LAB NOTES FOR EXAM 1 SECTION

Transcription

1 LAB NOTES FOR EXAM 1 SECTION EX. 2-1: DIVERSITY AND UBIQUITY OF MICROOGANISMS Purpose: Microorganisms are found everywhere in the environment around us. To demonstrate this and to get a taste of the different types of organisms in our environment, we can culture these microorganisms by collecting them on a swab and transferring them to an agar plate. Media & Materials: 1 Tryptic Soy Agar (TSA) plate, 1 sterile swab and 1 tube of sterile saline per person 1. We will use a simplified version of the lab manual protocol. The lab manual describes several methods for sample collection. Confer with your lab partners at your table so that each person uses a different method. Your table should have a total of 4 plates (5 if you have an extra person at your table). 2. Label the plate with your name, today's date, and the source of inoculum to be collected (for example, desktop, doorknob, faucet, hair, skin, etc.). You may also use any public body surface except for your mouth (this means any areas of skin that you might normally expose in polite company). Transfer any microbes you may have picked up to the agar plate by gently swabbing the surface of the plate with the swab. The instructor will demonstrate the method for spreading the cells on the plate. 3. The plates will be incubated at 37ºC until next lab period. 4. Observe your plates during our next lab meeting. On your data sheet, make note of the various colors, textures, and shapes that are produced when microscopic organisms are allowed to reproduce in large numbers. The individual areas of growth you see are colonies and consist of millions of identical cells that all arose from a single parent cell. 5. Be sure to dispose of your cultures in the Biohazard waste containers when your observations are completed. EX. 1-3: ASEPTIC TRANSFER AND INOCULATION TECHNIQUES Since this is the first time you will be working with bacterial cultures, the procedure for handling and transferring microorganisms will be described in detail. Aseptic technique, the procedure used to prevent contamination, is carried out so that you, your neighbors, and your belongings are not contaminated by the microbial culture and so that microbes from the environment do not contaminate your bacterial culture. Purpose: In this experiment you will be transferring living cells grown on two different types of media: broth and slant. You will use these cells to inoculate a fresh slant and a fresh broth culture. Cultures: a broth culture of Serratia marcescens and an agar slant culture of Serratia marcescens Media: 1 tryptic soy agar (TSA) slant and 1 tryptic soy broth (TSB) per student 1. Before beginning, label all tubes of sterile media with the name of the organism, the date, and your names or initials. ALWAYS label before inoculating. 2. Follow the protocol described in the manual. We will be using only one organism today, Serratia marcescens. This organism produces a red pigment when incubated under the appropriate 1

2 conditions. This red or pink color will help you to determine if your aseptic transfer has been successful when you look for growth next lab period. You should not see any pink or red color in your freshly inoculated tubes today. 3. Use the broth culture of Serratia marcescens to inoculate the slant. Use the slant culture to inoculate the broth. 4. Your instructor may demonstrate some techniques that are slightly different from the ones described in the lab manual. These variations are acceptable as long as they are safe and produce the desired results. If you feel confused by any differences, ask for clarification. 5. After you replace the screw top on a culture tube, back off the top about 1/2 turn before placing the tube in the incubator in order to allow air to circulate. Do not incubate cultures with the top screwed down tightly. Those bugs need to breath, just like you! 6. Incubate all cultures at 37ºC until next lab period. 7. During the next lab meeting, make your observations on the data sheet located near the back of your lab manual. Dispose of your cultures in the appropriate Biohazard container as directed by your instructor. EX. 1-4: STREAK PLATE ISOLATION OF PURE CULTURE (PART 1) Purpose: A pure culture is one that contains a single type of organism. You must first isolate single colonies in order to cultivate a pure culture. Single colonies contain identical cells that have arisen from a single cell and are genetic clones of each other. Single colonies can only be obtained by spreading single cells far apart from each other on the surface of an agar plate, then allowing them to grow. There are several ways to separate single cells, but we will only be using the streak plate method using an inoculating loop. Cultures: a mixed broth culture containing both Serratia marcescens and Staphylococcus aureus and a mixed broth culture containing both Escherichia coli and Micrococcus luteus. Media: 2 TSA plates per pair of students The Streak Plate Method 1. Each student will inoculate a mixed broth culture onto a separate agar plates according to the streak plate procedure described by the lab manual and demonstrated by the instructor. Work with your lab partner so that each of you streaks a different mixed culture. 2. When you make the streaks across the plate, do them gently so that you do not dig into the agar with the loop. This will cause growth to occur in streaks, rather than in single colonies. 3. Incubate your plates upside down (lid on the bottom, agar on top) in a wire basket at 37ºC for until next lab period. Baskets can be shared between lab partners. These plates are considered mixed cultures because they will contain two different types of colonies. 4. Next lab meeting: make your observations on the data sheet pages. Use blank paper if you need additional space. Include a drawing and use the information in Ex. 2-2 to write a description of a colony from each different type of organism. Observe the plates for visible differences in colony morphology (size, color, shape, elevation, margin, texture, optical characteristics). Did you get nice isolated single colonies? They should be spaced far enough apart so that you can pick up an without touching more than one colony using a loop. Serratia marcescens colonies should be pink or reddish. Staphylococcus aureus form smaller, white colonies. Escherichia coli produces beige colonies 2 to 3 mm in diameter, while Micrococcus luteus will produce smaller yellow colonies. 2

