LABORATORY MANUAL BTY557 LAB IN MICROBIOLOGY

Proteus flagella (Liefson’s stain)

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negative staining acid-fast staining Size measurement of purified bacterial strain by micrometry 5 7 9 11 6 7 8 9 10 To study motility of bacterial strain using hanging-drop and stab method Standard growth curve of purified bacterial strain IMVIC and TSI test To test for antibiotic sensitivity of bacterial sample MIC test for antibiotic sensitivity of bacterial strain against specific antibiotic 12 13 14 17 18 Text Book(s): 1. 3 2 3 4 5 Maintenance & purification of microbes by streak-plate method Gram-staining. Cummings publisher. Microbiology: A Laboratory Manual 3rd edition. Cappuccino.. Experiments in microbiology. J. Other Readings: Reference Book(s): 1. 1992. spore staining Capsule staining. 2. 2 .Table of contents S. plant pathology & Biotechnology: by KR Aneja. Practical Microbiology: by FC Garg. 1 Title of the experiment Isolation & culture of microbes from soil/ water/ air/ milk by spread plate and pour-plate method using serial dilutions and SPC techniques Page No. N.G and Sherman. No.

n. 1-ml pipettes. b) pour-plate and c)spread plate method using serial dilutions and SPC techniques Experiment No. Mixed culture of Serratia marcescens. the initial dilution is made by transferring 1 ml of E. Avoid splashing of medium up over the slides of Petri plate. boiling water bath Materials required: 24-hour 10ml nutrient broth culture of Escherichia coli.0 ml to one petri plate and 0. Inoculation loop. Learning Objectives: To isolate bacteria by streak plate technique.a. 1 a) Title of the experiment: Isolation and purification of microorganisms by streak plate method Equipments required: Autoclave. The 10-2 dilution is then shaken by grasping the tube between the palms of both hands and rotating quickly to create a vortex. incubator. Error analysis: . and 10-8.a.Experiment 1 Title of the experiment: Isolation & purification of microbes from soil/ water/ air/ milk by a) streak-plate. boiling water bath Materials required: Sterile Petri plates. Allow the medium into the plates to solidify. Allow it to cool and take a loopful of mixed culture aseptically.n. 6 petri plates. After pouring the medium into the plate. Streak the mixed culture on the solid surface of culture medium by lifting the cover of the Petri plate from one edge. Nutrient agar. water bath. 1 b) Title of the experiment: Isolation and purification of microorganisms (bacteria) from soil/water/air by pour plate method Equipments required: Autoclave. this second blank represents a 10-4 dilution of the original sample. Label the bottom surface of sterile Petri plates with your name and date. Required Results: Parameters: Single separate colony can be isolated Relationship: . Repeat the process once more to produce a 10 8 dilution. Using aseptic technique. This third dilution represents a 10-6 dilution of the original sample. This is a 1/100 or 10-2 dilution. Label four tubes of saline 10-2. Graphs: . This serves to distribute the bacteria and break up any clumps. 4 tubes of 5ml nutrient broths. 3 .4-5ml pipets and pi-pump. Do the same for the 10-6 and the 10-8 dilutions. with pi-pump. Cautions: Base plates should dried completely streaking should be proper so that single separate colonies can be isolated Experiment No. Be sure that you work near the flame of Bunsen burner in the asepic zone and flame the neck of the flask containing nutrient agar prior to pouring to destroy microorganisms present around the rim of the flask. gently rotate the plate to distribute the medium evenly. Hold the inoculation loop and heat in the fame of the burner to red-hot.a. 10-4. Immediately after the 10-2 dilution has been shaken. Outline of Procedure: Disinfect the surface of the working table and your hands with a disinfectant.n. Shake the 10-4 dilution vigorously and transfer 1ml to the third 99ml blank. uncap it and aseptically transfer 1ml to a second 99ml saline blank. coli sample to a 99ml sterile saline blank. Shake the 10-4 dilution again and aseptically transfer 1. incubator. water bath. Since this is a 10-2 dilution. 10 -6. Bunsen burner.1ml to another petri plate. 6 agar pour tubes of nutrient agar. 4 sterile 99-ml saline blanks. cool to 50°C and pour 20-25 ml medium into the bottom of the Petri plates. Micrococcus luteus and Escherichia coli. Liquify nutrient agar. Learning Objective: Isolation of Bacteria by serial Dilution To learn the spread plate and pour plate techniques Outline of Procedure: Label the bottom of six petri plates 1-6.

