S. Rodriguez BIOS242 Week 1 Lab 1 Culture Transfer Techniques

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Herzing University *

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242

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Mechanical Engineering

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Jan 9, 2024

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BIOS242 Lab 1 Name: Susy Rodriguez Lab 1: Culture Transfer Techniques Learning Objectives:  Identify the importance of aseptic technique in the field of microbiology  Apply the concept of aseptic technique and its importance in the field of microbiology.  Identify different forms of basic growth media  Transfer a pure bacterial culture from one growth media to another, a process called sub- culturing. Introduction: Microorganisms are everywhere in our environment; they are in the air, the soil, on surfaces (fomites), and on and within living things. In a hospital setting, contamination of clinical samples may have an impact on the diagnosis and treatment of patients. Experimental results from pure cultures, which contain a single type of microorganism, can be skewed and produce erroneous results when contaminated with exogenous microorganisms. Unwanted microorganisms can be introduced into samples by direct contact with contaminated surfaces or hands by touching either the growth media or the inner surfaces of the culture tube with objects that have not been sterilized. In addition, microbes in the air can enter tubes and plates of growth media by way of air currents. Thus, aseptic technique is an important concept to learn in the lab and in clinical settings. Aseptic techniques are designed to prevent unwanted microorganisms from contaminating either sterile materials or pure cultures. If bacteria are handled correctly, only the desired organisms will grow on transfer cultures. Using proper aseptic technique, the transfer of a sample from a pure culture will allow only that specific bacterium to grow. This process is called subculturing and is used to maintain the cells as well as keep them in an active growth phase for experiments. In this lab exercise and in future experiments, it is critical to apply aseptic technique to prevent the contamination of pure cultures. The growth and survival of microorganisms require a source of nutrients and a favorable environment. For example, bacteria that grow in the human gut may grow better at body temperature than at room temperature. While some microorganisms have very specific growth requirements, many bacteria can grow in media containing low molecular weight substances derived from powdered beef extract and enzyme derived short chains of amino acids. Cultures can be grown in tubes in a liquid broth medium. 1

BIOS242 Lab 1 Name: Susy Rodriguez Alternatively, the broth can be mixed with agar, a seaweed extract with no nutritional value, to form a semisolid medium. This can be put onto plates or in tubes. In tubes, the agar can either be allowed to harden in the upright position- known as an agar deep or, on a slant, depending on the application. Slanted tubes of agar provide a larger surface area for growth and are useful for storing specimens for an extended period of time. In contrast, upright agar tubes are used for stab cultures, which can provide information on the organism’s requirement for oxygen. In this laboratory exercise, Serratia marcescens , a bacteria, will be transferred into a broth culture and to slant and upright (stab) agar media. Both transfer loops and transfer needles will be used. Your instructor will demonstrate how to use the transfer loops and transfer needles using aseptic techniques. You will be using a gas burner so remember to keep hair back and hands away from the flame. The following transfers will be made, using aseptic technique. 1. Broth culture to sterile broth 2. Broth culture to sterile slant 3. Broth culture to stab culture 4. Slant culture to sterile broth 5. Slant culture to sterile slant 6. Slant culture to sterile stab Materials: Nutrient broth, Nutrient agar slants, Nutrient agar stabs, liquid and slant cultures of Serratia marcescens, inoculating loop, inoculating needle, incinerators Note to Students: 1. You may use metal loops/needles or disposable plastic loops/needles. The instructions may vary based on the type. Please check with your instructor regarding usage and disposal of loops and needles. 2. You may use incinerator or burner with flame to sterilize metal loops and needles. Please check with your instructor regarding how to safely use incinerators or burners to sterilize. 2

BIOS242 Lab 1 Name: Susy Rodriguez Method: 1. Remove extraneous materials from the lab bench and disinfect it with 10% bleach solution. 2. Obtain 24 hour cultures of Serratia marcescens (broth and slant). 3. Label the sterile tubes (broth, slant, and stab) with the name of the organism and your group designation. 4. Loop sterilization for those labs with reusable metal loops following the instructions below (a and b). If your lab uses sterile plastic loops, use a new loop for each transfer being mindful not to touch it on any surface. a. Sterilize the inoculating loop or needle by holding it in the flame of the gas burner, moving it through until the wire turns red. b. Allow the inoculating loop or needle to cool for 10 – 20 seconds. Do not wave it in the air or put it down, as it may pick up contaminating bacteria and will no longer be sterile . 5. Transfer from a broth culture to a new broth culture by following the steps outlined in a-h. a. Loosen the cap of the sterile broth tube, but do not remove it completely. b. Open the tube of broth culture. While holding the cap in your hand (do not put it down, as this action will compromise its sterility) pass the mouth of the tube through the flame a few times. c. Place the sterilized inoculating loop from step 4 into the culture, being careful not to touch the sides of the tube. d. Remove the loop, pass the mouth of the tube through the flame again, and cap it. e. Take the sterile, labeled broth tube, uncap it and flame the mouth of the tube. f. Insert the inoculating loop with the bacteria sample into the sterile broth and shake it a few times before removing it. g. Pass the mouth of the tube through the flame and cap it. h. Metal loops only: Flame the loop until it turns red to sterilize it. 6. Transfer from a broth culture to a slant culture 3