3 EX. 1-4 (and EX. 2-2): STREAK PLATE ISOLATION OF PURE CULTURE (PART 2) Purpose: Isolate a pure culture from mixed culture plates by restreaking a fresh streak plate from a single isolated colony Organisms: Last period s mixed culture plates with isolated colonies from Ex Media: 4 TSA plates per pair of students 1. Label fresh plates with the names of the four organisms found on your mixed culture plates, your initials, and the date. Look at the two streak plates that you and your lab partner prepared from mixed cultures the last lab period. Choose single colonies that are well separated from the others so that they are easy to pick up. 2. From a mixed culture plate, aseptically touch the edge of the sterile loop to the colony. It is not necessary to scoop up the entire colony. You should not transfer a visible quantity of inoculum to the fresh plate. Using the streak plate dilution method that you used last week, streak a fresh, labeled plate with the appropriate colony. Remember to flame the loop in between streaking each quadrant of the plate. If the colonies are very small and close together, you can use a needle to pick up cells. Note: Make sure that you pick up your inoculum from only one colony. 3. Use the same method to streak plates of all four organisms. 4. Place the plates inverted in a basket. Incubate the four plates at 37ºC until next lab period. 5. Next lab period: You will use these plates as part of Ex. 2-2, Colony Morphology. Record your observations on your data sheet. Use additional paper for drawings or other information. Each plate should have only one type of colony. EX. 2-2: COLONY MORPHOLOGY Purpose: Observe possible difference in colony morphology between different bacterial species grown on solid culture media. Organisms: broth cultures of Bacillus subtilis and Mycobacterium smegmatis Media: Two TSA plates per pair 1. With a sterile loop, inoculate Bacillus subtilis and Mycobacterium smegmatis onto separate TSA plates using the streak plate method. NOTE: Be careful to avoid cross-contamination between plates. Be especially conscientious in flaming your loop after transferring any Bacillus species, which produces spores. Make sure that the loops are flamed long enough to glow orange. Take the time to flame the loops and go through the procedures slowly and carefully. Report any spills to the instructor so that proper cleanup can occur. 2. You have already streaked plates with the other organisms from Ex Incubate all the plates 37ºC until next lab period. The Mycobacterium and Micrococcus cultures may require additional incubation time. Therefore, if the growth is scant and the colonies are very small, incubate these cultures for 1 to 2 more periods. 3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. There may be demonstration plates of other organisms to view as well. Be sure to include observations on these cultures in your results. 3

4 WHEN YOU ARE FINISHED MAKING ALL YOUR OBSERVATIONS, CULTURES SHOULD BE DISPOSED OF IN YOUR RED AUTOCLAVE BAGS AND THOSE BAGS PLACED IN THE LARGE GRAY BIOHAZARD WASTE CAN. EX. 2-3: GROWTH PATTERNS ON SLANTS Purpose: Observe possible differences in morphology between different bacterial species grown on slant media Organisms: Bacillus subtilis and Mycobacterium smegmatis Last period s mixed culture plates with isolated colonies from Ex. 1-4 Media: 6 TSA slants 1. Label each slant tube with the date, organism name, and your initials. 2. Using the aseptic technique for inoculating slants that we learned last week, inoculate each tube. For Serratia marcescens, Micrococcus luteus, Escherichia coli, Staphylococcus aureus, carefully pick up cells of a single colony from your mixed plates. Normally, pure cultures would be used for transferring organisms to slants or broths, but we are using the mixed plates due to time considerations. If you do not have sufficient single colonies of each organism, there will be some pure culture plates available as well. 3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. For your uninoculated control, use a fresh TSA slant, but return it to the cart when finished. EX. 2-4: GROWTH PATTERNS IN BROTH Purpose: Observe possible differences in morphology between different bacterial species grown in broth media Organisms: Bacillus subtilis and Mycobacterium smegmatis Last period s mixed culture plates with isolated colonies from Ex. 1-4 Media: 6 TSB broths 1. Label each broth tube with the date, organism name, and your initials. 2. Inoculate these tubes with the same cultures used in Ex Again, be especially careful when transferring cells from your mixed culture plates. 3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. For your uninoculated control, use a fresh TSB broth tube, but return it to the cart when finished. EX. 2-7: FLUID THIOGLYCOLLATE: ATMOSPHERIC OXYGEN REQUIREMENTS Purpose: Observe the growth patterns of different organisms according to their oxygen requirements 4