Cautions: The dilutions should be made properly The labeling should be done carefully.a. Record your results. marcescens. Cautions: The dilutions should be made properly The labeling should be done carefully. incubator. Repeat this process for the remaining five plates. Equipments required: Autoclave.Remove one agar pour tube from the 48 to 50C water bath. Relationship: n. Experiment:. making sure the entire surface of the plate has been covered. Invert the plates and incubate for 24 to 48 hours at room temperature or 30°C. At the end of the incubation period. 7. label the bottom of the agar medium plates with the name of the bacterium to be inoculated. Immerse the spreader in ethanol.test tubes. and date. your name. Repeat the procedure to inoculate the remaining two plates.a. Graphs: n. After the pour plates have cooled and the agar has hardened. Error analysis: n.1 ml of the respective bacterial culture onto the center of a tryptic agar plate. boiling water bath Materials required: Petri plates. select all of the petri plates containing between 30 and 300 colonies. 6. (b) one with S. Also make sure you do not touch the edge of the plate. With a wax pencil. Dip the L-shaped glass rod into a beaker of ethanol and then tap the rod on the side of the beaker to remove any excess ethanol. Three plates are to be inoculated: (a) one with B. Briefly pass the ethanol-soaked spreader through the flame to burn off the alcohol. The agar and sample are immediately mixed gently moving the plate in a figure-eight motion or a circular motion while it rests on the tabletop.a. and (c) one with the mixture. measure some representative colonies and carefully observe their morphology. 9.a. 4 . Graphs: n.1 (c) Title of the experiment: Isolation and purification of microorganisms (bacteria) from soil/water/air by spread plate method. they are inverted and incubated at 25C for 48 hours or 37C for 24 hours. Count the colonies on each plate. Error analysis: n. 3. nutrient agar medium Learning Objectives: To teach the students the basic culture techniques for microorganisms Procedure 1. 4. inoculation loop. 8. Plates with fewer than 30 colonies are designated too few to count (TFTC).a.a. and reflame. Plates with more than 300 colonies cannot be counted and are designated too many to count (TMTC). subtilis. 5. water bath. tap on the side of the beaker to remove any excess ethanol. Required Results: Parameters: Microbial colonies will be obtained and from this data the microbial load of the given sample can be ascertained Relationship: n. Spread the bacterial sample evenly over the agar surface with the sterilized spreader. and allow it to cool inside the lid of a sterile petri plate. After incubation. Pipette 0. Carefully remove the cover from the 10 -4 petri plate and aseptically pour the agar into it. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added to liquefied agar Required Results: Parameters: Microbial colonies will be obtained and from this data the microbial load of the given sample can be ascertained. 2.

