Tis spsi-rie| Tetniqee Mare Prveate Tn ese tater and Laboroory Exercises Cate Techniques Mizrobiooy. Fh Eton Totten opr 212 EXERCISE Spread-Plate Technique Materials per Student 24-to 48-hour tryptic soy broth cultures of Bacillus subtilis (ATCC 6051, white or cream colonies), Serratia marcescens (ATCC 13880, red colonies) or Micrococeus roseus (ATCC 186, red colonies), and a mixture of the two (S. marcescens [or M. roseus] and B. subtilis) Bunsen burner inoculating loop 95% ethyl alcohol L-shaped glass rod wax pencil '500-ml beaker pipettes with pipettor 3 teyptic soy agar plates, rulers Learning Objectives Each student should be able to 1, Understand the purpose of the spread-plate technique 2. Perform the spread-plate technique Suggested Reading in Textbook 1. Isolation of Pure Cultures, section 5.8. 2. The Spread Plate and Streak Plate, section 5.8 see also figures 5.7-5.9, 5.11 3. Colony Morphology and Growth, section 5.8. Pronunciation Guide Bacillus subtilis (bah-SILlus sub-tl-lus) Serratia marcescens (se-RA-she-ah mar-s = After this exercise, the student should be able to use the spread-plate technique (o separate a mixture of two or more bacteria into well-islated colonies. The bacteria (0 be used are Bacillus subnlis and Serratia marcescens ot M. roseus. B. subsilis is easy to culture since it grows on simple medium (e.g, syplic soy ager) and produces dull while to cream colonies that aze easy to zee, S. marcescens was used in the last experiment and produces large red, pink, or magenta colonies. By using color and colony mot phology, the student can see what a well-isolated colony of tach of the above bacteria looks like. The isolated bacteria can then be picked up and streaked onto fresh medium (o oblaia a pure culture, Why Are the Above in This Exercise? Bacteria Used & Medical Application In the clinical laboratory, growth of a pute culture is ab- solutely necessary before any biochemical tsts can be per- formed to identify a suspect microorganism, Principles Tn natural habitats, bacteria usually grow together in ‘populations containing a number of species. In order to adequately study and characterize an individual bacte- rial species, one needs a pure culture. The spread- plate technique is an easy, ditect way of achieving this result. In this technique, a small volume of dilute 93Tretia tian Coon, 212 Tin sescttortaryand | 15 Speedie Cate Totnigues Tetniqee Bacterial Colony Characteristics on Ager Media as Seen with the Naked Bye, The characeusesofbactetal colonies ae deserved wong the Gllowsng tenn oH eH uncer CreuarFlamerious —toguar Sone om se DX at ale 5 ‘Appearance: Shiny or dut pte preperty:Opeaue, vansueet,ansparent Plamentaton Pigments [purl oa Yon Nemplamented ream, tan white ‘Texture: Rough orsmoath bacterial mixture containing 100 to 200 cells or less is ‘cansfrred to the center of an agar plate and is spread evenly over the sutface with a sterile, L-shaped glass rod. The glass rod is normally sterilized by dipping in alcohol and flamed to bur off the alcohol. After ine bation, some of the dispersed cells develop into iso- lated colonies. A colony is a large number of bacterial cells on solid medium, which is visible to the naked ‘eye asa discrete entity. In this procedure, one assumes that a colony is derived from one cell and therefore represents a clone of a pure culture Aller incubation, the general form of the colony and the shape of the edge or margin can be deter- ‘mined by looking down at the top of the colony. The nature of the colony elevation is apparent when viewed from the side as the plate is held at eye level “These Variations are illustrated in figure 15.1. After a wel-isolated colony has been identified, it can then be picked up and stueaked onto a fresh medium to obtain a pure culture. 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 04, asic Laboratory and Culture Techniques be inoculated: (a) one with B. subs, (b) one with ‘S. marcescens, and (c) one with the mixture. Pipette 0.1 ml of the respective bacterial culture conto the center of a tryptic agar plate (figure 15.24). Dip the L-shaped glass rod into a beaker of ethanol (figure 15.26) and then tap the rod on the side of the beaker to remove any excess ethanol Briefly pass the ethanol-soaked spreader through the flame to bum off the alcohol (figure 15.2c), and allow it to cool inside the id ofa sterile peti plate Spread the bacterial sample evenly over the agar surface with the sterilized spreader (figure 15.24), ‘making sure the entire surface of the plate has been covered. Also make sure you do not touch the edge of the plate, Immerse the spreader in ethanol, tap on the side of the beaker to remove any excess ethanol, and rellame, Repeat the procedure to inoculate the remaining two plates, Invert the plates and incubate for 24 to 48 hours at room temperature or 30°C. After incubation, measure some representative colonies and carefully observe their morphology (figure 15.3). Record your results in the report for exercise 15.Matera Tin sescttortaryand | 15 Speedie Tete veo Laboratory Exercises Cate Techniques Tetniqee Compre, 22 Microbiology. Fth Eton Figuee 15.2 Spread-Plate Technique Figure 15.3. Spreed Plate, Macroscopic photomicrograph ofa spread ple, Notice the many wellslnted colonies Spread-Plate Technique 95Cray froceie—CiweTechaee Tet Congr. Laboratory Report 15 “= Lab Section Spread-Plate Technique 1, Make drawings of several well-isolated colonies from each plate and fill in the table OOO Colora ceonyies) 2. With your ruler, measure the diameter of the average colony appearing on each plate by placing the ruler on the bottom of the plate, Hold the plate and ruler against the light to make your readings and be sure to measure a well-separated colony. a. Size of B. subrili colony b. Size of S. marcescens colony 7