5 Organisms: Broth cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Neisseria sicca, and Clostridium butyricum Media: Four tubes of thioglycollate broths and one tube of supplemented thioglycollate per pair 1. Thioglycollate is a reducing agent that removes oxygen from the broth. Label the tube of SUPPLEMENTED thioglycollate for the organism Clostridium butyricum (don't forget your initials and the date). Label the other tubes for each of the remaining organisms. 2. After observing the instructor's demonstration, inoculate each broth using a sterile disposable bulb pipette: Transfer 0.25 ml (the first mark on the sterile pipette above the joint) of the appropriate culture by gently squeezing the bulb to expel the inoculum into the broth from the bottom of the tube to the top. DO NOT ALLOW ANY AIR TO BUBBLE INTO THE BROTH. 3. Discard the pipette in your autoclave bag. 4. Repeat the procedure for the other four organisms. Incubate the tubes at 37ºC until next lab period. 5. Next lab period: DO NOT SHAKE or disturb the broth cultures before you have the opportunity to make your observations. Make observations on the 6 cultures on your data sheets. Use the terms we covered in class and on the study guide in your descriptions. EX. 7-3: THE KIRBY-BAUER ANTIBIOTIC SENSITIVITY TEST PROCEDURE Purpose: Observe the effects of a variety of antibiotics on a Gram positive and a Gram negative organism using the Kirby Bauer antibiotic sensitivity test. We will be using a procedure similar to the one described in the lab manual, with some modifications. Follow the procedure described below: Organisms: broth cultures of Escherichia coli and Staphylococcus aureus Media and Materials: 2 tubes sterile TSB, 2 disposable 1 ml bulb pipets, 2 large Mueller-Hinton agar plates, 2 sterile cotton swabs, Antibiotic disks for streptomycin, tetracycline, penicillin, chloramphenicol, cephalothin, erythromycin, novobiocin, vancomycin 1) Dilute each stock culture 1:50 by adding 0.25 ml of culture to 5 ml sterile TSB. Be sure to label the TSB tubes before adding the stock culture. Mix well. 2) Label each plate with name of an organism. Inoculate each plate with a single organism by dipping a sterile swab into the diluted broth culture so that the swab is saturated. Remove excess inoculum by gently rolling the swab against the inner surface of the tube. The swab should be moist, but not dripping. 3) Use the swab to evenly cover the entire surface of the agar in a horizontal direction. Remoisten the swab, turn the plate 90 and streak the surface in the other direction. Make sure that you cover the entire plate and that the bacteria are spread all the way to the plate edge. 4) Dispose of the used swab in the autoclave bag. 5) Allow the plate to absorb the liquid for a few minutes, with the top on and right side up. 6) Inoculate the second plate with the other organism using the same procedure. Make sure to use a fresh swab for the second culture. 7) We will NOT be using a disk dispenser. Instead, use a pair of flamed and cooled of forceps to remove the antibiotic paper disk from the end of the cartridge. Place disks on the agar surface of 5

6 the plate, using the laminated guide to space them evenly around the plate. Gently touch the disk with the tip of forceps so that it adheres to the surface of the agar. Do not push the disk into the agar. Each disk is already marked, so you do not need to label the plate with the antibiotic names. Symbol Amount Antibiotic Intermediate Range (mm) S10 10 µg Streptomycin TE µg Tetracycline P10 10 µg Penicillin C µg Chloramphenicol CF µg Cephalothin E µg Erythromycin NB µg Novobiocin VA µg Vancomycin ) Invert the plates and incubate them 37ºC until next lab period. Since these plates should not incubate more than 48 hours, be sure to view them the next period. IF they are incubated too long, colonies may begin to grow in the zone of inhibition, making the interpretation of your results difficult. 9) Next lab period: examine the plates for the presence or absence of a zone of inhibition around each disk. 10) Measure the diameter of the zone of inhibition around each disk and determine the susceptibility (resistant, intermediate, or sensitive) of the organisms to the antibiotics. Consult the chart in the lab manual for interpretation of zone size. Consult the chart above for the intermediate range for the eight antibiotics. A measurement that is smaller than the intermediate range indicates resistance while a measurement larger than the intermediate range indicates susceptibility. EX. 2-9: TEMPERATURE These experiments will be done on plates, rather than broths, to reduce the amount of media required for each experiment. The instructor will demonstrate the changes in the procedure as written below. Purpose: Determine the optimum growth temperature of different bacterial species Organisms: Escherichia coli, Staphylococcus aureus, Bacillus stearothermophilus, Pseudomonas fluorescens Media: Four TSA plates per pair 1. Draw two perpendicular lines on the bottom of the plate to divide the plates into fourths. Write the incubation temperature on each plate: 4 C, 25 C, 40 C and 60 C. Label each sector of each plate with the name of one of the four organisms listed above. Label the plates with your name and date as usual. 2. Make a straight line inoculation of each organism on each of the four plates. The instructor will demonstrate. 6