A Quebec colony counter should be used. cool to 50°C and pour 20-25 ml medium into the bottom of the Petri plates (Be sure that you work near the flame of Bunsen burner in the asepic zone and flame the neck of the flask containing nutrient agar prior to pouring to destroy microorganisms present around the rim of the flask). Outline of Procedure: Label the bottom of six petri plates 1-6. Bunsen burner. the initial dilution is made by transferring 1 ml of E. Allow the medium into the plates to solidify.2 Title of the experiment: Maintenance & purification of microbes by streak-plate method Experiment No. Disinfect the surface of the working table and your hands with a disinfectant. Label four tubes of saline 10-2. Avoid splashing of medium up over the slides of Petri plate. The 10-2 dilution is then shaken by grasping the tube between the palms of both hands and rotating quickly to create a vortex. Plates with more than 300 colonies cannot be counted and are designated too many to count (TMTC).0 ml to one petri plate and 0. This third dilution represents a 10-6 dilution of the original sample. This serves to distribute the bacteria and break up any clumps. Label the bottom surface of sterile Petri plates with your name and date. Count the colonies on each plate. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added to liquefied agar. 6 agar pour tubes of nutrient agar (plate count agar). Shake the 10-4 dilution vigorously and transfer 1ml to the third 99ml blank. and 10-8. they are inverted and incubated at 25oC for 48 hours or 37oC for 24 hours. Since this is a 10-2dilution. Using aseptic technique. After pouring the medium into the plate. gently rotate the plate to distribute the medium evenly. Repeat this process for the remaining five plates. Liquefy nutrient agar. Allow it to cool and take a loopful of mixed culture aseptically. select all of the petri plates containing between 30 and 300 colonies. this second blank represents a 10-4 dilution of the original sample. 10-4. 10 -6. This is a 1/100 or 10-2 dilution. number of colonies (CFUs) = # of bacteria/ml dilution X amount plated Record your results. pipettes. 5 . Do the same for the 10-6and the 10-8dilutions. Materials required: 24-hour 10ml nutrient broth culture of Escherichia coli. Immediately after the 10-2dilution has been shaken. 1 a) Title of the experiment: Isolation and purification of microorganisms by streak plate method Equipments required: Water bath. test tubes.1ml to another petri plate. spectrophotometer. After the pour plates have cooled and the agar has hardened. cuvettes. Carefully remove the cover from the 10-4 petri plate and aseptically pour the agar into it. The agar and sample are immediately mixed gently moving the plate in a figure-eight motion or a circular motion while it rests on the tabletop. Hold the inoculation loop and heat in the flame of the burner to red-hot.Experiment :. Repeat the process once more to produce a 10-8 dilution. uncap it and aseptically transfer 1ml to a second 99ml saline blank. 1-ml pipettes with pi-pump. 6 petri plates. Plates with fewer than 30 colonies are designated too few to count (TFTC). coli sample to a 99ml sterile saline blank (figure below. Shake the 10-4 dilution again and aseptically transfer 1. At the end of the incubation period. Remove one agar pour tube from the 48 to 50oC water bath. 4 sterile 99-ml saline blanks. Learning Objectives: To isolate and purify bacteria by streak plate technique.

6 . 5. 4. Serial dilution should be done. Relationship: n. Error analysis: n.a. separate the different microorganisms present in sample.a. Cautions: Contamination should not occur. Required results: Parameters: Any sample consists of multiple microorganisms. 6.Streak the mixed culture on the solid surface of culture medium by lifting the cover of the Petri plate from one edge.a. Parameters and Plots: None. Graphs: n.

