LABORATORY MANUAL BTY557 LAB IN MICROBIOLOGY

Proteus flagella (Liefsons stain)

Table of contents

S. No. 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. 3

2 3 4 5

Maintenance & purification of microbes by streak-plate method Gram-staining, spore staining Capsule staining, 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

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Text Book(s): 1. Experiments in microbiology, plant pathology & Biotechnology: by KR Aneja. Other Readings: Reference Book(s): 1. Cappuccino, J.G and Sherman, N., Microbiology: A Laboratory Manual 3rd edition, Cummings publisher, 1992. 2. Practical Microbiology: by FC Garg.

Experiment 1 Title of the experiment: Isolation & purification of microbes from soil/ water/ air/ milk by a) streak-plate, b) pour-plate and c)spread plate method using serial dilutions and SPC techniques Experiment No. 1 a) Title of the experiment: Isolation and purification of microorganisms by streak plate method Equipments required: Autoclave, incubator, water bath, boiling water bath Materials required: Sterile Petri plates, Nutrient agar, Bunsen burner, Inoculation loop, Mixed culture of Serratia marcescens, Micrococcus luteus and Escherichia coli. Learning Objectives: To isolate bacteria by streak plate technique. Outline of Procedure: Disinfect the surface of the working table and your hands with a disinfectant. Label the bottom surface of sterile Petri plates with your name and date. Liquify nutrient agar, cool to 50C 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. After pouring the medium into the plate, gently rotate the plate to distribute the medium evenly. Avoid splashing of medium up over the slides of Petri plate. Allow the medium into the plates to solidify. Hold the inoculation loop and heat in the fame of the burner to red-hot. Allow it to cool and take a loopful of mixed culture aseptically. Streak the mixed culture on the solid surface of culture medium by lifting the cover of the Petri plate from one edge. Required Results: Parameters: Single separate colony can be isolated Relationship: - n.a. Graphs: - n.a. Error analysis: - n.a. Cautions: Base plates should dried completely streaking should be proper so that single separate colonies can be isolated Experiment No. 1 b) Title of the experiment: Isolation and purification of microorganisms (bacteria) from soil/water/air by pour plate method Equipments required: Autoclave, incubator, water bath, boiling water bath Materials required: 24-hour 10ml nutrient broth culture of Escherichia coli, 4 sterile 99-ml saline blanks, 1-ml pipettes, with pi-pump, 6 petri plates, 6 agar pour tubes of nutrient agar, 4 tubes of 5ml nutrient broths,4-5ml pipets and pi-pump. 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. Label four tubes of saline 10-2, 10-4, 10 -6, and 10-8. Using aseptic technique, the initial dilution is made by transferring 1 ml of E. coli sample to a 99ml sterile saline blank. This is a 1/100 or 10-2 dilution. The 10-2 dilution is then shaken by grasping the tube between the palms of both hands and rotating quickly to create a vortex. This serves to distribute the bacteria and break up any clumps. Immediately after the 10-2 dilution has been shaken, uncap it and aseptically transfer 1ml to a second 99ml saline blank. Since this is a 10-2 dilution, this second blank represents a 10-4 dilution of the original sample. Shake the 10-4 dilution vigorously and transfer 1ml to the third 99ml blank. This third dilution represents a 10-6 dilution of the original sample. Repeat the process once more to produce a 10 8 dilution. Shake the 10-4 dilution again and aseptically transfer 1.0 ml to one petri plate and 0.1ml to another petri plate. Do the same for the 10-6 and the 10-8 dilutions.

Remove one agar pour tube from the 48 to 50C water bath. 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. Repeat this process for the remaining five plates. After the pour plates have cooled and the agar has hardened, they are inverted and incubated at 25C for 48 hours or 37C for 24 hours. At the end of the incubation period, select all of the petri plates containing between 30 and 300 colonies. Plates with more than 300 colonies cannot be counted and are designated too many to count (TMTC). Plates with fewer than 30 colonies are designated too few to count (TFTC). 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 Required Results: Parameters: Microbial colonies will be obtained and from this data the microbial load of the given sample can be ascertained. Relationship: n.a. Graphs: n.a. Error analysis: n.a. Cautions: The dilutions should be made properly The labeling should be done carefully. Experiment:- 1 (c) Title of the experiment: Isolation and purification of microorganisms (bacteria) from soil/water/air by spread plate method. Equipments required: Autoclave, incubator, water bath, boiling water bath Materials required: Petri plates,test tubes, inoculation loop, nutrient agar medium Learning Objectives: To teach the students the basic culture techniques for microorganisms Procedure 1. With a wax pencil, label the bottom of the agar medium plates with the name of the bacterium to be inoculated, your name, and date. Three plates are to be inoculated: (a) one with B. subtilis, (b) one with S. marcescens, and (c) one with the mixture. 2. Pipette 0.1 ml of the respective bacterial culture onto the center of a tryptic agar plate. 3. 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. 4. Briefly pass the ethanol-soaked spreader through the flame to burn off the alcohol, and allow it to cool inside the lid of a sterile petri plate. 5. Spread the bacterial sample evenly over the agar surface with the sterilized spreader, making sure the entire surface of the plate has been covered. Also make sure you do not touch the edge of the plate. 6. Immerse the spreader in ethanol, tap on the side of the beaker to remove any excess ethanol, and reflame. 7. Repeat the procedure to inoculate the remaining two plates. 8. Invert the plates and incubate for 24 to 48 hours at room temperature or 30C. 9. After incubation, measure some representative colonies and carefully observe their morphology. Record your results. Required Results: Parameters: Microbial colonies will be obtained and from this data the microbial load of the given sample can be ascertained Relationship: n.a. Graphs: n.a. Error analysis: n.a. Cautions: The dilutions should be made properly The labeling should be done carefully.

Experiment :- 2 Title of the experiment: Maintenance & purification of microbes by streak-plate method Experiment No. 1 a) Title of the experiment: Isolation and purification of microorganisms by streak plate method Equipments required: Water bath, Bunsen burner, cuvettes, spectrophotometer, test tubes, pipettes. Materials required: 24-hour 10ml nutrient broth culture of Escherichia coli, 4 sterile 99-ml saline blanks, 1-ml pipettes with pi-pump, 6 petri plates, 6 agar pour tubes of nutrient agar (plate count agar). Learning Objectives: To isolate and purify bacteria by streak plate technique. Outline of Procedure: Label the bottom of six petri plates 1-6. Label four tubes of saline 10-2, 10-4, 10 -6, and 10-8. Using aseptic technique, the initial dilution is made by transferring 1 ml of E. coli sample to a 99ml sterile saline blank (figure below. This is a 1/100 or 10-2 dilution. The 10-2 dilution is then shaken by grasping the tube between the palms of both hands and rotating quickly to create a vortex. This serves to distribute the bacteria and break up any clumps. Immediately after the 10-2dilution has been shaken, uncap it and aseptically transfer 1ml to a second 99ml saline blank. Since this is a 10-2dilution, this second blank represents a 10-4 dilution of the original sample. Shake the 10-4 dilution vigorously and transfer 1ml to the third 99ml blank. This third dilution represents a 10-6 dilution of the original sample. Repeat the process once more to produce a 10-8 dilution. Shake the 10-4 dilution again and aseptically transfer 1.0 ml to one petri plate and 0.1ml to another petri plate. Do the same for the 10-6and the 10-8dilutions. Remove one agar pour tube from the 48 to 50oC water bath. 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. Repeat this process for the remaining five plates. After the pour plates have cooled and the agar has hardened, they are inverted and incubated at 25oC for 48 hours or 37oC for 24 hours. At the end of the incubation period, select all of the petri plates containing between 30 and 300 colonies. Plates with more than 300 colonies cannot be counted and are designated too many to count (TMTC). Plates with fewer than 30 colonies are designated too few to count (TFTC). Count the colonies on each plate. A Quebec colony counter should be used. 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. number of colonies (CFUs) = # of bacteria/ml dilution X amount plated Record your results. Disinfect the surface of the working table and your hands with a disinfectant. Label the bottom surface of sterile Petri plates with your name and date. Liquefy nutrient agar, cool to 50C 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). After pouring the medium into the plate, gently rotate the plate to distribute the medium evenly. Avoid splashing of medium up over the slides of Petri plate. Allow the medium into the plates to solidify. Hold the inoculation loop and heat in the flame of the burner to red-hot. Allow it to cool and take a loopful of mixed culture aseptically.

