Chapter 3 Skills for the Microbiology Laboratory

Skills for the Microbiology Laboratory Aseptic transfer, isolation streak plate, KOH test Skill One - Aseptic transfer of broth In this exercise, a transfer of sterile broth from one tube to another will be made. If the transfer is performed correctly, bacterial growth will not be evident in the tubes. Aseptic means that there is no contamination present. Question (to be answered before class): If bacterial growth occurs in a broth culture tube, what will you observe? Pipettors: Notice that that there are four pipettors in the laboratory bench drawer, two larger pipettors and two smaller pipettors. The larger pipettor is usually set at 1.0 mL (1000 µl) and the smaller pipettor is usually set at 0.1 mL (100 µl). The large pipettors use the blue pipet tips; the smaller pipettors use the yellow or white pipet tips. Pressing the plunger to the first stop measures the volume of the liquid. Pressing to the second stop expels the liquid. The pipette tips are kept in boxes; the inside of the boxes and the tips are sterile. When a tip is needed, open the box, press the pipettor to the tip, and close the box immediately. When you are finished with a tip, expel it into the ‘tips’ beaker on the laboratory bench. Press the plunger to the first stop prior to placing the pipet tip beneath the liquid surface. Slowly release the plunger after you have placed the pipette tip into the liquid. Liquid should move into the pipet tip. Observe the tip to ensure that liquid is present in the tip. Place tip beneath the liquid in the second tube. Pressing slowly to the second stop expels the liquid. Session One: Exercise: Each student will obtain two tubes of trypticase soy broth (TSB). Label Tube 1 and Tube 2, using the blue tape. DO NOT use your sharpie marker to write on the glass tubes. Transfer 1.0 mL of broth from Tube 1 to Tube 2 by using the large pipettor. Change the pipettor tip. Then transfer 1.0 mL of broth from Tube 2 to Tube 1. Incubate the tubes for 48 hours at 37° C. 1.0 ml Tube 1 1.0 ml Tube 1 Tube 2 1 Session two: Examine the two tubes. Is there growth in the tubes? If not, then you performed the transfer aseptically and correctly. If you ended up with growth due to contamination, your instructor may ask you to repeat the exercise. Skill Two - Isolation streak A turbid or cloudy broth indicates the presence of microorganisms, usually bacteria. There may be millions of organisms in a single loopful of broth. What kinds of bacteria are present? Do we have only one species, or several? Does the organism ferment certain sugars? Many questions can only be answered by growing bacteria on an agar plate. An isolation streak is performed to ensure that future work is performed with one species of bacteria. An isolation streak is done on agar, with the goal of obtaining10-20 isolated colonies. Remember that each colony on a bacterial plate comes from a single cell. The cells will pile up on top of each other to form a colony, which is visible to the naked eye. The microorganisms in the broth are spread out in such a fashion that single cells have room to multiply. Cultures of bacteria are described as follows: A pure culture contains one known bacterial species. A mixed culture contains two or more known bacterial species. A contaminated culture contains one or more unknown bacterial species. Know these terms. There are two techniques for doing isolation streaks, T-streak and Quadrant streak. Each student will perform both techniques. Session One: One pair of students at each bench will use a broth culture tube of Escherichia coli. The other pair of students at the bench will use Staphylococcus epidermidis. Students will share one tube of broth. Turn on the incinerators and let them heat until red-hot. Each pair of students will need: One tube of broth (either E. coli or S. epidermidis) Two TSA plates per student (four plates per pair) One inoculating loop per student Sharpie marker The T-streak: Divide the plate into three sections using a sharpie marker on the bottom (agar side) of the plate. Label the plate around the rim with your name, section, date, and organism. Sector one 2 Sector three Sector two Streak as follows: Sector one Sector two Sector three 1. Hold your loop like a pencil and place it in the incinerator until it glows red. Remove it. This is termed incinerating the loop. Allow the inoculating loop to cool by counting to five. 2. Dip the loop into the broth. With sector one towards your left (if you are right-handed), move your loop evenly back and forth across the agar surface in sector one. Keep your streaks close together. Stay between your lines. Do not dig your loop into the agar. When finished, incinerate the loop. 3. DO NOT DIP THE LOOP INTO THE BROTH AGAIN. Instead, rotate the plate so that sector two is towards your left. Go back into sector one 4-6 times with the loop. Then, staying between your lines, move your loop evenly back and forth across the agar surface in sector two. When sector two is completely streaked, incinerate the loop again. DO NOT DIP THE LOOP INTO THE BROTH AGAIN. 