Materials required:-48-72 hour nutrient slant culture of Bacillus cereus and thioglycollate culture of Clostridium butyricum. 2. 5. 7. Young broth cultures of Bacillus spp. Wash the slides immediately with tap water to avoid excess reaction of alcohol-acetone reagent.a. replenish stain as needed. 3 (b) Title of Experiment: To perform spore staining by Schaeffer Fulton method.3 Title of the experiment :. 2. Air dry and heat fix. Malachite green and safranin. record your result in the chart.a. Counterstain with safranin for 30 seconds. Equipments required: Bunsen burner. Wsh with tap water. Outline of the Procedure 1. or terminal on each preparation. ‘Blot dry with bibulous paper and examine under oil immersion. hot plate. Cover the smears with crystal violet-ammonium oxlate solution and allow to stand for one minute. crystal violet. Cautions: Culture used should be pure. Make individual smears in the usual 3. Do not rub. 8.a.. To learn the rationale and mechanism of Gram stain. Allow smear to air-dry.Experiment :. Examine the smears using high dry and oil immersion lens of microscope.To perform: a) Gram-staining b) Spore staining 3 (a) Title of experiment: To perform the Gram staining of the purified bacterial culture. Graphs: n. allowing the preparation to steam for 2 to 3 minutes. Subterminal. Relationship: n. Discribe the locations of the endospore within the vegetative cell as being central. Obtain two clean glass slide 2. Outline of the Procedure: Prepare thin smears of both the cultures on separate slides. 3. Graphs: n. Remove slides from hot plate. 4. Cover the smears with safranin reagent and keep for 30-60 seconds. blot dry the slides. Lugol’s iodine. Caution: Do not allow to evaporate.a. cool and wash under running tap water. Learning Objectives: 1. 3. Required results: Parameters: Following your observation of all slides under oil immersion. Equipment required: Microscope Materials Required: Clean slides. Prevent the stain from boiling by adjusting the hot plate temperature. To perform and interpret Gram stain. Error analysis: n. staining tray. 1. lens paper and microscope. bibulous paper. 7 . To differentiate between Gram positive and Gram negative reactions. inoculating loop. Flood smears with malachite green and place on a warm hot plate.ammonium oxalate solution. Indicate color of the spore and vegetative cell on each preparation. Apply alcohol-acetone reagent drop wise until the acetone solution flows off the smear free from crystal violet. and Escherichia coli. Wash the slides as above. glass slides. 6. Required Result: Parameters: Bacterial cells stained violets are described as Gram positive and those appearing pink are termed Relationship: n. Rinse the slides with Lugol’s iodine reagent for 30 seconds. Make drawing of a representative microscopic field of each preparation.a. and heat fix in the usual manner. Wash with gently running tap water. Safranin solution. Iodine-acetone/ Acetone alcohol solution.

Caution: Stain must be prepared fresh & Apply stain gently on smear 8 .Error analysis: n.a.

high dry and oil-immersion objectives. and 48 –hours-old skimmed milk cultures of Alcaligenes viscolacits. Examine microscopically. Apply stain gently on smear 4 (b) Title of experiment: To perform the negative staining of the purified bacterial culture Equipments required: Microscope Materials Required: Clean slides. Genlty blot dry and examine under oil immersion. Using sterile technique. To learn and prepare a negative stain. Materials required: inoculating loop or needle.a. To interpret the application and mechanism of negative staining technique. and Enterobacter aerogenes. 1% crystal violet and 20% copper sulfate (CuSO4. Take a loopful of broth culture with a sterile inoculation needle and mix into the nigrosinedrop. Caution: 1.a.5H2O). Let the smear air dry and examine microscopically using low. Outline of the Procedure: Clean the slide greese free with soap powder. Graphs: n. Learning Objectives: 1. Do not spread the drop or let it dry. Place a drop of nigrosine black at one end of the slide. bibulous paper. 2. Now spread the drop with the end edge of another slide to produce a smear of varying opacity. Relationship: n. Let stand for 5 to 7 minutes Allow smears to air dry. Nigrosine black dye.a.Experiment:-4 Title of the experiment :. Caurion: do not heat fix. Required Results: Parameters: In negative staining bacteria appear colorless against a background. Repeat steps 1 to 6 for each of the remaining test cultures. Required Results: Parameters: It helps to identify between capsular material and bacterial cell.To perform a) Capsule staining b) Negative staining d) Acid-fast staining 4 (a) Title of experiment: To perform capsule staining to distinguished between capsular material and bacterial cell Equipment required: Bunsen burner and microscope.a.a. Cautions: Broth should not be contaminated Culture should be fresh 9 . Lens paper. Sterile tooth picks.a. Graphs: n. rinse and dry. Stain must be prepared fresh 2. staining tray. Prepare another slide by scrapping the base of your teeth and gums with a sterile toothpick. Error analysis: n. add three loopfuls of a cultures to the stain and gently mix with the inoculating loop. Error analysis: n. Leuconostoc mesenteroides. With a clean glass slide spread the mixture over the entire surface of the slide to create a very thin smear. glass slide. Wash smears with 20% copper sulfate solution. Relationship: n. 24 hour old culture of Bacillus subtils and Staphylococcus spp. Outline of the Procedure Obtain one clean glass slide Place several drops of crystal violet stain on a clean glass slide.