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

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

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

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

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

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

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

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Experiment No:-7 Title: To prepare standard growth curve of a given bacterial culture. Equipments required: Autoclave, hot air oven, spectrophotometer, Shaking incubator Materials required: Minimal medium broth culture of Escherichia coli (log phase), Minimal medium broth (200 ml in 500 ml Erlenmeyers Flasks) Sterile water blanks (99 ml), Nutrient agar, sterile Petri dishes, Sterile pipettes (1 & 10 ml), spectrophotometer, Cuvettes. Learning objectives To acquaint with population growth dynamics of bacterial culture. To determine the growth rate constant and generation time of bacterial culture. Outline of Procedure Inoculate a loopful of Escherichia coli in 50 ml minimal broth and incubate on rotary shaker at 2530C for 24 hour. Next day, early in the morning inoculate 4 ml broth culture of E. coli(@ 2%) into each of the two 500 ml flasks containing 200 ml of minimal broth with the help of a sterile pipette. Withdraw a sample of 2-4 ml from each flask using a sterile pipette. Take care that you flame the neck of flask and empty test tube property to avoid contamination. 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. This will give you 10-2 dilution. Likewise prepare 10-4 and 10-6 dilutions. Using sterile pipettes place 0.1 ml from each dilution on the surface of plates containing nutrient agar and spread uniformly with the help of a sterile bent glass rod. Incubate the flasks inoculated in step 2 on an incubator rotary shaker at 25-30C. Withdraw sample after every one hour and determine absorbance. Also prepare dilutions and spread 0.1 ml on nutrient agar medium. Continue drawing samples till stationary phase reaches as shown by plateau. Required results: Parameters: Determine the number of cell ml -1 by counting the colonies that appear after 48 hrs of incubation. Record the absorbance and corresponding sell number in the table and plot a graph of absorbance verses incubation time on semi log paper. Calculate the generation time by direct and indirect method Relationship: n.a. Graphs: n.a. Error analysis: n.a. Cautions Bacterial culture must be fresh Before taking absorbance standardize the equipment with blank

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Experiment No:8 Title: To perform IMVIC test and TSI test for Coliforms Experiment 8a): To perform Indole Test Equipments Required: Autoclave, Incubator, weighing balance Materials required: 24 hour to 48-hour trypticase soy broth cultures of Escherichia coli, Proteus vulgaris, and Enterobacter aerogenes, peptone water, Kovacs reagent Learning objectives: To determined the ability of microorganisms to degrade the amino acid tryptophan. Outline of Procedure Inoculate the bacterium to be tested in peptone water, which contains amino acid tryptophan and incubate overnight at 37oC. Add 10 drops of Kovacs 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. Relationship: n.a. Graphs: n.a. Error analysis: n.a. Cautions: Label the tubes properly. Experiment 8b): To perform Methyl Red Test Equipments Required: Autoclave, Incubator, weighing balance Materials required:24 hour to 48 hour trypticase soy broth cultures of Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumonia, MR-VP broth, Methyl red indicator. 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. Using sterile technique inoculate each experimental organism into its appropriately labeled tube of medium by mean of a loop inoculation. Incubate all cultures for 24 to 48 hours at 37 degrees C. Add five drops of the methyl red indicator to these tubes. Examine all cultures as to their color.

