In this article, I'd like to introduce the remarkable connection between Gram staining and various properties of microorganisms that will be incredibly useful for beginners in this field. When studying microbiology, one of the first things we learn is Gram staining. You might think that this article will be a just another explanation of Gram staining(borering!), but you're in for a surprise. By reading this article, you'll gain a deep understanding of the relationship between Gram staining and essential microbial properties that you'll encounter in the field of food microbiology, such as resistance to chemical agents, secondary contamination in the environment, principles of selective media, the infectivity and toxin types of foodborne pathogens, and more, all connected!
In this article, I'm not simply explaining Gram staining. Countless blogs and websites are dedicated to describing the technique itself. Instead, I’m here to delve into how Gram staining relates, almost like a domino effect, to every other crucial property for those studying food microbiology. You’ll find this article truly one of a kind, offering insights you won't find anywhere else.
A Brief Understanding of Gram-Negative and Gram-Positive Bacteria
During the rainy season, puddles form here and there. In raindrops, there are generally no bacteria unless dust or dirt is mixed in. However, when rainwater falls to the ground and forms puddles, bacteria multiply rapidly. I investigated the number of bacteria in empty bottles during the rainy season and detected 2 × 105 cfu (colony forming units, a unit for measuring the number of viable microorganisms using the plate culture method) per milliliter of water. It is believed that bacteria proliferated by utilizing trace amounts of organic matter adhering to the bottle walls. Thus, during the rainy season, puddles form in places with limited sunlight, such as terraces, roadside shoulders, surfaces of plants, or shaded areas of buildings, where many bacteria that are not typically found in dry soil or on plant surfaces thrive. Gram-negative bacteria are water-loving aquatic bacteria that prefer such moist environments.
Gram-Negative Bacteria
Gram-negative bacteria include Escherichia coli and Salmonella, among others. These bacteria can adhere to surfaces such as shoe soles after using the toilet and are transported to humid soil or puddles during the rainy season, where their survival rate is significantly higher compared to dry, solid surfaces. Therefore, it can be said that these Gram-negative bacteria, including foodborne pathogens, are present in our surroundings during the rainy season, posing a higher risk of secondary contamination of food. For instance, pseudomonas and the Enterobacteriaceae family, such as Escherichia coli, thrive vigorously in spills and puddles, such as liquid spills in food factories or pools of water after cleaning.
Gram-Positive Bacteria
On the other hand, on land, there are many bacteria called Gram-positive bacteria with thick cell walls. Life that originated in the intertidal zone of the sea is believed to have branched and evolved into marine organisms and land organisms, and bacteria are thought to have evolved similarly into marine bacteria and land bacteria. Gram-positive bacteria, which have adapted to life on land, characterized by dryness and rapid temperature changes, include Staphylococcus epidermidis inhabiting the skin of animals and plants, and Micrococcus species, which are resistant to drying. In particular, two genera, Bacillus (including Bacillus subtilis and Bacillus cereus) and Clostridium (including Clostridium botulinum and Clostridium perfringens), produce heat-resistant spores, making them the ultimate land-adapted evolution bacteria.
In short, in wet environments, Gram-negative bacteria, which are water-loving, thrive and pose a higher risk of foodborne illnesses due to their presence. However, their susceptibility to environmental stressors reduces the potential for secondary contamination. Conversely, Gram-positive bacteria, with their thick cell walls, are more resilient to dryness and rapid temperature changes, making them well-suited to survive in dry environments such as factories. Understanding the characteristics of these bacteria can help us better comprehend their behavior and the potential risks they pose in various environments.
Survival in Dry and Wet Environments: Characteristics of Gram-Positive and Gram-Negative Bacteria
Now, we will discuss the characteristics and differences between Gram-positive and Gram-negative bacteria. Specifically, we will focus on the most significant point. Gram-negative bacteria, such as Escherichia coli, are not well-suited for dry solid surfaces. They are more closely related to aquatic bacteria that thrive in watery environments. As an example, when I occasionally investigate the presence of Escherichia coli contamination on the doorknobs of university restrooms, I rarely detect Escherichia coli. Instead, I find predominantly Gram-positive bacteria such as Staphylococcus and Bacillus. Unlike Gram-positive bacteria, Gram-negative bacteria like Escherichia coli are generally not well-adapted to dry surfaces, such as rubber or stainless steel in food factories.
