Cell Culture:
What are colonies?
Why do they matter?
Bacteria are
Yeasts are
The importance of classification to microbiology
Microbes and other single celled organisms are classified and identified to distinguish them from other similar organisms by using specific criteria. The most important level of classification is the name of a species which need to be recognized consistently by all scientists.
Some of the things classification is based on are:
The science of naming and classifying organisms is called taxonomy and its based on organisms with similar characteristics, like the phylogenetic trees you saw on the last slide!
Taxonomy
Taxonomists use a structural hierarchy
Levels of classification
The common classification system has 8 levels of taxa; domain, kingdom, phylum, class, order, family, genus and species.
Domain: Highest rank in biological classification (see example image below)
Kingdom: Second level in biological classification.
Levels of classification
Phylum: Third rank in biological classification.
Class: Fourth level in biological classification.
Levels of classification
Order: Fifth level in biological classification.
Levels of classification
Family: Sixth level in biological classification.
Genus: Second last level in biological classification.
Levels of classification
Species: The last level in biological classification.
Species
Species: Important criteria!
Species
Species: Important criteria!
Classification in Microbiology
Due to the small size of bacteria and the visual appearance being similar by the naked eye there are different classification techniques needed!
What are colonies?
Each of these round dots are colonies of e. coli!
Why do organisms form colonies?
How do colonies communicate?
How do autoinducers participate in QS?
What is gram negative vs positive?
Gram-positive bacteria have a thicker outer membrane than gram-negative bacteria have.
What qualifies as an autoinducer?
How do you identify colonies?
Colony formation isn’t just fascinating—it’s also a key tool for scientists! By observing a colony’s visual characteristics, scientists can figure out what organism is growing.
Some common features used to identify colonies include:
These traits make it easier to identify and study different microorganisms!
Spot the Differences!
Take a look at these two cultures—they’re from different species. Can you identify the differences in their characteristics? Look closely at features like shape, color, size, surface appearance, and texture!
Bacteria Characteristics: K-12 E. coli
Physical Characteristics of Non-Transformed K-12 E. coli in Culture:
Bacteria Characteristics: K-12 E. coli
Can you guess why the culture temperature is 37℃?
Fungi Characteristics: Saccharomyces cerevisiae (Baker’s Yeast)
Physical Characteristics of Non-Transformed S. cerevisiae in Culture:
Fungi Characteristics: Saccharomyces cerevisiae (Baker’s Yeast)
Did you know that yeast grows on the surface of fruit? This is why breweries have to sterilize their fruit juices before they inoculate with their own alcohol-producing yeast!
Fungi Characteristics: Yeast
More on Colony Characteristics
Microorganisms can have diverse colony characteristics, also known as their colony phenotype. Here are some key features to observe:
More on Colony Characteristics
More on Colony Characteristics
Microorganisms exhibit a variety of colony phenotypes. Here are some additional features to observe:
Understanding these traits helps scientists identify and study different microorganisms
Identifying Characteristics: Practice
Take a closer look at the physical characteristics of the cells on the petri dishes on the next slides. Fill in the details based on your observations:
Use these characteristics to practice identifying and describing colonies!
Identifying Characteristics: Practice
Take a closer look at the physical characteristics of the non-transformed yeast strain. Fill in the details based on your observations:
Shape:
Side View:
Texture:
Surface:
Size:
Color:
Identifying Characteristics: Practice
Take a closer look at the physical characteristics of this transformed (engineered) bacteria strain. Fill in the details based on your observations:
Shape:
Side View:
Texture:
Surface:
Size:
Color:
Gram staining: Gram-positive bacteria vs Gram-negative bacteria
Another way to differentiate bacteria is gram staining which works by exploiting the differences in their cell walls. This can be used with other methods to identify bacteria (like molecular techniques i.e. PCR, quantitative PCR, genome sequencing and mass spectrometry).
Gram staining allows identification by:
This places microbes into a broad group which can narrow down the possible identity of the organisms.
Gram staining: Gram-positive bacteria vs Gram-negative bacteria
Gram-staining is useful to identify the structure of bacterial cell walls. The walls of cells will color differently depending on if they are positive or negative. Positive cells will be a purple or blue and negative will be pink or red!
However, this can also be used on other microbes! Like yeast!
Yeast cells (Candida albicans (left) and candida spp. (right)) gram stained.
Gram staining: Gram-positive bacteria vs Gram-negative bacteria
Gram-positive bacteria have a thick peptidoglycan cell wall and have no outer lipid membrane. These are called monoderms. These bacteria are typically staphylococci, streptococci and some listeria species.
The structure of the walls affect the cells ability to retain the crystal violet that is used for gram staining. This is because the peptidoglycan retains it!
Gram staining: Gram-positive bacteria vs Gram-negative bacteria
Gram-positive bacteria retain the complex that is created by the primary stain crystal violet and iodine. This binds to this negatively charged membrane (charge due to the presence of phosphodiester bonds between teichoic acid monomers) because this dye is positively charged. The iodine traps this crystal violet so it cannot be removed, this is termed a mordant! This complex is strong enough to stay even after they are washed with water or alcohol! The alcohol is termed a decolorizer in this procedure and will also remove the complexes from the gram-negative cells. These cells have 20% of their cell wall made of peptidoglycan so they are unable to retain the crystal violet and iodine complex. This is in comparison to the 80% composition of the peptidoglycan in the cell wall of gram-positive bacteria.
Gram staining: Gram-positive bacteria vs Gram-negative bacteria
Gram-negative cells will color pink and this is because of the counterstain called safranin! This is another positively charged dye which binds to the cell membrane.