Flood smear with carbol fuchsin containing Turgitol for 3 to5 minutes. Also.4 (c) Title of experiment: To study the Acid-fast Stain technique of bacterial culture Equipments required: Bunsen burner.a. bibulous paper. staining tray.a.aureus. Heated slide must be cooled prior to washing. inoculating loop. The chemical basis of the acid-fast stain. lens paper.a. Wash with lap water. Error analysis: n. Wash smear with tap water. Using sterility technique. Carbol fuchsin. glass slides. adding the reagent drop by drop until the alcohol runs almost clear with a slight red tinge. Caution: Stain must be prepared fresh 10 . prepare a bacterial smear of each organism plus a third mixed smear of M. For heatless method. prevent stain from boiling by adjusting the hot plate temperature. Learning Objective: To become familiar with 1. 2. hot plate. allowing the preparation to steam for 5 minutes Caution: Do not allow stain to evaporate: replenish stain as needed. Decolorize with acid-alcohol. and methylene blue. Outline of Procedure Obtain three clean glass slides. Required results: Parameters: acid fast bacteria will be differentiated from non-acid fast bacteria Relationship: n. acid-alcohol. smegmatis and S. Wash with tap water. Graphs: n. Counterstain with methyl blue for 2 minutes. and microscope Materials Required:72-96 hour trypticase soy broth culture of Mycobaterium smegmatis and 18-24 hour cultures of Staphylococcus aureus. Performance of the procedure for differentiation of bacteria into acid-fast and non-acid fast groups. Allow smears to air-dry and then heat fix in the usual manner: Flood smears with carbol fuchsin and place over a beaker of water on a warm hot place.

Outline of Procedure: Carefully place the ocular micrometer in to the eyepiece. Equipments required: Ocular micrometer.a.Experiment 5 Title of experiment: Size measurement of purified bacterial strain by micrometry. microscope Materials required: inoculating loop or needle. 24 hour old culture of Bacillus Learning objective: it helps students to know about size of bacterial strain. stage micrometer. Required results: Parameters: Helps to measure the size of bacterial strain. Error analysis: n. Relationship: n. Cautions: Handle the microscope carefully 11 .a.a. Place the stage micrometer on the microscope stage. Graphs: n. Determine the size by multiplying the average by your calibration factor and record it. bring to fine adjustment Determine the value of calibration factor Remove the stage micrometer from stage. Clear the focus under low power objective Add the drop of immersion oil to stage micrometer.

25°C for an additional 5 days. 24-hour broth culture of Pseudomonas aeruginosa.a.a. microscope Materials required: Inoculating loop.Experiment No. For microscopy examination. Quickly turn the slide right slide up so that the drop continues to adhere to the inner surface of the coverslip.a. Required results Parameters: Motility is observed visually by diffuse growth spreading from the line of inoculation. Press the slide gently to form a seal between the slide and coverslip.a. appearing as nodular growths along the stab line. If negative. Cautions: Prepare the stab carefully. depression slide. Using sterile technique. and Staphylococcus aureus Learning objective: Microscopic observation of living bacterial samples. first focus in on the drop of culture under the low-objective with reduced light.a. 12 . and Staphylococcus aureus Learning objective: Motility of living bacterial cultures in agar medium will be observed Outline of Procedure Prepare semi-solid nutrient agar medium and transfer 5 ml of molten nutrient agar medium in each test tube. Inoculate tubes by stabbing through center of the medium with inoculating needle to approximately one-half the depth of the medium Incubate at the proper temperature for the organism under consideration and examine at 18 – 48 hours. over the coverslip so that the depression cover the drop of the culture. continue incubation at 22 . 24-hour broth culture of Pseudomonas aeruginosa. Required results Parameters: Examine the hanging-drop preparation as to the size. Non-motile organisms grow only along the line of inoculation Relationship: n. place a loopful of the Mixed culture in the center of a clean coverslip Place the depression slide. Bacillus cereus. Certain strains of motile bacteria will show diffuse growth throughout the entire medium. nutrient agar. petroleum jelly. test tubes. Cautions: Handle the microscope carefully 6b): To study the motility of bacterial strain using stab method Equipments required Bunsen burner Materials required: Inoculating needle. Outline of Procedure Apply a ring of petroleum jelly around the concavity of the depression slide.a. Graphs: n. Error analysis: n. Cover slip. Autoclave the prepared tubes and subsequently allow them to solidify. while others may show diffusion from one or two points only. Bacillus cereus. shape and motility of different bacteria Relationship: n. 6 Title of the experiment: To study the motility of bacterial strain using: a) hanging drop technique b) stab method 6a): To study the motility of bacterial strain using hanging drop technique Equipments required Bunsen burner. Error analysis: n. Place a drop of oil on the cover slip and use the oil-immersion objective for detailed observation. with the concave surface facing down. Graphs: n.

