Laboratory exercise vii micro

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Laboratory exercise vii micro

  1. 1. Laboratory Exercise # 7: Aseptic TechniquePurpose: The purpose of this laboratory exercise is to acquaint the student with the proceduresof aseptic transfer of microbiological cultures.Introduction: In order for a microbiologist to identify a bacterium to genus (first name) andspecies (second name) the bacterium must be in the form of a pure culture. Unfortunately,bacteria in their natural environment are always found in close association with othermicroorganisms. A pure culture may be obtained by two different methods: pour plate and the streakplate method. This exercise will instruct the students in the “Four Quadrant Streak PlateMethod”. To isolate a pure culture of bacteria one must first have a sterile media/agar plate.To be sterile means that the media is devoid of any live organisms, such as bacteria. Thesterile media is streaked with the bacteria using a method that dilutes the number of bacteriaas you proceed through the streaking technique. Upon completion of the proper techniqueand the incubation of the plate, individual colonies should be observable on the plate. Colonies are formed by a single bacterium setting onto the agar and starting toutilize the nutrients within the media. The bacterium uses the media as a nutritionalfoundation for replication. Within 24 hours, a colony will appear on the agar that ismacroscopically observable. Each individual colony represents a single bacterium that hasreplicated at this location and has approximately several hundred bacterial cells within it. Finally in order to maintain a pure culture, we must use a method of transferringbacteria from one media to the next without contaminating it with other bacteria. Asepticallytransferring bacteria from one media source to another is essential. The student will learntechniques that enable them to transfer bacteria from one medium to another withoutcontaminating the culture.Materials:Trypticase soy agar plates of the following: Bacillus subtilis Escherichia coli Staphylococcus epidermidis Serratia marcescens Pseudomonas aeruginosa Micrococcus luteusTrypticase soy agar slant, TSA plate and Motility Test Medium - 1/studentInoculating loopInoculating needleBunsen burner 1
  2. 2. Procedure: Day # 11. The student will select one of the following microorganisms to demonstrate aseptictransfer: Bacillus subtilis Escherichia coli Staphylococcus epidermidis Serratia marcescens Pseudomonas aeruginosa Micrococcus luteus2. Using the methods shown in the following diagrams, aseptically inoculate your selectedmicroorganism to the following media: Trypticase Soy agar slant Motility Test Medium Trypticase Soy Agar Plate3. Incubate all tubes and plates at 37°C, except for those of Serratia, which are incubated atroom temperature, until the next laboratory period. Remember petri dishes must bestored inverted to prevent condensing water from falling on the growing colonies andcausing them to run together.4. Start Gram stains of each of the bacteria used within this exercise. Eventually you willcomplete six (6) stains, but not all of them need to be completed today.Procedure Day # 21. Observe the Motility Test medium tube and record results for each bacterium in the DataArea provided.2. Observe the results of the agar slant media and record pigment results only for eachbacteria in the Data Area provided.3. Record Colonial morphology results from the Trypticase Soy agar plates in the DataChart provided. Make sure to use a single, well isolated colony for your observations.4. Complete gram stains on all six bacteria and record your results in the Data Chartprovided. 2
  3. 3. Laboratory # 7: Aseptic Technique Fig. 7-AInoculation of Motility Test Medium Fig 7-B Inoculation of an agar slant 3
  4. 4. Fig. 7-C Inoculation of an agar plate I II I Streak the culture back and forth Rotate the plate 90° and touch In quadrant I. Flame loop. Loop in an uninoculated area to cool it. Streak second quadrant. Then flame loop. III III IV II II I I Rotate plate 90°. Cool loop. After cooling the loop. Streak Streak the third quadrant. the fourth quadrant making Flame the loop. sure not to enter the first quadrantA practice plate: Practice streaking technique using a pencil on this plate, before trying tostreak on real media. Remember to use a loop and a light touch on the media so as to notdig up the agar (Please do not turn the agar plate into confetti!). 4
  5. 5. Data: Each student is responsible for all six microorganisms.1. Describe the pigment of the growth in each of the slant tubes. Bacillus subtilis: _____________________________ Escherichia coli: _____________________________ Micrococcus luteus: _____________________________ Pseudomonas aeruginosa: _____________________________ Serratia marcescens: _____________________________ Staphylococcus epidermidis: _____________________________2. Draw and describe the growth seen in each of the Motility Test Medium tubes. Label eachof the drawings as to whether the microorganism is motile or non-motile. Bacillus subtilis Escherichia coli ______________ _______________ Staphylococcus epidermidis Serratia marcescens ________________________ _________________ Pseudomonas aeruginosa Micrococcus luteus ____________________ ___________________ 5
  6. 6. 3. Fill in the following chart on the colonial characteristics of each of the 6 microorganisms. Characteristic Bacillus subtilis Micrococcus luteus Form of colony Elevation Margin Surface Pigmentation Gram Stain Characteristics Escherichia coli Staphylococcus epidermidis Form of colony Elevation Margin Surface Pigmentation Gram stain Characteristics Pseudomonas aeruginosa Serratia marcescens Form of colony Elevation Margin Surface Pigmentation Gram stainQuestions:1. Explain the technique of streak plate isolation.2. What part of the flame is the best part to use to sterilize an inoculating loop or needle?3. Explain how a motile microorganism looks when it grows in a Motility Test Mediumculture.4. Why was the Serratia marcescens incubated at room temperature and not at 37°C? 6

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