2.1: Introduction to Streak Plate Techniques — Read

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Learning Objectives

Discuss the importance of creating streak plates in microbiology.
Define "colony" and understand how isolated colonies are formed.
- Discuss binary fission.
- Learn how to calculate generation times.
Characterize colony morphology based on colony shape, elevation, margin, size, and color.
Complete a Quadrant Streak, "Quad Streak", and describe the results.

How Microbes Grow

Microbes, like bacteria, are ubiquitous and found everywhere throughout life. We can support the growth of microbes through growth media, as described in "1.4: Aseptic Technique", and control their growth cycles. Manipulation of bacterial growth allows for clinical diagnosis and microbiology research.

Bacteria increase their cell population via a replication cycle called binary fission. Binary fission is characterized by 1 parent cell increasing their cell size, duplicating structural components, and copying their bacterial genome (DNA). Once a parent cell has copied all cell components and DNA, the center of the large parent cell will begin to constrict until a physical wall of separation, called a septum, is formed. The septum will cleave the cytoplasm, cell membrane, and cell wall forcing the enlarged parent cell into two new, separate daughter cells. Binary fission will increase a bacteria's population on growth media, creating colonies. .

A) A micrograph of two rod shaped cells attached at their ends. B) A diagram of binary fission. First a cell replicates its DNA and elongates. Then, as the cell continues to elongate, each loop of DNA travels to one end or the other. The cell then starts to constrict in the center. This results in two cells each containing a loop of DNA.

Figure 1. A) Bacteria replicate through a process called binary fission. B) One parent cell copies its genome and internal structures, splits its cell body with a septum, and produces two identical daughter cells.

A colony is a macroscopic mass of bacterial cells undergoing binary fission that is easily visible with the human eye. A single colony will be produced by a single bacterial cell replicating itself. Bacterial colonies from the same species should all have the same appearance as they contain exact copies of the same bacterial genome. Observation of these colony characteristics, or colony morphology, assists with identification of bacterial species in clinical samples. To properly observe colony morphologies, pure, isolated colonies are necessary. A pure, isolated colony is a single colony that does not touch other cell growth and is

not contaminated with other microbial species. Pure colonies can be produced by adhering to the rules of aseptic technique and using proper streak plate techniques.

isolated and non-isolated colonies on a petri plate

Figure 2. This plate shows bacteria streaked across 4 different quadrants. The bacteria at the top of the plate are in an area of lower cell density and are capable of forming colonies. Pure, isolated colonies should not touch other bacterial growth or be contaminated.

The rate of bacterial growth, binary fission, in a bacterial colony can be determined by a formula called Generation Time. Generation time in bacteria is the amount of time is takes for a bacterial population to go through one round of binary fission and double the total population number (which is why it may also be called the doubling time). All bacteria species have their own, unique generation time. For example, E. coli's generation time is 20 minutes whereas M. tuberculosis' generation time is 15 to 20 hours. The speed of population doubling has impacts on the pathogenicity of the bacterial species and potential host colonization. The rate of population growth and generation time are determined with the following formula:

Step 1:

Total Growth Time/Generation Time= Number of Generations

Step 2:

2^Number of Generations X (Number of Original Cells)= Total Number of Cells

For Example:

Escherichia coli, under optimum conditions, has a generation time of 20 minutes. If an experiment started with only 10 E. coli cells and allowed them to grow for 2 hours, what would be the total cell population at the end of the experiment?

**Make sure units of time match, hours to hours, minutes to minutes, etc. Convert units of time prior to using the growth formula.

2hours x 60mins = 120mins

120mins / 20mins = 6 generations

2^6 x 10 cells = 640 cells, The total cell population after 2 hours in 640 E. coli cells.

Colony Morphology

As bacteria replicate and produce colonies, these colonies will have unique growth patterns that can be used to identify the bacteria's species. The characteristics of these growth patterns are called the colony morphology and include:

Colony Form: overall general shape of the colony from an overhead view.
Colony Elevation: shape of the colony from a side-view.
Colony Margin: magnified observation of the colony's edge.
Additional Characteristics: size, color, and smell.

Figure 3. Colony morphology describes the appearance of one macroscopic bacterial colony. The above illustrations show common colony morphologies for overall colony shape (colony form), height of the colony (elevation), and specific pattern of the colony's edge (margin).

Figure 4. The above images show examples of real life colony morphologies. We can also compare colony color and size in addition to form and elevation to best identify unknown species.

Quadrant Streak Plate Technique

In order to produce pure, isolated colonies so colony morphologies can be observed, streak plate techniques are used. The purpose of streak plating is to spread bacteria across a media surface, decrease cell density as the bacteria are spread, and produce individual colonies. There are multiple methods of streak plating including zig-zag streaks, T-streaks, and quad streaks. In this course, we will complete the quadrant or quad streak plate technique.

The quad streak technique decreases bacterial cell density by streaking 4 separate zones on an agar plate.