1 ml from each dilution on the surface of plates containing nutrient agar and spread uniformly with the help of a sterile bent glass rod. This will give you 10-2 dilution. Cuvettes. Equipments required: Autoclave. Also prepare dilutions and spread 0. Next day. Graphs: n. Incubate the flasks inoculated in step 2 on an incubator rotary shaker at 25-30°C. hot air oven. Measure absorbance with the help of a spectrophotometer at 600 nm using minimal medium as the control. Transfer 1 ml of culture from each flask into 99 ml water blanks.a.1 ml on nutrient agar medium. Learning objectives To acquaint with population growth dynamics of bacterial culture. Minimal medium broth (200 ml in 500 ml Erlenmeyer’s Flasks) Sterile water blanks (99 ml). Continue drawing samples till stationary phase reaches as shown by plateau. Withdraw a sample of 2-4 ml from each flask using a sterile pipette. Withdraw sample after every one hour and determine absorbance. Take care that you flame the neck of flask and empty test tube property to avoid contamination. spectrophotometer. Outline of Procedure Inoculate a loopful of Escherichia coli in 50 ml minimal broth and incubate on rotary shaker at 2530°C for 24 hour. Using sterile pipettes place 0. sterile Petri dishes. Sterile pipettes (1 & 10 ml).a. early in the morning inoculate 4 ml broth culture of E.Experiment No:-7 Title: To prepare standard growth curve of a given bacterial culture. Calculate the generation time by direct and indirect method Relationship: n. Cautions Bacterial culture must be fresh Before taking absorbance standardize the equipment with blank 13 . To determine the growth rate constant and generation time of bacterial culture. spectrophotometer. Required results: Parameters: Determine the number of cell ml -1 by counting the colonies that appear after 48 hrs of incubation. coli(@ 2%) into each of the two 500 ml flasks containing 200 ml of minimal broth with the help of a sterile pipette. Error analysis: n. Shaking incubator Materials required: Minimal medium broth culture of Escherichia coli (log phase). Likewise prepare 10-4 and 10-6 dilutions. Nutrient agar.a. Record the absorbance and corresponding sell number in the table and plot a graph of absorbance verses incubation time on semi log paper.

Add five drops of the methyl red indicator to these tubes. Learning Objective: To detecting mixed acid producers and hence differentiate between various coliforms Outline of Procedure Label each of the MR-VP broth media with the name of the bacterial organism to be inoculated.a. Required results: Parameters: Development of red color is taken as positive. Incubate all cultures for 24 to 48 hours at 37 degrees C. Graphs: n. MR negative organism produce yellow color. and Enterobacter aerogenes. weighing balance Materials required:24 hour to 48 hour trypticase soy broth cultures of Escherichia coli.a.a. Outline of Procedure Inoculate the bacterium to be tested in peptone water.a. Methyl red indicator. Kovac’s reagent Learning objectives: To determined the ability of microorganisms to degrade the amino acid tryptophan. Experiment 8b): To perform Methyl Red Test Equipments Required: Autoclave. Experiment 8c): To perform Voges-Proskauer Test Equipments Required: Autoclave.Experiment No:8 Title: To perform IMVIC test and TSI test for Coliforms Experiment 8a): To perform ‘Indole Test’ Equipments Required: Autoclave. Incubator.a. weighing balance 14 . Proteus vulgaris. Relationship: n. Enterobacter aerogenes. Relationship: n. Cautions: Label the tubes properly. Using sterile technique inoculate each experimental organism into its appropriately labeled tube of medium by mean of a loop inoculation. Cautions: Label the tubes properly. Examine all cultures as to their color.a. MR-VP broth. and Klebsiella pneumonia. which contains amino acid tryptophan and incubate overnight at 37oC. peptone water. Incubator. weighing balance Materials required: 24 hour to 48-hour trypticase soy broth cultures of Escherichia coli. Error analysis: n. Incubator. Error analysis: n. Add 10 drops of Kovac’s reagent to all deep tube cultures and agitate gently Required results: Parameters: Formation of a red or pink colored ring at the top is taken as positive. Graphs: n.

