Mare Prveate Laboroory Exercises Cate Techniques Mizrobiooy. Fh Eton Totten opr 212 EXERCISE Pour-Plate Technique Materials per Student 24-10 48-hour mixed uyptic soy broth culture of Escherichia coli (ATCC 11229), Serratia marcescens (ATCC 13880; Micrococcus roseus ATCC 186 also can be used), and Bacillus subtilis (ATCC 6051) 3 tryptic soy agar pour tubes 3 9-ml sterile 0.9% NaCl (saline) blanks 48° (o 50°C water bath boiling water bath wax pencil 3 petri plates inoculating loop Bunsen burner 43 sterile 1-ml pipettes with pipettor Learning Objectives Hach student should be able to 1. Understand the pour-plate technique 2. Perform a pour-plate technique to obtain isolated colonies Suggested Reading in Textbook 1. ‘The Pour Plate, section 5.8. Pronunciation Guide Escherichia coli (esh-er-L-ke-a KOH-lee) Bacillus subtilis (bab-SIL-lus sub-til-lus) Serratia marcescens (se-RA-she-ah mat-SES-sens) ay Why Are the Following Bacteria Used in Tis Exercise? Aoerpocedue tat is wed to obain well slated, poe colonies is the pour pate technique, Since Serratia marcescens, Bai ul, and Escherichia col wee wed nthe pst tre execs, andthe pus clas plates shold fave Ben eves sme cues are ed in hone cis, Remon, marcescens produces vod cokes, subi white cteam coli: and Ecol white elnies. Principles The pour-plate technique also will yield isolated colonies and has been extensively used with bacteria and fungi. The original sample is diluted several times to reduce the microbial population sufficiently to ob- tain separate colonies upon plating (figure 17.1). The small volumes of several diluted samples are added to sterile peti plates and mixed with liguid tryptic soy agar that has been cooled to about 48° to 50°C. Most bacteria and fungi will not be killed by the brief expo- sure to the warm agar. After the agar has hardened, cach cell is fixed in place and will form an individual colony if the sample is dilute enough, Assuming no chaining or cell clusters, the total number of colonies are equivalent to the number of viable microorgan- isms in the diluted sample. To prepare pure cultures, colonies growing on the surface or subsurface can be {inoculated into fresh medium, Procedure 1, With a wax pencil, label three sterile saline blanks 103 2. Melt the tryptic soy agar deeps in a boiling water bath and cool in a 48° to 50°C bath for at least 10, to 15 minutes (see figure 13.2) 105Matera Tn ese tater and Laboratory Exercises Cate Techniques Microbiology. Fth Eton Tr.Pou-Piae echique ] Tretia tian Coon, 212 Figure 17.1 ‘The Powr-Plate Technique, The ongialsazple edited sever tne o decease or die the popuiion salient 1 al ofeach dlason 1 then dipensed ato the bottm of sper plate. Aga pours ae then added to cach pte. Loated ele row nto colonies and can be wied to establih pure clrates, The sutie colonies ae euculse and lange, ubsfce cole te lenicla o&le eae) ve] fe P| Je me | he cot ea x a howpoe jin jo fea c , oo = 4 \ 4 casey = ress rors or oe 3. With a wax pencil, label the bottoms of three petti plates 1 to 3, and add your name and date. 4, Tnoculate saline tube I with 1 ml of the MIXED bacterial culture using aseptic technique (see {figure 14.3) and MIX thoroughly. This represents 2 10-'dilution, 5. Using aseptic technique, immediately inoculate tube 2 with 1 ml from tube 1; a 10-* dilution. 6. Using aseptic technique, mix the contents of tube 2and use it to inoculate tube 3 with 1 ml; a 10 dilution, 7. After tube 3 has been inoculated, mix its contents, remove the cap, flame the top, and aseptically transfer I ml into peti plate 3, Then inoculate plates 1 and 2 in the same way, using 1 ml from tubes 1 and 2, respectively, 8, Add the contents of the melted tryptic soy agar pours to the peti plates. Gently mix each agar plate with a circular motion while keeping the plate flat on the bench top. Do not allow any agar 106 Basic Lahoratory and Culture Techniques to splash over the side of the plate! Set the plate aside to cool and harden. 9. Incubate the plates at 30° to 37°C for 24 (0 48, ‘hours in an inverted position. 10. Examine the pour plates and record your results in the report for exercise 17.LabormoryExerczosin Clare Techni * Coon, 212 Laboratory Report 17 “= Dat Lab Se Pour-Plate Technique 1, Examine each of the agar plates for colony distribution and amount of growth, Look for discrete surface colonies and record your results, Do the same for the subsurface colonies. Color each species of bacterium a different color or label each. Fill in the table, OO Elevation Massa Numer 107