6: STERILE TECHNIQUE
- Page ID
- 157065
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Differentiate between nutrient broth, nutrient agar slants, and deeps, and describe the purpose of each medium.
- Use an inoculating loop and needle correctly for transferring microorganisms to various media types.
BACKGROUND
A central challenge in microbiology is maintaining the purity of microbial cultures while accurately tracking where organisms have been introduced, despite the fact that individual microorganisms are not visible to the naked eye. It is essential to develop consistent habits that allow for reliable identification of inoculated versus uninoculated media throughout experimental procedures.
Maintaining a systematic approach is one of the most effective strategies for achieving this goal. For example, always flame sterilize the inoculating instrument before returning it to the test tube rack or work area unless it has already been sterilized. This practice minimizes the risk of unintentionally spreading viable organisms to laboratory surfaces, personal items, or other individuals. To further support accuracy, uninoculated media should be consistently positioned on one side of the workbench or test tube rack, and inoculated media should be placed on the opposite side.
In addition to tracking inoculations, it is essential to avoid contamination. Contamination occurs when unwanted microorganisms are inadvertently introduced into a culture. This can happen through exposure to airborne particles, contact with the laboratory bench or hands, use of improperly sterilized instruments, or even from exhaled breath. Avoiding contamination is especially critical in clinical and diagnostic applications. For instance, if a patient sample becomes contaminated during processing, it may lead to an incorrect identification of the pathogen and inappropriate treatment. Such errors can result in prolonged illness, treatment failure, or serious complications for the patient.
The prevention of contamination relies on the use of aseptic technique, a set of practices designed to minimize the introduction of extraneous microorganisms into cultures and the surrounding environment. Even for individuals not pursuing careers in microbiology, aseptic technique has practical benefits. These include limiting the transmission of illness in everyday settings, reducing the risk of foodborne disease, and extending the shelf life of perishable items.
This exercise introduces fundamental aseptic techniques used to transfer microorganisms between different types of culture media. In this lab, you will work with nutrient broth (a liquid medium) and nutrient agar (a solid medium). Nutrient agar is commonly prepared in three configurations: slants, deeps, and Petri plates.
When nutrient agar is dispensed into a test tube and allowed to solidify at an angle, it forms a slant. The slanted surface provides a larger area for microbial growth than if the medium had solidified in a vertical position. Slants are commonly used for storing cultures over extended periods. Once growth is established, the slant can typically be stored under refrigeration for several weeks or months, depending on the organism. Although Petri plates can also be used for storage, they are more susceptible to contamination and tend to dry out more quickly than slants.
When nutrient agar solidifies in an upright position within a test tube, it forms a deep. Deeps are used to support the growth of anaerobic organisms—microorganisms that do not require oxygen—and to assess whether an organism can grow in the presence or absence of oxygen. The top of the deep provides an oxygen-rich environment, while the bottom is largely anaerobic. Inoculation of deeps is performed using an inoculating needle, which is similar to a loop but lacks the circular wire end. The needle is used to insert cells deep into the agar while minimizing the introduction of air.
Proficiency in these foundational techniques is essential for future exercises, particularly those involving pathogenic organisms. The types of media used in this activity represent standard tools in microbiological practice: slants for maintaining cultures, broths for generating large cell populations, and deeps for investigating anaerobic growth.
MATERIALS
1 nutrient agar slant
1 nutrient agar deep
1 nutrient agar broth
Inoculating loop
Bunsen burner
Sterile nutrient broth tube (liquid medium)
Source culture (agar plate or broth tube)
Test tube rack
Permanent marker
One of the following: (Instructor will hand out)
Escherichia coli Saccharomyces cerevisiae
Penicillium roqueforti Micrococcus luteus
METHODS/PROCEDURES
SAFETY REMINDERS
Never place the cap of the test tube on the bench.
Always carry test tubes in a rack.
Be aware of the open flame – never leave a flame unattended.
Always rotate the tube not the cap
Use a permanent marker (Sharpie) to label your tubes of media
First initial
Full last name
Genus, species and strain (if designated)
Exercise #
Date
👉 Only write on the glass - do not write on the white portion of the tube.
👉 You can abbreviate the genus but not the species
Arrange your workspace
Place your materials within easy reach. Position the sterile broth tube on one side of your test tube rack and the source culture on the other side to help avoid cross-contamination.
1. Light your Bunsen burner
- Inoculate the broth:
- Sterilize the inoculating loop
Hold the loop in the flame until it becomes red-hot. Be sure the entire wire is sterilized. Then, allow the loop to cool for 10–15 seconds. Do not wave the loop in the air or blow on it to cool it. - Obtain the source culture
- If the culture is on a plate: slightly lift one side the lid and gently touch the loop to a colony on the surface of the agar. Close the lid immediately.
- If the culture is in a broth tube: remove the cap with the little finger of your loop hand. Flame the lip of the tube. Insert the cooled loop and collect a small amount of culture. Flame the lip again and recap the tube.
- Remove the cap of the sterile broth tube using the same hand that holds the loop.