MR-VP broth. Then add 0. Error analysis: n.Materials required:24-hour to 48 hour trypticase soy both cultures of Esherichia coli. Cautions: Label the tubes properly. and Klebsiella pneumoniue. Incubator. Add 0.a. Using sterile technique. Using sterile technique inoculate each experimental organism into its appropriately labeled tube of medium by mean of a loop inoculation. medium changes its color from green to blue.a. 40% KOH. α. Enterobacter aer genes. Incubate all cultures for 24 to 48 hours at 37 degrees C. Required Results: Parameters: Appearance of red color is taken as a positive test Relationship: n. Cautions: Label the tubes properly.2 ml of 40% KOH to the broth and shake.naphthol. weighing balance 15 .a.a. Error analysis: n. Learning Objective: To enable students to differentiate further among enteric organisms. Outline of Procedure Label each of the MR-VP broth media with the name of the bacterial organism to be inoculated. weighing balance Materials required:24 hour to 48 hour trypticase soy broth cultures of Escherichia coli. Outline of Procedure Label each of simmons citrate agar slants with the name of the bacterial organisms to be inoculated. Experiment 8d): To perform Citrate Utilization test Equipments Required: Autoclave.a. Required results: Parameters: If the organism has the ability to utilize citrate. Experiment 8e): To perform ‘Triple Sugar Iron (TSI) Test’ Equipments Required: Autoclave. Incubator. Enterobacter aerogens and Kleb-siella pneumonia. Simmons citrate agar slants Learning Objective To enable students to differentiate among enteric or-ganisms on the basis of their ability to ferment citrate as a sole carbon source.6 ml of alpha-naphthol to test broth and shake them. inoculate each organisms into its appropriately labeled tube by means of a stab or-streak inoculation Incubate all cultures for 24 to 48 hour at 37 degree C. Graphs: n.a. Allow the tubes to stand for 15 minutes. Relationship: n. Graphs: n.

Materials required: TSI agar medium. Cautions: Label the tubes properly. Using sterile technique.a. Hydrogen sulfide production results in a black precipitate in the butt of the tube Relationship: n. Outline of Procedure: Label each of TSI agar slants with the name of the bacterial organisms to be inoculated. Required results: Parameters: Carbohydrate fermentation is indicated by the production of gas and a change in the color of the pH indicator from red to yellow. Graphs: n. Error analysis: n. inoculate each organisms into its appropriately labeled tube by means of a stab or-streak inoculation Incubate all cultures for 24 to 48 hour at 37 degree C. test tubes.a.a. inoculation needle etc. 16 .

