Lab 5 Pure Culture techniques

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101

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Feb 20, 2024

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1 LABORATORY EXERCISE #5: Pure culture technique Background information This exercise will introduce students to the streak plate method, by which pure cultures of most bacteria, and some algae and fungi can be obtained. Pure cultures of protozoa are uncommon because of their sophisticated nutritional requirements, i.e ., their reliance on living microscopic prey. Pure culture techniques for viruses are similar in theory to those described in this exercise but are more complex in practice because of the nature of viruses as obligate parasites. Learning objectives • Aseptically handle tubes and plates • Properly label tubes and plates • Follow proper disposal procedures for biological discard • Explain the basic steps of streak plating. • Interpret general growth results of microbes on different media. • Describe colony morphologies using proper terminology. Lab activities 1. Streak plate technique (Part A) 2. Colony descriptions (Part B) Laboratory notebook o Title: Pure culture technique o Purpose: Obtain a pure culture of a microorganism using streak plating. o Materials & Methods: Lab#/pages (outline and any deviations) o Results: Descriptions and drawings of plates and colony morphologies. o Discussion: Did you obtain a pure culture from a mixedculture? How could your streak technique be improved? What are the colony morphologies of the microbes in your mixed culture?

2 A. Streak plate technique 1. MIXED CULTURE In this technique, separation of cells in a mixture is achieved by physically moving (and hence, diluting) the cells across a solid agar surface. The dispersed cells then multiply to form colonies--in theory a colony develops from repeated divisions of a single cell, but in actuality, colonies can originate from multiple and like cells ( e.g ., a sarcinal packet or a chain of rods, cocci) and from multiple and unlike cells (e.g., dissimilar cells trapped in heavy slime produced by another organism). 1. Each pair of students will streak 2 nutrient agar plates following the pattern in Figure 1 (one plate each). The inoculum is a saline mixture of Escherichia coli and Serratia marcescens. 2. Flame sterilize your loop and using aseptic technique, obtain a loopful of your mixed culture. Place this loopful of inoculum at but not touching the edge of the Petri dish. Flame the loop, cool it in the agar away from the inoculum and spread the inoculum as shown in streak #1. Flame the loop again, cool and streak #2 at right angles to #1. 3. Run the loop into the edge of #1 two or three times to obtain inoculum for #2 but then do not return to streak #1. Use very tight streaks and continue to streak as shown in Figure 1. Flame the loop, cool and perform #3 just as for streak #2. Remember, do not return to streak #2 after sufficient inoculum is obtained to make streak #3. Finally, flame the loop to decontaminate it. Figure 1. Pattern for streaking a plate to achieve colony isolation. ***Note, all streaking should be done without gouging the agar surface. Gouging is avoided by making certain the inoculating loop wire has a smooth surface and by applying gentle or no downward pressure on the loop during the streaking process. Also, use "loose and rapid" strokes--slow, tense and laborious strokes inevitably result in gouges. After streaking the first plate, have your instructor evaluate your streak pattern before streaking your unknown (see below). 4. Incubate one plate at room temperature and one at 37 ⁰ C. Remember to invert the plates. Why?

3 2. SEMESTER UNKNOWN Each student will be given an unknown organism and perform a series of tests to identify the organism over the course of the semester. All results from your unknown should be recorded under lab 7 as described by your instructor. After your instructor approves your streak plate technique, streak for colony isolation as described in Lab 7. . B. Colony descriptions: Lab period 2 or 3 Observe the streak and pour plates you prepared. Did both techniques give well-isolated colonies of the bacteria in the original suspensions? Note the lenticular shape (lens-like) of the subsurface colonies in the pour plates. Can you account for this shape? Why do you think one streak plate was incubated at room temperature and one at 37 deg C? What is a major difference between the colony pigmentation on these plates? It is traditional to describe colonies (Figure 3) grown on nutrient agar and after they reach maturity. Therefore, retain one plate of your mixed culture which has well-separated colonies of both bacteria. After 5-7 days of total incubation, record a colony description for both organisms. Do the same for your unknown and record the colony morphology for your unknown under Lab 7. Colony descriptions can be an aid to the ultimate identification of a bacterium and normally include the appearance of the whole colony; the type of edge the colony possesses; what it looks like in profile, i.e. , elevation; how the colony surface appears, surface; and how it "feels" when touched with an inoculating loop, consistency. See Figure 3 for examples. Colonies also possess optical qualities and sometimes pigmentation. Important optical characters are whether the colony is opaque to transmitted light and its appearance in reflected light (fluorescent, iridescent, bioluminescent, etc.). Pigmentation, if present, should be noted as to color and whether the pigment has spread into the agar adjacent to the colony indicating a diffusible pigment. Finally, the presence of a pigment may occur under some growth conditions and not others. For example, Serratia marcescens will be pigmented brilliant reddish-orange at room temperature but at higher temperatures, even though growth occurs, it will not be pigmented. What would your hypothesis be to explain the different pigmentation at different temperatures? Observe the plates on demonstration. Each is accompanied by a card on which is given the colony description appropriate for the organism. Use this demonstration as a guide to describing the colonial features of E. coli and S. marcescens and ultimately, of your semester bacterial unknown.

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4 Figure 3. Selected characteristics that are used to describe bacterial colonies. (Hendrix) Surface: Smooth Glistening Rough Dull Wrinkled Dry Powdery Optical: Opaque Translucent Transparent Iridescent Color (often dependent upon specific agar grown upon): White Red Purple Blue Pink Buff Yellow etc C. Clean up When you have finished working: • TURN OFF YOUR FLAME. • Place plates and slants in your section’s spot in the incubator (for 37°C) or where your TA indicates on the counter (for 25°C). Make sure all plates are labeled with your initials, date, medium, temperature, and contents. • Wipe down your bench with 70% ethanol: Spray surface with ethanol, spread with a Kim wipe, and allow to air-dry. Toss Kim wipe in the normal trash . Return ethanol spray bottle to its secondary container in the back of the sink. • Toss gloves in the large red biohazard bin . • Follow the general lab clean-up instructions to exit the lab. After recording your data: • Toss Mixed Culture plates in the large red biohazard bin . • Your Unknown plates will be used to prepare working slants in the next lab. Afterwards, you will store them in the refrigerator for future use.

5 D. Pre-lab questions: 1. What is the proper way to work aseptically with tubes and bottles? (In particular, how should you handle the lid, and why?) 2. Where should you label your Petri dishes? Explain why that is the best place for a label. 3. What is the purpose of making a streak plate? (That is, what are we hoping to accomplish?) 4. When making a streak plate, when (or how often) should you flame your inoculating loop? Why do we want to flame it at those specific times in the protocol? E. Applying Concepts: 1. You found a bright blue growth on a tomato you had left on your kitchen counter. You’ve never seen a microorganism that color, and you want to know what it is. You take the tomato into the lab, sterilize an inoculating loop, transfer a sample from the tomato to a Nutrient Agar Petri dish, and incubate it at 37°C. But when you come back two days later, your Petri dish is empty. Why might this be? Be specific about what might have happened. How would you test your hypothesis? References: Seeley, H. and P. Van Demark. 1972. Microbes in Action, 2nd ed., W. H. Freeman and Company, San Francisco. Hendrix, J.D., Kennesaw State University

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