Survival and Proliferation of Gram-Negative Bacteria
Environments where Gram-negative bacteria, including Escherichia coli, are more likely to survive and proliferate include cooling water tanks, wet floors, drains, and conveyor belts in food production lines where meat processing involves working for several hours with meat juices. While modern food factories with well-implemented sanitation practices can effectively eliminate Gram-negative bacteria like Escherichia coli, Gram-positive bacteria like Bacillus species can persist tenaciously.
Evolution of Gram-Positive Bacteria
Bacteria transitioned from water to land and evolved accordingly. Gram-positive bacteria can be considered to have adapted to survive temperature variations and dryness on land. Examples of Gram-positive bacteria are Staphylococcus species, which inhabit the skin of terrestrial mammals, and Bacillus species, which reside on the surfaces of plants on land. Another example, which we will discuss in detail in a separate article, is Clostridium botulinum, which can be considered a bacterium that has adapted to land environments. A common characteristic of Gram-positive bacteria is their ability to withstand harsh environmental conditions specific to land, such as high temperatures, dryness, and high salt concentrations, unlike Gram-negative bacteria such as Escherichia coli and Salmonella.
Regarding the relationship between high salt concentrations and land environments, some may question this connection. However, for instance, Staphylococcus species thrive on our skin as sweat evaporates, resulting in concentrated sweat that is exposed to salt concentrations much higher than seawater.
In short, Gram-negative bacteria, such as Escherichia coli, prefer wet environments and are less adapted to dry surfaces, while Gram-positive bacteria, such as Staphylococcus and Bacillus, have evolved to survive and thrive on land, with better tolerance to dryness and temperature variations. Understanding the characteristics and preferences of these bacteria can help us comprehend their behavior and the potential risks they pose in different environments.
Gram-Negative Bacteria as Seaweed and Gram-Positive Bacteria as Land Trees
To better grasp the differences between Gram-positive and Gram-negative bacteria, let's imagine the contrast between seaweed and trees on land. Land trees have a solid and sturdy image, while seaweed is physically flexible but covered with a slimy mucous membrane.
Whether it's bacteria or seaweed, it's important for aquatic organisms to have chemical barriers rather than just physical strength. The water contains various substances, including those harmful to living organisms. For aquatic organisms, it can be problematic if these substances easily enter their cells. Gram-negative bacteria, instead of simply developing a physically strong cell wall, have chosen to envelop themselves with an outer membrane as a chemical barrier, which can be easier to understand.
In contrast, land-dwelling bacteria don't face the same urgent need for a chemical barrier since their environment is not water-based. Understanding this difference can be easier by realizing that the necessity for a chemical barrier is not as crucial for land bacteria as it is for aquatic bacteria. Of course, this metaphorical explanation is aimed at helping beginners visualize the difference between Gram-positive and Gram-negative bacteria. Strictly speaking, the land environment is not always dry for bacteria. However, by imagining the contrast between seaweed and land trees, we can better comprehend the structures of Gram-positive and Gram-negative bacteria.
Differences in Cell Surface Structures
Next, I would like to briefly discuss the characteristics and differences in cell structures between Gram-positive and Gram-negative bacteria. Understanding their structural differences is closely related to their habitat, characteristics, and considerations for food hygiene management.
Gram-positive bacteria, which have a thick peptidoglycan layer in their cell walls, stain strongly in the Gram staining process. On the other hand, Gram-negative bacteria, which have a thin peptidoglycan layer but are covered with an outer membrane composed of lipopolysaccharides, do not stain as strongly.
Let's take a closer look at the cell surface structures of microorganisms. Bacteria have a cell membrane composed of phospholipids, which is common to all living organisms. While the cell membrane provides fluidity and convenience, it is fragile against physical impacts. By comparing animals, which have only a cell membrane, with plants, which have thick cell walls, we can quickly understand the difference in their resilience to physical impacts.
