Mare Prveate Th Totten asic aborstory and [a6 The Seat Poe Laboroory Exercises Cate Techniques Techique an Dioremt opr 212 Mizrobiooy. Fh Eton Mois EXERCISE The Streak-Plate Technique and Differential Media Materials per Student (Streak-Plate Technique) 24. to 48-hour tryptic soy broth cultures of Escherichia coli (ATCC 11229, white colonies), Serratia marcescens (ATCC 13880, red colonies; Micrococcus roseus ATCC 186 can also be used), and Bacillus subtilis (ATCC 6051, white or cream colonies) 3 tryptic soy agar pours boiling water bath 48° to 50°C water bath Bunsen burner petri plates inoculating loop wax pencil (itferential Media) 24- to 48-hour tryptic soy broth culture of a mixture of Escherichia coli (ATCC 11229), Proteus vulgaris (ATCC 13315), and Staphylococcus aureus (ATCC 25903) smanpitol salt agar pour cosin methylene blue agar pour Learning Objectives Hach student should be able to 1. Understand the purpose of the streak-plate technique and differential media 2. Perform a streak-plate technique and isolate discrete colonies for subculturing Suggested Reading in Textbook 1, Isolation of Pure Cultures, section 5.8. 2. The Spread Plate and Streak Plate, section 5.8. 3. Colony Morphology and Growth, section 5.8 4. Differential Media, section 5.7. Pronunciation Guide Escherichia coli (esh-er Bacillus subtilis (bab-SU = Another procedure that is used to obtain well-isolated, pure colonies is the streak-plate technique. Since Serratia marcescens, Bacillus subtilis, and Escherichia coli were used in the past few exercises, shese same cultures are used in this exercise. Remember, 5. marcescens produces red colonies; B. subtilis, white to cream colonies; and E. co ‘white colonies. These same cultures will also be used in the next exercise (number 17) } Medical Application In the clinical laboratory, growth of a pure culture is ab- Why Are the Following Bacteria Used in This Exercise? solutely necessary before any biochemical tests can be per- formed to identify a suspect microorganism, Principles of the Streak-Plate Technique Isolated, pure colonies can also be obtained by the streakeplate technique. In this technique, the bacterial mixture is transferred to the edge of an agar plate with an inoculating loop and then streaked out over the sur- face in one of several patterns. At some point on the streaks, individual cells will be removed from the loop as it glides along the agar surface and will give rise to separate colonies (gure 16.1). Again, one assumes that fone colony comes from one cell. The key principle of this method is that by streaking, a dilution gradient is es- tablished on the surface of the plate as cells are de- posited on the agar surface. Because of this gradient, 99Matera Tn ese tater and Te te Steat-Pote Tretia tian Laboratory Exercises Cate Techniques Techique an Diem Coon, 212 Microbiology. Fth Eton Mois Figure 16:1 Streak Plate, Notice che welsobted colonies of| Evol (whit) and 5 manevens Fed), confluent growth occurs on part of the plate where the cells are not sufficiently separated, and individual, well- isolated colonies develop in other regions of the plate Where few enough cells are deposited to form separate colonies that can be seen with the naked eye. Cells from the new colony can then be picked up with an inoculat- ing needle and transferred to an agar slant or other suit- able medium for maintenance of the pure culture. Procedure 1. Melt three sterile, capped tubes of tryptic soy agar by heating them in a boiling water bath until melted (see figure 13.28,b. 2. Cool the tubes in a 48° to SO°C water bath for 10-15 minutes 3. Remove the cap, flame the lip of the tube, and pour the agar into a pets plate (see figure 13.2c-D). Be careful to keep the lid of the plate covering the bottom and the mouth of the tube while pouring the agar. Do the same for the other two plates. 4, After pouring the plates, allow them to cool for a {few minutes on the bench top. With a wax pencil, ‘mark on the bottom of the plate the name of the bacterium to be inoculated, your name, and date ‘Also draw four quadrants on the bottom of the plate, as illustrated in figure 16.2c, to aid you in keeping track of your streaks. 5. Aseptically remove a loopful ofthe bacterial mixture (see figure 14.3), 6. Streak out the loopful of bacteria on the agar place that you have prepared as follows a. Carefully lit the top of the pete plate just ‘enough to insert your inoculating loop easily 100 Basic Lahoratory and Culture Techniques (Gigure 16.2a). The top should cover the agar surface as completely as possible at all times in order to avoid contamination. Insert the inoculating loopful of bacteria and spread it cover a small area (area 1) at one edge of the plate as shown in figure 16.2b in order to make effective use of the agar surface. This is accomplished by letting the loop rest ‘gently on the surface of the agar and then ‘moving it across the surface each time without digging into the agar. bb. Remove the inoculating loop and kill any remaining bacteria by flaming them, Then insert the loop under the lid and cool it atthe edge of the agar near area 1 ©. Rotate the plate while carefully keeping in ‘ind where the initial streaks ended (use the marked quadrants as a guide) and cross lover the streaks in area I (figure 16.26). ‘Streak out any bacteria picked up as shown 4d. Remove the loop, flame it, cool in the agar as belore, and repeat the streaking process (Gigure 16.2, area 3) e. Ifnecessary, you can repeat this sequence ‘once more to make a fourth set of streaks {area 4), Use fewer cross-streaks here than in the previous quadrant. £, Repeat the above procedure (a~e) for the other two bacteria on two new peti plates. 