Laboratory Exercise # 7: Aseptic Technique Purpose: The purpose of this laboratory exercise is to acquaint the student with the procedures of aseptic transfer of microbiological cultures. ntroduction: n order for a microbiologist to identify a bacterium to genus (first name) and species (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 other microorganisms. A pure culture may be obtained by two different methods: pour plate and the streak plate method. This exercise will instruct the students in the Four Quadrant Streak Plate Method. 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. The sterile media is streaked with the bacteria using a method that dilutes the number of bacteria as you proceed through the streaking technique. Upon completion of the proper technique and 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 to utilize the nutrients within the media. The bacterium uses the media as a nutritional foundation for replication. Within 24 hours, a colony will appear on the agar that is macroscopically observable. Each individual colony represents a single bacterium that has replicated 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 transferring bacteria from one media to the next without contaminating it with other bacteria. Aseptically transferring bacteria from one media source to another is essential. The student will learn techniques that enable them to transfer bacteria from one medium to another without contaminating the culture. Materials: Trypticase soy agar plates of the following: Bacillus subtilis Staphylococcus epidermidis Pseudomonas aeruginosa Escherichia coli Serratia marcescens Micrococcus luteus Trypticase soy agar slant, TSA plate and Motility Test Medium - 1/student noculating loop noculating needle Bunsen burner 1
Procedure: Day # 1 1. The student will select one of the following microorganisms to demonstrate aseptic transfer: Bacillus subtilis Staphylococcus epidermidis Pseudomonas aeruginosa Escherichia coli Serratia marcescens Micrococcus luteus 2. Using the methods shown in the following diagrams, aseptically inoculate your selected microorganism to the following media: Trypticase Soy agar slant Motility Test Medium Trypticase Soy Agar Plate 3. ncubate all tubes and plates at 37 C, except for those of Serratia, which are incubated at room temperature, until the next laboratory period. Remember petri dishes must be stored inverted to prevent condensing water from falling on the growing colonies and causing them to run together. 4. Start Gram stains of each of the bacteria used within this exercise. Eventually you will complete six (6) stains, but not all of them need to be completed today. Procedure Day # 2 1. Observe the Motility Test medium tube and record results for each bacterium in the Data Area provided. 2. Observe the results of the agar slant media and record pigment results only for each bacteria in the Data Area provided. 3. Record Colonial morphology results from the Trypticase Soy agar plates in the Data Chart 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 Chart provided. 2
Laboratory # 7: Aseptic Technique Fig. 7-A noculation of Motility Test Medium Fig 7-B noculation of an agar slant 3
Fig. 7-C noculation of an agar plate Streak the culture back and forth n quadrant. Flame loop. Rotate the plate 90 and touch Loop in an uninoculated area to cool it. Streak second quadrant. Then flame loop. V Rotate plate 90. Cool loop. Streak the third quadrant. Flame the loop. After cooling the loop. Streak the fourth quadrant making sure not to enter the first quadrant A practice plate: Practice streaking technique using a pencil on this plate, before trying to streak on real media. Remember to use a loop and a light touch on the media so as to not dig up the agar (Please do not turn the agar plate into confetti!). 4
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 each of 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
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 stain Questions: 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 Medium culture. 4. Why was the Serratia marcescens incubated at room temperature and not at 37 C? 6