Now, bacteria, like plants, have evolved to have a cell wall outside the cell membrane. However, in the case of bacteria, there are different developmental processes of the cell wall evolution between aquatic Gram-negative bacteria and land-adapted Gram-positive bacteria.
Land-adapted Gram-positive bacteria have developed a strong and thick cell wall outside the cell membrane. On the other hand, aquatic Gram-negative bacteria do not have a significantly thick cell wall but have developed an outer membrane that can be considered as an additional layer of cell membrane.
Resistance to Chemical Substances: Differences in Cell Surface Structures
The differences in cell surface structures between Gram-positive and Gram-negative bacteria also directly correlate with their resistance to chemical substances. In general, Gram-positive bacteria are more susceptible to chemical substances, while Gram-negative bacteria are more resistant. In Gram-negative bacteria, the lipopolysaccharides in the outer membrane act as barriers to prevent the invasion of various chemical substances. On the other hand, the cell walls of Gram-positive bacteria are thick and physically strong but, structurally, they are relatively porous and provide little chemical barrier.
To illustrate this, imagine the cell wall of Gram-positive bacteria as a solid steel frame without concrete, like an unfinished building that allows for good ventilation. In contrast, Gram-negative bacteria can be compared to wearing a sturdy trench coat (the outer membrane) with a waterproof lining, even though it is thin and physically less robust.
Therefore, generally speaking, Gram-positive bacteria can be relatively easily inhibited in growth by antimicrobial agents compared to Gram-negative bacteria. This property is applied in selective culture media for the detection of bacteria in food, such as Escherichia coli and Salmonella. These selective media take advantage of the weaker nature of Gram-positive bacteria towards certain drugs.
To understand the resistance to chemical agents, let's review the cell surface structure of Gram-negative bacteria in more detail. As mentioned earlier, Gram-negative bacteria have a thin cell wall and are covered by an outer membrane that can be considered as an additional layer of cell membrane. Strictly speaking, this outer membrane is not simply a phospholipid bilayer like the cell membrane. The outer surface of the outer membrane contains polysaccharide chains. The outer membrane of Gram-negative bacteria consists of lipids and polysaccharides and is called lipopolysaccharide (LPS). These polysaccharide chains have a hydrophilic character. Therefore, LPS plays a role in repelling chemical agents with hydrophobic functional groups.
For Gram-negative bacteria, LPS is crucial in protecting themselves from chemical compounds. In the Gram staining process, the phenomenon where Gram-negative bacteria stain less with crystal violet means that the outer membrane of Gram-negative bacteria prevents the penetration of the chemical agent crystal violet. Hence, the fact that Gram-negative bacteria stain less with crystal violet indicates their resistance to chemical compounds with hydrophobic functional groups. Simply by observing the Gram staining results, we can understand the differences in resistance to chemical substances between these two types of bacteria.
In short, the differences in cell surface structures between Gram-positive and Gram-negative bacteria directly affect their resistance to chemical substances. Gram-positive bacteria have a thick but relatively porous cell wall, while Gram-negative bacteria possess an outer membrane that provides a strong chemical barrier. Understanding these differences helps us comprehend the varying degrees of resistance to chemical agents exhibited by different bacteria.
Infection-Type and Toxin-Type Foodborne Pathogens
The difference between Gram-positive and Gram-negative bacteria is also relevant in terms of foodborne pathogens. Foodborne pathogens can be divided into two types: toxigenic bacteria and infectious foodborne pathogens. Toxigenic bacteria cause illness through toxins they produce, which accumulate in the food and induce illness when consumed. In contrast, infectious foodborne pathogens harm humans directly. These bacteria invade the human body through the mouth, stomach, and intestines, attempting to penetrate the intestinal epithelial cells, which may lead to symptoms such as fever and diarrhea.
As the mechanisms of onset differ between toxigenic and infectious foodborne pathogens, the time and symptoms of onset also vary. In the case of toxigenic food poisoning, it typically takes several hours to a day after consuming food contaminated with toxins for symptoms to appear, with nausea being a primary symptom.
For instance, if you had a meal at a restaurant and unknowingly consumed food contaminated with Staphylococcus aureus enterotoxin, you might begin to feel nauseous within about three hours. This rapid onset within a few hours is characteristic of toxigenic food poisoning.