inoculate all agar plates with their respective test organisms as follows: Dip a sterile cotton swab into a well mixed saline test culture and remove excess inoculation by pressing the saturated swab against the inner wall of the culture tube. Do not press the discs into the agar. Label the covers of each of the plates with the name of the test organism to be inoculated. swab the entire agar surface horizontally. distribute the individual disc at equal distances with forceps dipped in alcohol and flamed. Using it. Antimicrobial Sensitivity Disc:Penicillin G.1 at 600n m. Error analysis: n. sterile cotton swabs. Bacterial culture must be fresh 2. Incubate all plate cultures in an inverted position for 24 to 48 hours at 37°C.9 Title of the experiment:. tetracycline. 30 μg. Cautions: 1. depositing the disc simultaneously onto the agar surface. vertically and around the outer edge of the plates to ensure a heavy growth over the entire surface. apply the antibiotic discs by placing the dispenser over the agar surface and pressing the plunger. 30 μg. Chloramphenicol. Proteus vulgaris. Antibiotic must be added aseptically 17 . Streptomycin 10 μg. 10μg.a.Experiment No. Scope of results: Parameters: Resistance of bacterial culture is determine by measuring the length of inhibition zone with simple scale and compare with standard chart available in the laboratory Relationship: n. 30 μg. Bunsen burner. Mueller hinton agar plates. and sulfanilamide. vancomycin. Outline of Procedure Place agar plates right side up in an incubator heated to 37°C for 10 to 20 minutes with the covers adjusted so that the plates are slightly opened.:. glassware Learning objectives: Differentiate between taxonomically important groups of bacteria. Allow the culture plates to dry for about 5 minutes. Using sterile technique.a. weighing balance Materials required: 0. gentamicin. Incubator. 300 μg Sensi-disc dispensers or forceps.85% saline suspension of Escherichia coli. If dispensers is not available. Graphs: n.a.D of 0. 10 μ g.To test the antibiotic Sensitivity test of a given bacterial culture Equipments Required: Autoclave. Gently press each disc down with the wooden end of a cotton swab or sterile forceps to ensure that the discs adhere to the surface of the agar.Mycobacterium smegmatics Bacillus cereus and Enterococcus faecalis adjusted to an O. Staphylococcus aureus Pseudomonas aeruginosa. Using the sensi-disc dispenser.

Incubator. 10 Title of the experiment:. Caution: Bacterial culture must be fresh Antibiotic must be added aseptically 18 . place a set of 10 sterile test tubes labeled 1 through 10. Error analysis: n. To demonstrate the reversal of penicillin inhibition against the test organism in the presence of penicillinase. Using a sterile 2 ml pipette. weighing balance Spectrophotometer Materials required:1:1000 brain heart infusion (BHI) broth dilution of 24 hour BHI broth cultures of Staphylococcus aureus ATCCtm 27661 (penicillin-sensitive strain) and Staphylococcus aureus ATCC 27659 ( penicillinase-producing strain. glassware Learning objective To employ a broth culture system for the determination of the minimal inhibitory concentration (MIC) of penicillin. Label one rack set I-penicillin-sensitive and the other rack set II-penicillin resistant Using a sterile 10 ml pipette and mechanical pipetting device add 2 ml of BHI broth to the tubes labeled 2 through 10 in sets I and II With a 2 ml sterile pipette.a. Using a sterile 2 ml pipette add 2 ml of the 1:1000 dilution of the S.aureus ATCC 27659.a. Graphs: n. repeat step for set II.aureus ATCC 27661 to all tubes in set I. Repeat step to inoculate all the tubes in set II with the 1:1000 dilution of S.Experiment No. Incubate both sets of tubes for 12 to 18 hours at 37°C Required results: Parameters: The MIC will be determined Relationship: n. Tube 10 receive no antibiotic and serves as a positive control. transfer 2ml from tube to tube.a. 40 ml of brain-heart infusion broth in a 100-ml Erlenmeyer flask and 10 ml of sterile aqueous crystalline pencillin G solution (100 μg/ml). Mix well and transfer 2 ml from tube three to tube four. Continue this procedure through tube 9 into beaker. Set I serial dilution: Using a sterile 2ml pipette. Outline of Procedure Into each of two test-tube racks.To determine the minimal inhibitory concentration (MIC) for antibiotic sensitivity of bacterial strain Equipments Required: Autoclave. and 2 ml of the penicillin solution to tubes 1 and 2 in set I and II and mix the content of the tube well. Discard 2 ml from tube 9.

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