Required results: Parameters: Development of red color is taken as positive. MR negative organism produce yellow color. Relationship: n.a. Graphs: n.a. Error analysis: n.a. Cautions: Label the tubes properly. Experiment 8c): To perform Voges-Proskauer Test Equipments Required: Autoclave, Incubator, weighing balance

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Materials required:24-hour to 48 hour trypticase soy both cultures of Esherichia coli, Enterobacter aer genes, and Klebsiella pneumoniue, MR-VP broth, - naphthol, 40% KOH, 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. Using sterile technique inoculate each experimental organism into its appropriately labeled tube of medium by mean of a loop inoculation. Incubate all cultures for 24 to 48 hours at 37 degrees C. Add 0.6 ml of alpha-naphthol to test broth and shake them. Then add 0.2 ml of 40% KOH to the broth and shake. Allow the tubes to stand for 15 minutes. Required Results: Parameters: Appearance of red color is taken as a positive test Relationship: n.a. Graphs: n.a. Error analysis: n.a. Cautions: Label the tubes properly. Experiment 8d): To perform Citrate Utilization test Equipments Required: Autoclave, Incubator, weighing balance Materials required:24 hour to 48 hour trypticase soy broth cultures of Escherichia coli, 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. Outline of Procedure Label each of simmons citrate agar slants with the name of the bacterial organisms to be inoculated. Using sterile technique, 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. Required results: Parameters: If the organism has the ability to utilize citrate, medium changes its color from green to blue. Relationship: n.a. Graphs: n.a. Error analysis: n.a. Cautions: Label the tubes properly. Experiment 8e): To perform Triple Sugar Iron (TSI) Test Equipments Required: Autoclave, Incubator, weighing balance

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Materials required: TSI agar medium, test tubes, inoculation needle etc. Outline of Procedure: Label each of TSI agar slants with the name of the bacterial organisms to be inoculated. Using sterile technique, 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. 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. Hydrogen sulfide production results in a black precipitate in the butt of the tube Relationship: n.a. Graphs: n.a. Error analysis: n.a. Cautions: Label the tubes properly.

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Experiment No.:- 9 Title of the experiment:- To test the antibiotic Sensitivity test of a given bacterial culture Equipments Required: Autoclave, Incubator, weighing balance
Materials required: 0.85% saline suspension of Escherichia coli, Staphylococcus aureus Pseudomonas aeruginosa, Proteus vulgaris,Mycobacterium smegmatics Bacillus cereus and Enterococcus faecalis adjusted to an O.D of 0.1 at 600n m, Mueller hinton agar plates, Antimicrobial Sensitivity Disc:Penicillin G. 10 g; Streptomycin 10 g; tetracycline, 30 g; Chloramphenicol, 30 g; gentamicin, 10g; vancomycin, 30 g; and sulfanilamide, 300 g Sensi-disc dispensers or forceps, Bunsen burner, sterile cotton swabs, glassware Learning objectives: Differentiate between taxonomically important groups of bacteria. Outline of Procedure Place agar plates right side up in an incubator heated to 37C for 10 to 20 minutes with the covers adjusted so that the plates are slightly opened. Label the covers of each of the plates with the name of the test organism to be inoculated. Using sterile technique, 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. Using it, swab the entire agar surface horizontally, vertically and around the outer edge of the plates to ensure a heavy growth over the entire surface. Allow the culture plates to dry for about 5 minutes. Using the sensi-disc dispenser; apply the antibiotic discs by placing the dispenser over the agar surface and pressing the plunger; depositing the disc simultaneously onto the agar surface. If dispensers is not available, distribute the individual disc at equal distances with forceps dipped in alcohol and flamed. 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. Do not press the discs into the agar. Incubate all plate cultures in an inverted position for 24 to 48 hours at 37C. 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.a. Graphs: n.a. Error analysis: n.a. Cautions: 1. Bacterial culture must be fresh 2. Antibiotic must be added aseptically

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

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