On the other hand, in the case of infectious foodborne illness, symptoms often take more than 10 to 24 hours to appear after consuming food contaminated with bacteria, with diarrhea being a common symptom. While toxins in toxigenic food poisoning cause immediate symptoms, in infectious cases, the invading bacteria first encounter the powerful sterilizing effect of stomach acid. Surviving bacteria that reach the intestines then compete with the resident intestinal microbiota for nutrients. Finally, they attach to the intestinal epithelial cells, causing harm. This extended process results in a delayed onset of symptoms. The intestinal epithelial cells, sensing the presence of these intruders, respond by reducing water absorption, leading to diarrhea as a form of self-defense.
As a typical example, imagine you had a meal and accidentally consumed food contaminated with infectious foodborne pathogens. In the case of toxigenic food poisoning, such as Staphylococcal intoxication, you might experience symptoms like nausea about three hours after eating. However, with infectious foodborne illness, symptoms typically do not appear within this short time frame. You could still enjoy going to karaoke or attending an after-party without feeling unwell.
Then you would return home feeling fine and go straight to bed. Symptoms would start to appear in the morning, and by the time you wake up to go to work, you might be suffering from severe diarrhea and fever. This pattern is typical of the onset time for infectious foodborne illness. As you can see, it differs from toxigenic food poisoning.
Now, let's explore the relationship between the differences in Gram-positive and Gram-negative bacteria and the categories of toxigenic and infectious foodborne pathogens.
In broad terms, Gram-positive bacteria are associated with toxigenic foodborne pathogens, while Gram-negative bacteria are more commonly linked to infectious foodborne pathogens. For example, bacteria such as Staphylococcus aureus and Clostridium botulinum, both Gram-positive, are toxigenic foodborne pathogens. Gram-negative bacteria like Salmonella, Escherichia coli, and Vibrio cholerae are typical infectious foodborne pathogens.
Let’s consider the reasons behind this distinction. As mentioned earlier, Gram-positive bacteria have primarily evolved to adapt to terrestrial environments. While their cell walls are physically robust, they are less frequently exposed to various water-soluble substances, unlike bacteria in aquatic environments. Consequently, their cell walls are relatively porous. This is a significant difference from Gram-negative bacteria, which are more suited to water-rich environments. The cell wall of Gram-positive bacteria is susceptible to the invasion of certain chemical agents but, at the same time, facilitates the secretion of extracellular enzymes and toxins. It is plausible that Gram-positive bacteria in terrestrial environments secrete enzymes or toxins into their surroundings without concern of them being washed away, especially when breaking down organic matter or attacking other organisms.
On the other hand, Gram-negative bacteria, which typically inhabit aquatic environments, generally do not secrete digestive enzymes or toxins readily into the extracellular space. Instead, they tend to adhere to their targets directly, whether attacking other organisms or decomposing dead matter. As mentioned previously, the lipopolysaccharides in the outer membrane of Gram-negative bacteria repel chemical agents, providing protection to the cells. In other words, they lack an efficient mechanism for exporting large proteins, such as toxin proteins, outside their cells.
In short, the differences between Gram-positive and Gram-negative bacteria are also relevant to their roles as foodborne pathogens. Gram-positive bacteria are often associated with toxigenic foodborne poisoning, while Gram-negative bacteria are commonly linked to infectious foodborne illness. Understanding these differences aids in comprehending the varying mechanisms of pathogenesis, the time it takes for symptoms to appear, and the nature of symptoms in foodborne diseases caused by these two types of bacteria.
Understanding the Differences between Gram-Positive and Gram-Negative Bacteria: A Summary of Key Learnings
In this article, I explained the characteristics of Gram-positive and Gram-negative bacteria and their relevance as foodborne pathogens. I discussed how Gram-negative bacteria are physically vulnerable but chemically resistant, while Gram-positive bacteria exhibit the opposite characteristics. I also mentioned that most infection-type foodborne pathogens are Gram-negative bacteria. In this section, we will summarize and organize this information.
