this procedure with the remaining two organisms, using a different stain for each. 6. Examine all stained slides under oil immersion. OBSERVATIONS AND RESULTS In the space provided 1. Draw a representative field for each organism 2. Describe the morphology of the organisms with reference to their shapes(bacilli,cocci,spirilli) and arrangements(chains,cluster,pairs). 17Experiment No: GRAM STAINING PURPOSE: To become familiar with 1. The chemical and theoretical bases for differential staining procedures. 2. The chemical basis of the Gram stain. 3. Performance of the procedure for differentiating between the two principle groups of bacteria: gram-positive and gram-negative. PRINCIPLE Differential staining requires the use of at least three chemical reagents that are applied sequentially to a heat-fixed smear. The first reagent is called the primary stain. Its function is to impart its color to all cells. In order to establish a color contrast, the second reagent used is the decolorizing agent. Based on the chemical composition of cellular components, the decolorizing agent may or may not remove the primary stain from the entire cell or only from certain cell structures. The final reagent, the counterstain, has a contrasting color to that of the primary stain. Following decolorization, if the primary stain is not washed out, the counterstain cannot be absorbed and the cell or its components will retain the color of the primary stain. If the primary stain is removed, the decolorized cellular components will accept and assume the contrasting color of the counterstain. In this way, cell types or their structures can be distinguished from each other on the basis of the stain that is retained. The most important differential stain used in bacteriology is the Gram stain, named after Dr.Christian Gram. It divides bacterial cells into two major groups, gram-positive and gram-negative, which makes it an essential tool for classification and differentiation of microorganisms. The Gram stain uses four different reagents. Descriptions of these reagents and their mechanisms of action follow. Primary stain Crystal Violet This violet stain is used first and stains all cells purple. Mordant Gram's iodine This reagent serves as a mordant, a substance that forms an insoluble complex by binding to the primary stain. The resultant crystal violet-iodine (CV-I) complex serves to intensify the color of the stain, and all the cells will appear purple-black at this point. In gram-positive cells only, this CV-I complex binds to the cell wall. The resultant magnesium-ribonucleic acid-crystal violet-iodine (Mg-RNA-CV-I) complex is more difficult to remove than the smaller CV-I complex. Decolorizing Agent Ethyl Alcohol, 95% This reagent serves a dual function as a lipid solvent and as a protein- dehydrating agent. Its action is determined by the lipid concentration of the microbial cell walls. In gram-positive cells, the low lipid concentration is important to retention of the Mg-RNA-CV-I complex. Therefore, the small amount of lipid content is readily dissolved by the action of the alcohol, causing formation of minute cell wall pores. These are then closed by alcohol's dehydrating effect. As a consequence, the tightly bound primary stain is difficult to remove, and the cells remain purple. In gram-negative cells,the high lipid concentration found in outer layers of the cell wall is dissolved by the alcohol, creating large pores in the cell wall that do not close appreciably on dehydration of cell wall 18proteins. This facilitates release of the unbound CV-I complex, leaving these cells colorless or unstained. Counterstain Safranin This is the final reagent, used to stain red those cells that have been previously decolorized. Since only gram-negative cells undergo decolorization, they may now absorb the counterstain. Gram-positive cells retain the purple color of the primary stain The preparation of adequately stained smears requires that you bear in mind the following precautions: 1. The most critical phase of the procedure is the decolorization step, which is based on the ease with which the CV-I complex is released from the cell. Remember that over- decolorization will result in loss of the primary stain, causing gram-positive organisms to appear gram-negative. Under- decolorization, however, will not completely remove the CV-I complex, causing gram-negative organisms to appear gram-positive. Strict adherence to all instructions will help remedy part of the difficulty, but individual experience and practice are the keys to correct decolorization. 2. It is imperative that slides be thoroughly washed under running tap water between applications of the reagents. This removes excess reagent and prepares the slide for application of the subsequent reagent. 3. The best Gram stained preparations are made with fresh cultures , that is, not older than 24 hours. As cultures age, especially in the case of gram-positive cells, the organisms tend to lose their ability to retain the primary stain and may appear to be gram-variable; that is, some cells will appear purple, while others will appear red. Materials Cultures 24-hour nutrient agar slant cultures of Escherichia coli, Staphylococcus aureus, and Bacillus cereus. Reagents Crystal violet, Gram's iodine, 95% ethyl alcohol, and safranin. Equipment Bunsen burner, inoculating loop or needle, staining tray, glass slides, bibulous paper, lens paper, and microscope. PROCEDURE The steps are pictured in Figure 1. Obtain four clean glass slides. 2. Using sterile technique, prepare a smear of each of the three organisms and on the remaining slide prepare a smear consisting of a mixture of S.aureus and E.coli. Do this by placing a drop of water on the slide and then transferring each organism separately to the drop of water on the slide with a sterile, cooled loop. Mix and spread both organisms by means of a circular motion of the inoculating loop. 3. Allow smears to air dry and then heat fix in the usual manner. 4. Flood smears with crystal violet and let stand for 1 minute. 5. Wash with tap water. 6. Flood smears with the Gram's iodine mordant and let stand for 1 minute. 7. Wash with tap water. 198. Decolorize with 95% ethyl alcohol. Caution: Do not over-decolorize. Add reagent drop by drop until crystal violet fails to wash from smear. 9. Wash with tap water. 10. Counterstain with safranin for 45 seconds. 11. Wash with tap water. 12. Blot dry with bibulous paper and examine under oil immersion. OBSERVATIONS AND RESULTS Following your observation of all slides under oil immersion, record your results in the chart. 1. Make a drawing of a representative microscopic field. 2. Describe the cells according to their morphology and arrangement. 3. Describe the color of the stained cells. 4. Classify the organism as to the gram reaction: Gram-positive or gram-negative. Refer to photo numbers 2-4 in the color-plate insert for illustration of this staining procedure. 20

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