The safranin binds to both types of cell but the crystal violet and iodine complex causes a different color (the dark purple or blue).
Gram staining: Gram-positive bacteria vs Gram-negative bacteria
Gram-negative bacteria have a thin peptidoglycan cell wall and they also have an outer layer membrane containing lipopolysaccharides. These cells are called diderms. These bacteria typically include enterobacter species, salmonella, escherichia and pseudomonas.
Gram staining: Gram-positive bacteria vs Gram-negative bacteria
Gram-negative bacteria have a thin peptidoglycan cell wall and they also have an outer layer membrane containing lipopolysaccharides. These cells are called diderms. These bacteria typically include enterobacter species, salmonella and pseudomonas.
Gram staining: Gram-positive bacteria vs Gram-negative bacteria
Gram staining has limitations because it does not actually allow you to reliably assess the phylogenetic relationships. This is because of convergent evolution, the second membrane characteristic of gram negative bacteria evolved many times over different lineages.
The Importance of Sterility in Cell Culture
Microorganisms love their growth media, like YPD agar for yeast and LB agar for E. coli—but so do other unwanted organisms! That’s why maintaining sterility is crucial to avoid contamination.
While E. coli and yeast are hardy and can often grow even with some contamination, other cell lines can be much more sensitive. In some cases, contamination can wipe out an entire culture. Even worse, accidentally growing harmful organisms can pose serious risks.
To stay safe and protect your experiments, always deactivate contaminated plates. Use bleach and an inactivation bag to safely dispose of them. Need help? Check out our instructions on how to deactivate plates!
The Importance of Colonies in Genetic Engineering
Colonies play a crucial role in genetics research and biotechnology. They allow scientists to introduce new genes into bacteria for various purposes, like studying gene function or producing valuable proteins.
A key reason colonies are so important is their growth phase! After hours of culture, colonies enter the log phase (or exponential phase), where cells reproduce quickly and are at their healthiest. This is the ideal stage for cell transformation.
If cells move beyond this phase, they may begin to burst or die, reducing the chances of a successful transformation. Older cells are also more vulnerable to DNA damage, making them less reliable for genetic engineering.
By working with colonies, scientists ensure that cells are in the same growth phase, which helps avoid contamination and improves the consistency and success of their experiments!
Steps to Growing and Identifying Colonies
1
Scientists start by preparing growth media, which is often selective to minimize contamination. Different microorganisms need specific “food,” so the media is tailored to their unique requirements.
Preparation of growth media
Streaking the organism
Next, scientists streak their organism onto the media—either re-streaking from a previous culture or using a stab. There are various streaking methods to maximize colony formation, and we’ll explore a few later!
2
3
4
Microorganisms grow at different rates and temperatures, so it’s essential to incubate them under the right conditions for optimal growth.
Microorganism incubation
After incubation, colonies should appear on your plate! Instead of long streaks, the colonies will look like small, dot-like formations.
Colony formation!
Example: Streaked Magenta E. coli
What is a Lawn?
A lawn is a dense, continuous growth of cells spread across the surface of the agar plate. It needs to be diluted using inoculation loops to isolate individual colonies. In the figure on the right, A shows an example of a lawn.
What Does a Colony Look Like?
A colony is a small, dot-like cluster of cells that forms when individual cells grow and divide in isolation. Colonies are typically obtained through streaking techniques. In the figure on the right, B shows an example of a colony.
A
B
Example: Streaked Cyan S. Cerevisiae
Example Organism: Saccharomyces cerevisiae, genetically engineered to fluoresce green and resist G418.
Culture Conditions: Grown on G418-selective YPD agar at 30℃.
Steps:
Plate 1
Plate 2
B
A
Why Use Streaking Patterns?
Streaking patterns dilute the cells to lower concentrations, allowing individual colonies to form. An example streaking pattern is shown on the stencil. This is called the four-quadrant pattern.
This is an example of the four-quadrant streaking method!
Beyond Temperature: Key Factors for Successful Cell Incubation
Humidity
Humidity is crucial for preventing the evaporation of growth media. Higher humidity helps keep your cell growth media (often with a jell-o-like texture) from drying out, ensuring the cells have the moisture they need to thrive.
Air Composition
Proper humidity and air composition, alongside temperature, are key to creating the perfect environment for your cells to grow!
Beyond Temperature: Key Factors for Successful Cell Incubation
Not all incubators are equipped with advanced components to regulate air composition and humidity—but don’t worry! There are simple and effective ways to manage these factors:
Invert Your Plates: Flip your plates upside down to prevent condensation from forming on the lid. Condensation can lead to water dripping onto your culture, which may compromise its growth.
Use a Humidity Chamber: Placing a humidity chamber over your petri dish helps minimize moisture loss from the agar, keeping your culture in optimal condition.
These easy adjustments ensure successful results, even with basic incubator designs!
The Streak-it Kit Experiment
Your Experiment
In this experiment, you’ll use the Streak-it Kit to:
This experiment will teach you fundamental microbiology techniques and the importance of genetic engineering tools in research!
Before Starting:
E. coli
Yeast
The Streak-it Kit Co-Culture
At this stage of the experiment, you’ll start your culture from either a stab or a solid culture. If you choose to work with a solid culture, make sure to save your best culture plates for future use! These plates can be stored in the incubator or placed in a clean plastic bag in the fridge to preserve them.
How to Select the Best Colony:
Selecting the right colony is key to ensuring the success of your co-culture experiment!
Glossary