However, it's worth noting that while we have described Gram-negative bacteria as inhabiting aquatic environments, in reality, most infection-type foodborne pathogens do not reside in the sea, rivers, or lakes. Instead, they colonize the intestines of mammals and birds. These animals' intestines can be considered as environments rich in water.
Let's clarify this point further. Although we have described Gram-negative bacteria as residing in water, many readers may know from experience that these bacteria can also be detected in meat and vegetables. Meat and vegetables, unlike water, are not part of aquatic environments but exist on land. Therefore, some readers may question the notion that Gram-negative bacteria live in water. To address this, let's delve into the explanation of terrestrial environments.
Terrestrial environments encompass a variety of conditions. Gram-negative bacteria do inhabit terrestrial environments as well. Even in terrestrial environments, such as densely developed forests, there are ample water droplets present. Similarly, meat and vegetables that contain a significant amount of moisture also serve as habitats for Gram-negative bacteria.
For instance, consider the scenario of a volcanic eruption. Immediately after the volcanic ash settles, the terrestrial environment becomes extremely dry, making it a harsh place for organisms to survive. Over time, mosses and lichens start to grow, followed by herbaceous plants. Subsequently, shrub forests emerge, and then the development progresses to sun-demanding trees in the form of deciduous forests. As the forest matures, sunlight gradually becomes scarce in the inner parts. In such forests, trees capable of growing with minimal sunlight, like shade-tolerant trees, thrive, ultimately transforming the forest into a shade-tolerant forest.
As a result, the interior of a mature forest becomes a water-rich environment, with various areas resembling the image of leaves in the diagram. The forest environment is moist, creating a favorable condition for Gram-negative bacteria to thrive.
To summarize, what readers should understand is that Gram-negative bacteria can be detected in vegetables and meat because these food items contain moisture. The initial explanation of Gram-negative bacteria inhabiting water environments was to provide a comprehensible introduction. However, in reality, Gram-negative bacteria can reside in areas on land that have a high moisture content.
In conclusion, we have discussed the differences in habitat, characteristics, and mnemonic devices for remembering Gram-positive and Gram-negative bacteria. Understanding these distinctions helps us grasp the unique properties of these bacteria as foodborne pathogens. While Gram-negative bacteria are associated with infection-type foodborne illness, it's important to note that their habitat extends beyond aquatic environments, encompassing moist terrestrial environments as well. By comprehending these aspects, we can enhance our understanding of the diverse ecological niches of Gram-positive and Gram-negative bacteria.
Gram Staining: Principles, Methods, and Procedures (Appendix)
In this section, we will briefly discuss the principles, methods, and procedures of Gram staining. While this information can be found in manuals and textbooks, beginners in food microbiology may find it helpful to understand the practical aspects of Gram staining. Please note that this presentation aims to provide an outline of the technique and is not an exhaustive experimental manual. Some procedural details will be omitted for brevity.
Principles of Gram Staining:
Gram staining is a fundamental staining technique used in bacteriology, including the field of food microbiology. Its principle is based on differentiating bacteria into two groups based on the thickness of their cell walls. By performing a series of staining and decolorization steps, we can distinguish between these two groups.
Procedure:
Initially, the bacterial cells are stained with a purple-colored dye called crystal violet, which stains the cell walls. At this stage, both Gram-positive and Gram-negative bacteria appear purple. However, it is important to note that Gram-negative bacteria have thinner cell walls and are less intensely stained.
The crystal violet dye is then fixed onto the Gram-positive bacteria's cell walls by applying iodine solution (Gram's iodine). This step helps to prevent the dye from being easily removed during subsequent washings.
Next, the stained cells are decolorized using alcohol. Gram-positive bacteria retain the purple color due to the thick peptidoglycan layer in their cell walls, making them less susceptible to decolorization. In contrast, Gram-negative bacteria are easily decolorized as their thinner cell walls allow the dye to be washed away.
To distinguish between the two groups more clearly, a counterstain is applied. The cells are stained with a pink-colored dye called safranin.
Gram-negative bacteria, which have been decolorized, take up the pink stain, while Gram-positive bacteria retain the original purple color.
Results:
As a result of the Gram staining procedure, Gram-positive bacteria appear purple, while Gram-negative bacteria appear pink.