- Briefly flame the lip of the broth tube.
- Insert the loop into the broth and gently swirl or stir to release the cells.
- Remove the loop, flame the lip of the tube again, and replace the cap loosely by rotating the tube not the cap.
- Re-sterilize the loop by holding it in the flame until it is red-hot. Return it to the instrument block when cool.
- Place the inoculated broth tube back in the test tube rack on the same side as the source culture.
- Sterilize the inoculating loop
- Inoculate the slant
- Sterilize the inoculating loop
Hold the loop in the flame until it becomes red-hot. Be sure the entire wire is sterilized. Then, allow the loop to cool for 10–15 seconds. Do not wave the loop in the air or blow on it to cool it. - Obtain the source culture
- If culture is on a plate: slightly lift the lid on one side and gently touch the loop to a colony on the surface of the agar. Close the lid immediately.
- If culture is in a broth tube: remove the cap with the little finger of your loop hand. Flame the lip of the tube. Insert the cooled loop and collect a small amount of culture. Flame the lip again and recap the tube.
- Hold the slant in your non-dominant hand and remove the cap with the little finger of your dominant hand (the one holding the loop). Do not set the cap down.
Flame the lip of the slant tube - Quickly pass the open mouth of the tube through the flame.
Inoculate the slant - Insert the loop and gently touch the surface of the agar at the bottom of the slant. Slowly draw the loop upward in a zigzag or wavy line along the surface of the slant to the top. Avoid gouging or cutting the agar.
Finish the transfer - Carefully remove the loop. Flame the mouth of the tube again and replace the cap loosely. Do not tighten the cap to allow for gas exchange during incubation.
Flame the loop - Re-sterilize the loop by holding it in the flame until it is red-hot. Return it to the instrument block when cool.
- Place the inoculated slant tube back in the test tube rack on the same side as the source culture.
- Sterilize the inoculating loop
- Inoculate a nutrient agar deep
Sterilize the inoculating loop- Hold the loop in the flame until it becomes red-hot. Be sure the entire wire is sterilized. Then, allow the loop to cool for 10–15 seconds. Do not wave the loop in the air or blow on it to cool it.
Obtain the source culture - If culture is on a plate: slightly lift the lid on one side and gently touch the loop to a colony on the surface of the agar. Close the lid immediately.
If culture is in a broth tube: remove the cap with the little finger of your loop hand. Flame the lip of the tube. Insert the cooled loop and collect a small amount of culture. Flame the lip again and recap the tube. - Open the sterile deep tube
Hold the deep tube in your non-dominant hand. Remove the cap with the little finger of your dominant hand, which is also holding the inoculated needle. Do not set the cap down. - Flame the lip of the deep tube
Quickly pass the open mouth of the tube through the flame to maintain sterility. - Inoculate the deep
Insert the needle straight down into the center of the agar. Push the needle almost to the bottom of the tube in a single, steady motion. Do not let the handle touch the agar. Then withdraw the needle along the same path without wiggling or disturbing the agar. This technique minimizes oxygen introduction into the stab line. - Finish the transfer
Flame the mouth of the tube again and loosely replace the cap to allow for gas exchange during incubation. - Flame the needle
Re-sterilize the needle by heating it until red-hot. Return it to the rack or holder once cooled.
- Hold the loop in the flame until it becomes red-hot. Be sure the entire wire is sterilized. Then, allow the loop to cool for 10–15 seconds. Do not wave the loop in the air or blow on it to cool it.
- Incubate the inoculated media
Transfer the 3 inoculated test tubes in to the appropriate colored test tube racks for your section then place the rack in the class incubator.
The following is to be completed during lab then turned in on Canvas as a PDF
*If you are using an iPad or tablet you will need to take screen shots of your competed work, save the screenshots as one PDF then submit them on Canvas by the due date designated on Canvas.
*You can also print out the entire exercise to bring to lab with you. If you choose to complete the lab on paper, take pictures of the completed results and conclusions sections only, save them as one PDF, then submit to Canvas by the due date designated on Canvas.
STERILE TECHNIQUE
NAME ______________________
EXPECTATIONS
Do you think you were able to transfer cells from the original culture to the new medium without introducing contamination? Yes or No ___________
How will you be able to tell if you were successful?
RESULTS
Draw your observations of the growth in the tubes after incubation. Label each tube separately. Draw the broths as they appear when you remove them from the incubator then again after shaking them. Make sure everyone in your group has had the opportunity to draw the broths before shaking.
CONCLUSIONS
Were you successful in transferring the organisms without introducing contamination? ____________
Explain how you know.
2. Why is it important to keep culture tube caps slightly loosened during incubation?
3. Which of the three culture conditions (broth, deep, slant) is the only one in which you can make a judgment about oxygen use? Explain your answer.
4. If you accidentally introduced air bubbles while inoculating the deep how might that interfere with interpreting the organism’s oxygen requirements?
5. Which, if any, of the four organisms was able to grow anaerobically?
6. Which, if any, of the four cultures is an absolute aerobe?
7. Which, if any, the cultures are(is) facultative anaerobes?