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BIOS242 Lab 1 Name: Susy Rodriguez a. Follow steps 5a – 5d described above. b. Take a sterile, labeled slant culture, uncap it and flame the mouth of the tube. c. Insert the inoculating loop with the sample and touch the loop on the surface of the slant. Drag the loop gently across the top of the agar, starting from the bottom of the slant to the top. Be careful not to dig into the agar . d. Remove the loop, flame the mouth of the slant culture and cap it. e. Metal loops only: Flame the loop to sterilize it. 7. Transfer from a broth culture to a slab culture a. Stab cultures use an inoculating needle instead of a loop. b. Follow steps 5a – 5d described above, using an inoculating needle. c. Take a sterile, labeled stab culture, uncap it and flame the mouth of the tube. d. Insert the needle containing the bacteria into the tube in a straight line and rapidly withdraw it. e. Flame the mouth of the stab culture, recap it, and flame sterile the inoculating needle if you are using a metal needle. 8. Transfer from a slant culture to a broth, slant, or stab culture a. All of the steps described previously are the same, except for obtaining the sample. b. After obtaining the sterile loop, open the cap of the slant culture, flame the mouth of the tube and obtain a sample by gently touching the surface of the slant where there is bacterial growth. c. Remove the loop, flame the mouth of the tube and recap it. d. Once you have the sample, follow the procedures described above to transfer to broth, slant, and stab tubes. 9. When finished, incubate the tubes at approximately 25 o C for 24 to 48 hours. Next lab class: 4

BIOS242 Lab 1 Name: Susy Rodriguez 10. Examine the cultures for appearance of growth. Broth cultures should appear turbid (cloudy). Slant and stab cultures should have orange-red growth on the surface of the slant and along the line of inoculation in the slant culture. 11. Record your findings in the Lab Report. Lab Report Purpose: Please describe in complete sentences and in your own words, the purpose of this experiment. Observations: Broth Stock Culture: Broth Subculture Slant Subculture Stab Subculture Growth (+) or (-) + Cloudy Orange-Red Pigmentation (+) or (-) _ 5

BIOS242 Lab 1 Name: Susy Rodriguez Visual Growth Patterns 37* celcus for 24hr incubation Slant Stock Culture: Broth Subculture Slant Subculture Stab Subculture Growth (+) or (-) Orange-Red Pigmentation (+) or (-) Visual Growth Patterns 6

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BIOS242 Lab 1 Name: Susy Rodriguez Questions: 1. Why is proper aseptic technique important in microbiology? In microbiology, using the right aseptic method is crucial to avoid contaminating oneself or the culture. Maintaining the culture's sterility is the objective. 2. What is the importance of flaming the inoculating loop or needle before and after each inoculation? Flaming the inoculating loop or needle before and after each inoculation is crucial because it helps remove any contaminants that may have gotten into the air or on the loop needle. It also helps remove any bacteria that may have colonized the loop needle. 3. If you do not wait 10 – 20 seconds after flame sterilizing the inoculating instruments before obtaining the sample, what might be the consequences? If the inoculating devices are not flame sterilized for 10 to 20 seconds before retrieving the sample, this could lead to the sterilization of the bacteria, which could kill the microbes we are trying to culture. 7

BIOS242 Lab 1 Name: Susy Rodriguez 4. Why is it important to flame neck of the tubes immediately after uncapping and before recapping the tubes? In order to avoid contamination from air that could become caught inside the tubes or come from us and land inside, it is crucial to flame neck the tubes as soon as they are uncapped and before they are recapped. 5. The stab tube was inoculated with a needle. Why was this used instead of the inoculating loop? 6. The Serratia marcescens cultures were accidently incubated at 37 o C instead of 25 o C. You observed growth but the slant and stab cultures were white, not orange-red. Does this mean your sample was contaminated? No, this does not imply that there was contamination in the sample. Because the sample was put in a different incubator with a temperature that was too high for it to be effective, the observed slant and stab cultures were white rather than orange-red. 8

BIOS242 Lab 1 Name: Susy Rodriguez Grading Rubric: Activity Deliverable Points Experimental Set up Set up the cultures as directed in the lab or by instructor 4 Observation Observe the results and record them in lab report 4 Lab Report and Questions Complete lab report and answer questions  Purpose (1 point)  Questions (6 points) 7 All Lab Deliverables Complete ALL lab work and lab report 15 9

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