7. Incubate the plates at 30° to 37°C for 24 to 48 ‘ours in an inverted position, Afterwards, examine cach of the agar plates to determine the distribution and amount of growth in the three or four stteaked areas and record your results in the report for exercise 16, Principles for the Use of Differential Media Many kinds of media can be used with streak plates, ‘The first part of this exercise employed typtic soy agar, a general purpose complex medium. Often it is ‘most advantageous (o prepare streak plates with selec~ tive and/or differential media, Selective media favor the growth of particular microorganisms. For exam- ple, bile salts or dyes like basic fuchsin and crystal vi- let favor the growth of gram-negative bacteria by in- hibiting the growth of gram-positive bacteria without affecting gram-negative organisms. Differential media are media that distinguish between different groups of bacteria and even permit tentative identiti- cation of microorganisms based on their biologicalMatera Tn ese tater and Te te Steat-Pote Tretia tian Laboratory Exercises Cate Techniques Techique an Diem Coon, 212 Microbiology. Fth Eton Mois Figure 162. Pr ation ofa Streak Plate, Aows inicate motion ofthe loop. Taf fame and cool the loop between 1 and 22 and 3, nd andthe cad ofthe ese” The goals to thin the numben of batena grown i cach successive ates ofthe plate mt rotted and ‘read 0 thar well wolited colonies wil appear in guadean 3, ” characteristics. Blood agar is both a differential ‘medium and an entiched one. It distinguishes between hemolytic and nonhemolytic bacteria. Hemolytic bac- teria (e-g., many streptococci and staphylococti iso- lated from throats) produce clear zones around their colonies because of red blood cell destruction. ‘Two very important differential and selective media that are used to isolate and partially identify bacteria are mannitol salt agar and cosin methylene blue agar. Mannitol salt agar is used (o isolate staphy- lococei from clinical and nonclinical samples. It con- tains 7.5% sodium chloride, which inhibits the growth ‘of most bacteria other than staphylococei. Staphylo- ccoccus aureus will ferment the mannitol and form yel- ow zones in the reddish agar because phenol red be- comes yellow in the presence of fermentation acids (see figure 54.5). This differentiates it from S. epider- ‘midis, which forms colonies with red zones or both zones (see figure 54.6). Bosin methylene blue (EMB) agar is widely used for the detection of E. coli and xe- lated bacteria in water supplies and elsewhere. It con- 0 tains the dyes eosin Y and methylene blue that par- tially suppress the growth of gram-positive bacteria, ‘The dyes also help differentiate between gram-nega- tive bacteria, Lactose fermenters such as Escherichia coli will take up the dyes and form blue-black colonies with a metallic sheen, Lactose nonfermenters such as Salmonella, Proteus, and Pseudomonas form colorless to amber colonies In this exercise, we will combine the streak-plate technique with differential and selective media to iso- late and parlly identify Staphylococcus aureus and Es- cherichia coli Procedure 1. Mela sterile, capped tube of mannitol salt agar and a tube of EMB agar in a boiling water bath. 2. Cool the two tubes in a 48° to 50°C water bath for 10-15 minutes. 3. Remove the cap, flame the lip of the tube, and pour the agar into a sterile peti plate (see figure 13.2c-D) StreakPlate Technique 101Matera Tin sescttortaryand | 1. The suet-Piate Tretia tian Laboratory Exercises Cate Techniques Techique an Diem Corpor, 202 Microbiology. Fth Eton Mois Be careful o keep the ld of the plate covering the bottom and the mouth ofthe tube while pouring the ‘agar. Do the same forthe second plate. Allow the plates to cool for a few minutes on the bench top. Matk on the bottom of each plate your rams, the date, and the agar used. Asoptically remove a loopful ofthe bacterial mixture containing E.coli, S. aureus, and Proteus vulgaris. Prepare mannitol salt agar and EMB agar streak plates following the procedure described in step 6 on page 100. Incubate the plates at 35-37°C for 2448 hours in an inverted position, Examine them and evaluate the type of colony growth. Compare the colonies ‘on the two plates and try to determine which bacteria are growing on each, Record your “observations inthe report for Exercise 16,LabormoryExerczosin Clare Techni * Trig an Diferomil Compre, 22 Laboratory Report 16 “= Dat Lab Se The Streak-Plate Technique and Differential Media 1, Make a drawing of the distribution of the colonies on each peti plat. OOS 2. Select one discrete colony, describe it (see figure 15.1), and identify the bacterium it contains. Colony tevin Coley ctor 3. Draw your streaking patterns, Did you obtain isolated colonies? If not, what went wrong? If you carried ‘out the differential medium experiment, comment on the differences in growth on mannitol salt agar and EMB agar, 103