7 3. Put your plates in the appropriate baskets on the cart. These baskets will be incubated at the appropriate temperature. The 4 incubator is the refrigerator. The plates are incubated at the appropriate temperature until next lab period. 4. Next lab period: examine the plates for the presence or absence of growth and record your observations. Use a scale from 0-3 (0 being no growth and 3 being heavy growth) to describe the degree of growth. Note the temperature or temperature range that supports the growth of each organism. Is the organism a psychrophile, a mesophile, or a thermophile? In what possible environments would you find these organisms? EX. 2-11: OSMOTIC PRESSURE Purpose: Demonstrate the effect of osmotic pressure on microbial growth Organisms: Escherichia coli, Staphylococcus aureus, Bacillus subtilis Media: 5 beef heart infusion (BHI) plates, one with each of the following salt concentrations: 0.85%, 5%, 7.5%, 10% and 20% NaCl Work in pairs 1. Divide the plates into thirds. Label each sector with the name of one of the three organisms listed above. Label the plates with your name and date as usual. 2. Make a straight line inoculation of each organism on each of the five plates. 3. Incubate your plates at 37ºC until next lab period. 4. Next lab period: examine the plates for the presence or absence of growth and record your observations. Note the salt concentration that supports the growth of each organism. Which organism or organisms are halophiles? In what possible environments would you find these organisms? EX. 6-1: STANDARD PLATE COUNT Purpose: Determine the cell concentration of a concentrated broth culture. Use of serial dilution and spread plate techniques. Organisms: Escherichia coli Media: 4 TSA plates, 4 tubes of 4.5mL saline solution. Work in pairs. Spread plates are similar to streak plates except that the cells are delivered to the agar surface in a measured volume of liquid instead of with the inoculating loop. A spreader is used to spread the liquid evenly around the dish before it is absorbed into the agar. Often times, the original culture is too concentrated to perform a direct spread plate with 100 µl. In such cases, serial dilutions need to be performed first. The culture is diluted by 10 fold sequentially several times. Each dilution is then spread onto plates. The goal is to find one plate that has a countable number of colonies. The method for serial dilutions is described below. After colonies have grown, plates with countable number of cells can be used to calculate the original culture s concentration. The general formula for the calculation is: # colonies X dilution factor volume plated The dilution factor is the amount you diluted the culture. Therefore if you spread 0.1 ml of a culture you diluted by 1/100,000 onto a plate, and 25 colonies appeared, you would calculate concentration in ml as follows: (25 colonies X 100,000)/ 0.1ml = 25,000,000 cells/ml 7

8 0.5mL Saline 4.5 ml Saline Saline Saline 4.5 ml 4.5 ml 4.5 ml 0.5mL 0.5mL 0.5mL 1/10 E. coli 1/100 Dilution 1/1,000 Dilution 1/10,000 Dilution 1/100,000 Dilution 0.1 ml 0.1 ml 0.1 ml 0.1 ml 1/100 1/1,000 1/10,000 1/100, Label the four plates with the following dilutions: 1/100; 1/1,000; 1/10,000 and; 1/100, Label the saline tubes with the same dilution names. You will be given a tube of saline that has already been diluted to 1/10. Transfer 0.5 ml of the culture into the tube labeled "1/100." 3. Mix by suctioning in and out several times, then transfer 0.5mL of the mixed 1/100 into the tube labeled "1/1,000." 4. Mix by suctioning in and out several times, then transfer 0.5mL of the mixed 1/1,000 into the tube labeled "1/10,000." 5. Mix by suctioning in and out several times, then transfer 0.5mL of the mixed 1/10,000 into the tube labeled "1/100,000." 6. Using a fresh transfer pipet each time, transfer 0.1 ml of each of the diluted saline cultures onto the appropriate plate. Using a new disposable spreader each time, touch the spreader to the cell suspension and gently drag it back and forth, turning the plate slightly each time, to evenly distribute the cells over the agar surface. 7. Close the petri dish lid and dispose of the spreader. Let plates stay upright for 5 minutes. 8. Tape your agar plates together and place upside-down in the 37 C incubator. They will be incubated until the next lab period. 9. Next lab period: count the total number of colonies on each plate. If a plate has a confluency of colonies, record it as a lawn. You should find that the greater the dilution, the fewer colonies should appear. Choose the best plate (highest number of countable colonies) to plug into the formula above. Calculate your cell concentration and compare it to other groups. 8