4. Rotate the plate so that sector three is towards your left. Again, go back into the previous sector 4-6 times, stay between your lines as you streak evenly and closely back and forth across the agar in sector three. When finished, incinerate the loop. 5. Invert your plate and incubate for 48 hours at 37o C. The Quadrant streak: 1. When doing a quadrant streak, you mentally divide your plate into four sections. 2. Incinerate the loop. Start with section one on your left, dip your loop and then streak back and forth 6-8 times across the agar in sector one. Incinerate the loop. DO NOT DIP THE LOOP INTO THE BROTH AGAIN. 3. Rotate the plate. Streak section two going back into section one 4-6 times. Incinerate the loop. 4. Rotate the plate. Streak section three, going back into section two 4-6 times. Incinerate the loop. 3 5. Rotate your plate. Streak section four, going back into section three 4-6 times. Finish off with a squiggle down into the middle of the plate. Incinerate the loop. Please see the diagram and plate on pages 9-10 of the LeBoffe and Pierce laboratory atlas. 6. Invert your plate and incubate for 48 hours at 37° C. Session two: Examine the streak plates and evaluate them (see results page). Also, have your instructor look at your streak plates. If the plates are not satisfactory, you may be asked to repeat an isolation streak. Skill three – KOH (string) test The KOH (potassium hydroxide) or string test distinguishes between Gram-positive and Gramnegative bacteria. Gram-positive bacteria have a thick cell wall that resists the action of the KOH. However, the thinner cell wall of Gram-negative bacteria gets broken down by the KOH. When the membrane dissolves, DNA is released from inside the cells. If you add KOH to a large number of Gram-negative cells, the released DNA becomes very slimy and cohesive, and can be seen with the naked eye. KOH test: Put 1-2 drops of KOH on a clean glass slide. Incinerate the loop. Transfer a small amount of one colony with the inoculating loop to the slide. Mix the colony with the KOH drops, stirring well, for about 60-90 seconds. Slowly raise the loop above the slide. If the bacteria are Gram-positive, nothing will happen. If the bacteria are Gram-negative, a slimy “string” between the loop and the slide will be visible. Note: this test only works for colonies from agar plates, not for broth cultures. After you have completed the observations of the isolation streak plates, perform a KOH test on Escherichia coli and Staphylococcus epidermidis. Please see page 61, figure 6-11, in the laboratory atlas for a photograph of a positive KOH test. 4 Results and questions – Microbiology laboratory skills 1. Results for aseptic transfer Tube 1 Appearance of tube (record “clear” or “turbid”) Tube 2 Clear Clear Did you successfully perform an aseptic transfer? Explain fully using the results above. Yes, because there was no bacterial growth in the tubes. There was no contamination. Suppose you were transferring a pure bacterial culture in broth to a different TSB tube. Would aseptic transfer be important? Why? Explain fully. Yes, aseptic transfer would still be important because it will still prevent contamination of the pure bacterial culture from the environment. 2. Analyze your streak plates (Y / N) Do you have colonies in all sections? Did you dig into the agar? Are your streaks even and closely spaced? Did you obtain 10-20 isolated colonies? Number isolated colonies T-streak No No Yes Yes 1-3 Quadrant No Yes No No 1 Repeat yes no yes yes About 30 How could you improve your streak plate technique? I could improve my streak plate technique by making the streaks more even, taking more from the other quadrants when streaking. I also can do better by not hitting the agar. 3. KOH test (string test) E. coli Results (record ‘string’ or string ‘no string’) S. epidermidis No string What can you conclude from the results of the string test? Do these conclusions match with the Gram-stain? The E. Coli is gram negative and S. epidermidis is gram positive. The KOH has broken down the cell wall of the E. coli., while the S. epidermidis cell wall resisted the KOH. The gram- stain test results did not match with the KOH test results. 5