9: ISOLATION BY STREAK PLATE
- Page ID
- 157069
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Demonstrate correct use of the streak plate technique to isolate individual bacterial colonies
- Evaluate culture purity based on colony separation and appearance.
BACKGROUND
When working with an unknown microorganism, the first step is to obtain a pure culture (a population of cells growing in the absence of any other species). On solid media, such as a nutrient agar plate, a pure culture often produces isolated colonies (visible clusters of cells that originate from a single cell). Because all the cells in an isolated colony come from one parent cell, they are considered genetic clones.
Our environment is full of microorganisms. Even a single stray cell accidentally introduced into a pure culture can grow and multiply until it outnumbers the intended organism. This is why microbiologists must carefully check that a culture is not mixed, but truly pure.
In medical settings, obtaining a pure culture is especially important. When samples are collected from patients, it is nearly impossible to pick up only the infectious agent without also collecting normal flora (the body’s natural microorganisms) or environmental organisms. To correctly identify the infectious agent, mixtures of organisms must be separated into pure cultures.
The classic way to demonstrate culture purity is to spread a sample on nutrient agar so that individual cells are deposited far enough apart to form isolated colonies. Each isolated cell divides repeatedly to form a visible colony. If colonies are separated from one another and all look alike, the culture is considered pure. To ensure purity, microbiologists select a single colony and transfer it to a fresh plate. Colonies that appear on this second plate are all derived from the original colony, meaning the cells are genetically identical.
To achieve this, microbiologists use a method called the streak plate technique. A sterile loop is dipped into a sample and drawn across the surface of a nutrient agar plate. Where the loop first touches the agar, many cells are deposited, and the resulting colonies will grow too close together to be isolated. As the loop is moved into new sections of the plate, fewer and fewer cells are deposited. Eventually, the cells are spread out enough that individual colonies can form without touching each other.
Your instructor will demonstrate the streak plate technique before you attempt it. Practicing the hand motion with an empty loop can be very helpful before working with live organisms.
Key point: It does not matter in which quadrant an isolated colony forms—what matters is that it is clearly separated from other colonies, with the full perimeter visible.
MATERIALS
(Per Group of 1)
1 Tube of mixed bacteria per bench
4 Nutrient agar plates (1 poured last class period, 3 poured today to use for next class period)
METHODS/PROCEDURES
1. Divide the bottom of your Petri dish into three sections (in the shape of a “T”), as demonstrated by your instructor.
2. Label your plate clearly,
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3. Using a sterile loop, pick up cells from the mixed culture and spread them carefully across the large top section of the plate. Avoid crossing back over areas you have already streaked.
4. Flame-sterilize your loop. Do not return to the culture tube. Instead, lightly drag the sterile loop once through the edge of section one to pick up a very small number of cells.
5. Spread these cells evenly across section two. Avoid crossing back over areas you have already streaked
6. Flame-sterilize your loop again. Drag it lightly once across section two to pick up a few cells.
7. Spread these cells across section three. Avoid crossing back over areas you have already streaked This section should contain very few cells, allowing isolated colonies to form.
8. Incubate the plate agar up until the next class meeting.
Next Class: You will select isolated colonies of each of the three colony types you observe and prepare a new T-streak plate with each one.
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.
ISOLATION BY STREAK PLATE
NAME ______________________
EXPECTATIONS
Do you think you'll have too many colonies or too few? In which quadrant of the plate do you think you will get the best isolated colonies, one, two or three?
RESULTS
Draw your T streak of the mixed culture after incubation.
At the following lab draw the three streak plates you made from the isolated colonies below. (Be careful to correctly label all your drawings.)
CONCLUSIONS
1. Were you successful at isolating all three cultures? If not, what could you do differently in the future to get better isolation?
2. Describe everything you can see that is different about the three types of colonies. Think about things like color, size, texture, shape, edges, and transparency.
3. If you had a broth mixture of three kinds of cells and one kind had capsules (or other sticky substances) on its surface, how might the capsules alter the effectiveness of a T-streak.
4. Imagine you are working with a broth culture into which you have just put exactly the same number of three different kinds of bacteria. In this growth medium one of the three bacteria grows much faster than the others. You make a T-streak from the broth right after you added the three cell types. The broth is left in an incubator and then four hours later you take another sample of the broth and make another T-streak. How would you expect the two T-streak plates to differ?