Figure 5. To decrease bacterial cell density and produce isolated colonies, the quad streak can be completed following these 4 steps: Step 1: Bacteria from a stock plate should be spread in 1 quadrant of the plate in a back-and-forth (zig- zag) pattern. Step 2: A sterile loop is used to drag a small amount of bacteria from Quad 1 into Quad 2 and spread in a zig-zag pattern. Step 3: A sterile loop is used to drag a small amount of bacteria from Quad 2 to Quad 3 and spread in a zig-zag pattern. Step 4: A sterile loop is used to drag a small amount of bacteria from Quad 3 to Quad 4 and spread in a zig-zag pattern. Quad 4 should have the lowest bacterial density and produce isolated colonies.

To complete a Quad Streak, use a sterile inoculation loop or swab to collect a small sample of bacteria from a stock plate and spread it in 1 area (quadrant 1) of an agar plate in a tight zig-zag pattern. No more bacteria should be collected from the stock plate. Sterilize the inoculation tool and drag a small area of bacteria from quadrant 1 into a new, un-inoculated area of the plate (quadrant 2). Spread the bacteria across quadrant 2 in a tight zig-zag pattern. Sterilize the inoculation tool again, drag a small amount of bacteria from quadrant 2 into the new quadrant 3 area, and spread the bacteria in a less-tight zig-sag pattern to start and create space for individual colonies to form. Finally, sterilize the inoculation tool one last time, drag a small amount of bacteria from quadrant 3 into the new quadrant 4, and spread the bacteria in a loose zig-zag pattern to ensure proper space for isolated colony growth. After incubation, there should be a lot of bacterial growth in quadrant 1 as this region has the highest cell density (coming from the original stock plate sample).

Quadrant 4 should have the least amount of bacterial growth as cell density has been significantly decreased by streaking throughout the other quadrants and individual, isolated colonies should form.

Real-World Connections

Diagnosis of Strep Throat

Strep throat is the most common cause of bacterial pharyngitis in humans. This disease is caused by Streptococcus pyogenes, a Group A strep bacterium. This bacterium thrives in the warm, moist environment of the throat, and often induces sore throat, difficulty swallowing, and fever in patients. Despite being more common in children, it can affect people of all ages.

But how does a doctor determine whether a sore throat is caused by strep? There are two main types of tests that are used to check for strep A infections: the Rapid Strep Test and the Throat Culture Test. The Rapid strep test looks for strep A antigens, or the substances in the bacteria that initiate an immune response. This test can provide results in 10-20 minutes, but it is less reliable than the throat culture test. The throat culture test is considered the “gold standard” diagnostic test for strep throat. This test involves swabbing the back of a patient’s throat and streak plating the swab on an agar plate. Doctor’s can then determine if the patient is infected with Streptococcus pyogenes, based on the characteristics of the bacterial culture that grows in the plate. This often results in more accurate diagnosis; however, the culture must be incubated for 24 to 48 hours resulting in a slower return time for the diagnosis. This highlights the importance of microbiology techniques when diagnosing patients with a common bacterial infection, such as strep.

Figure 6. The above image shows Streptococcus pyogenes colonies grown on a blood agar plate. Observing the characteristics of the bacterial colonies can help identify an organism.

Attributions

"Microbiology Laboratory Manual: Labs, 1.8 Plating on Petri Plates for Isolation" by Dr. Rosanna Hartline, West Hills College Lemoore is licensed under, LibreTexts: Biology is licensed under CC BY-NC-SA 4.0

"Microbiology Labs II: Microbial Growth, Generation Time" by Dr. Gary Kaiser, Community College of Baltimore County, Catonsville Campus, LibreTexts: Biology is licensed under CC BY 4.0

"Microbiology Lab Manual: Experiment 4, Colony and Cell Morphology; Simple Stains" by Sheri Miraglia, City College of San Francisco, LibreTexts: Biology is licensed under CC BY-SA 4.0

"Microbiology Labs 1: 8, Bacterial Colony Morphology " by Jackie Reynolds , Dallas College, Richland Campus, LibreTexts: Biology is licensed under CC BY-NC-SA 4.0

"Medical Tests: Strep A Test" by MedlinePlus, Use of MedlinePlus Materials Statement is in the Public Domain

"Group A Strep Infection: Testing for Strep Throat or Scarlet Fever" by Center for Disease Control, CDC Material Statement is in the Public Domain

"Group A Strep Infection: Clinical Guidance for Group A Streptococcal Pharyngitis" by Center for Disease Control, CDC Material Statement is in the Public Domain

Image Citations

Figure 1, "Microbiology Textbook: Chapter 9, Microbial Growth" by OpenStax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0

Figure 2, "Microbiology Laboratory Manual: Labs, 1.8 Plating on Petri Plates for Isolation" by Dr. Rosanna Hartline, West Hills College Lemoore is licensed under, LibreTexts: Biology is licensed under CC BY-NC-SA 4.0

Figure 3, Modified From: