Environmental Sampling
Bacterial Sampling
In the realm of microbiology, bacterial sampling serves as a foundational technique across various disciplines. In public health, the identification of pathogenic bacteria present in water, soil, and air is paramount to preventing disease outbreaks. By meticulously analyzing these environmental samples, health professionals can implement timely interventions to safeguard communities. Ecological studies leverage bacterial sampling to unravel the intricate roles bacteria play within ecosystems, shedding light on their interactions with other organisms and their contributions to environmental balance. Furthermore, the biotechnology sector taps into bacterial sampling to discover strains with beneficial properties, aiming to harness them for industrial innovations and medical applications. Each of these endeavors underscores the indispensable nature of accurate and systematic bacterial sampling.
Dilution Series
Creating a dilution series stands as a pivotal procedure in microbiology, enabling researchers to reduce bacterial concentrations in samples to manageable levels. This systematic approach ensures that when samples are plated on agar surfaces, individual colonies can be accurately counted, typically aiming for a range of 30 to 300 colonies. The dilution series unfolds as a stepwise process: a measured portion of the original sample is mixed with a sterile solvent—often water or saline—to produce a less concentrated solution. This diluted sample then becomes the source for the next dilution step, and the process repeats, resulting in progressively lower bacterial concentrations. Such meticulous dilution is crucial; overly concentrated samples can lead to overlapping colonies, rendering accurate counts impossible. By controlling the dilution levels, microbiologists can back-calculate to determine the original bacterial concentration in the sample, ensuring precise assessments and fostering reliable research outcomes.
Figure 34 Dilution Series Creation
Colony Counting
Colony counting is a straightforward method used to estimate the number of bacteria in a sample. After creating a dilution series, where a bacterial sample is progressively diluted, small volumes from each dilution are spread onto agar plates.
Once these plates are incubated, each viable bacterium forms a visible colony. Counting these colonies helps determine the concentration of bacteria in the original sample. Ideally, you want to count colonies on a plate with 30 to 300 colonies. This range ensures accuracy; fewer than 30 colonies might mean the sample is too diluted, while more than 300 can result in overlapping colonies, making it hard to count them accurately.
To find the concentration of bacteria in the original sample, simply multiply the number of colonies by the dilution factor. For example, if you count 150 colonies on a plate that was a 1:1,000 dilution, the original concentration is 150,000 CFU/mL (colony-forming units per milliliter). This method is essential for understanding bacterial levels in everything from water quality testing to food safety.
Factors Affecting Bacterial Growth
Bacterial growth is highly dependent on several environmental and nutritional factors. Nutrient availability is one of the most crucial elements. Bacteria require essential nutrients like carbon, nitrogen, and various minerals to fuel their growth and reproduction. Without these key nutrients, bacterial proliferation is limited.
Temperature is another significant factor that influences bacterial activity. Different species of bacteria thrive in specific temperature ranges, leading to classifications such as psychrophiles, which prefer cold environments, mesophiles that thrive at moderate temperatures, and thermophiles that favor hot conditions. The optimal temperature range for each type directly affects their growth rates and overall survival.
The pH of the environment also plays a critical role. Most bacteria prefer a neutral pH, but some can adapt to acidic or alkaline conditions. For example, acidophiles thrive in low pH environments, while alkaliphiles prefer high pH conditions.
Oxygen levels further determine bacterial growth. Aerobic bacteria require oxygen to survive, while anaerobic bacteria thrive in its absence. Facultative anaerobes, however, have the unique ability to grow in both oxygen-rich and oxygen-deprived environments.
Water is necessary for metabolic processes and the transport of nutrients within bacterial cells. Without adequate moisture, bacteria may become dormant or die off. Understanding these factors is key to controlling bacterial populations in various settings, from healthcare to food production.
Controlling Microbial Growth
Principles of Control
Controlling microbial populations requires a thorough understanding of the principles behind microbial management. Sterilization is the most stringent method, aiming to remove or destroy all microorganisms and viruses from an object or environment. Achieving sterilization ensures that even the most resilient forms of microbes, like endospores, are eliminated, though prions are often not considered in this process.
Disinfection, on the other hand, aims to eliminate most pathogens but does not guarantee the removal of all microbial life. After disinfection, some microbes may still remain viable, though their numbers are significantly reduced. Disinfectants, which are chemical agents used on inanimate objects, play a key role in this process. These chemicals are designed to target specific types of microorganisms, such as bactericides for bacteria, fungicides for fungi, and viricides for viruses. When similar chemicals are formulated for use on living tissues, they are referred to as antiseptics, providing a safe way to reduce microbial presence on sensitive areas like skin and wounds.
Techniques for Control
A variety of techniques are employed to control microbial growth effectively. Decontamination is a broad approach that reduces pathogen numbers to safe levels, using methods like washing, heating, or applying chemical treatments. This process is vital in settings where complete sterilization is not necessary but where reducing microbial presence is essential for safety.
Sanitization takes decontamination a step further by substantially lowering microbial populations to meet health standards. This process is widely used in food preparation and public spaces to minimize the risk of disease transmission. It’s important to note that sanitization does not achieve a specific level of microbial control but ensures that standards are met to prevent outbreaks.
Preservation techniques are used to delay the spoilage of perishable products. This can be accomplished by adjusting storage conditions to slow microbial growth or by adding bacteriostatic preservatives, which inhibit bacterial proliferation. One of the most well-known preservation methods is pasteurization. This process involves briefly heating a product to reduce the number of spoilage organisms and eliminate pathogens without altering the product’s taste or texture.
Methods of Microbial Control
Physical Methods
Physical methods are the cornerstone of microbial control, providing reliable and often straightforward ways to reduce or eliminate microorganisms. Heat is the most widely used method, with moist heat techniques like autoclaving, boiling, and pasteurization being particularly effective. Moist heat works by denaturing proteins in the microorganisms, leading to their destruction. On the other hand, dry heat methods, such as incineration and hot air sterilization, sterilize by oxidizing cellular components.
Filtration is another essential physical method, particularly useful for removing microorganisms from both liquids and air. High-Efficiency Particulate Air (HEPA) filters are integral to air purification systems, especially in environments requiring sterility, like hospitals and laboratories. Radiation offers a powerful tool as well, with ionizing radiation (e.g., gamma rays and X-rays) causing direct DNA damage, while non-ionizing radiation, such as ultraviolet (UV) light, induces thymine dimers in DNA, disrupting replication processes. Additionally, controlling temperature through refrigeration or freezing can effectively slow microbial metabolism, limiting their growth. Desiccation and osmotic pressure methods, involving the removal of water through drying or the use of high sugar or salt concentrations, further inhibit microbial proliferation by depriving cells of the moisture they need to thrive.
Chemical Methods
Chemical control methods provide a versatile and targeted approach to managing microbial populations. Disinfectants and antiseptics are critical tools; disinfectants like alcohols, phenolics, halogens, and quaternary ammonium compounds are used on inanimate surfaces, while antiseptics are applied to living tissues to prevent infection. Antibiotics play a central role in combating bacterial infections, specifically targeting bacterial cell wall synthesis, protein synthesis, or other critical functions.
Sterilants and high-level disinfectants, such as ethylene oxide and glutaraldehyde, are indispensable in sterilizing medical equipment, ensuring that instruments are free from all forms of microbial life before use. Heavy metals, though used in smaller quantities, are also effective; silver and mercury, for instance, exhibit antimicrobial properties that disrupt microbial cell functions even in tiny amounts.
Mechanisms of Action
The effectiveness of antimicrobial agents lies in their specific modes of action against microbial cells. For example, cell wall-targeting agents like penicillins interfere with cell wall synthesis, making the bacterial structure weak and leading to cell lysis. Alcohols and detergents, on the other hand, disrupt cell membranes, causing the contents of the cell to leak out and leading to cell death.
Some agents inhibit essential biological processes, such as protein and nucleic acid synthesis, by binding to ribosomes or DNA, preventing replication and transcription. Others, like sulfa drugs, inhibit critical metabolic pathways, such as folic acid synthesis, which is vital for bacterial growth. By understanding these mechanisms, the application of antimicrobial agents can be optimized for maximum efficacy.
Evaluating Antimicrobial Agents
Evaluating the effectiveness of antimicrobial agents is crucial to ensure they perform as expected in various settings. The Phenol Coefficient Test is a traditional method that compares the effectiveness of a disinfectant to phenol, providing a standard benchmark. The Disk Diffusion Method, commonly known as the Kirby-Bauer Test, involves placing disks infused with antimicrobial agents on bacterial cultures and measuring the zones of inhibition, which indicate susceptibility.
The Minimum Inhibitory Concentration (MIC) is another critical measure, determining the lowest concentration of an antimicrobial agent that can prevent microbial growth. The Use-Dilution Test assesses the effectiveness of disinfectants on specific surfaces, ensuring that they can effectively sanitize materials in real-world conditions.
Resistance to Antimicrobial Agents
Microbial resistance presents a significant challenge in the realm of infectious disease management. Intrinsic resistance arises from a bacterium's inherent structural or functional traits, rendering certain agents ineffective. In contrast, acquired resistance develops through genetic mutations or horizontal gene transfer mechanisms like conjugation, transformation, or transduction, enabling bacteria to adapt and survive in the presence of previously effective antimicrobial agents.
Application in Healthcare and Industry
In healthcare, microbial control is vital to preventing healthcare-associated infections (HAIs). Sterilization and disinfection protocols are strictly enforced to maintain patient safety. Additionally, antibiotic stewardship programs are critical in managing the use of antibiotics, helping to slow the development of antibiotic resistance.
The food industry relies heavily on microbial control methods such as pasteurization, proper cooking, and preservation to ensure the safety of food products. Chemical preservatives are often used to inhibit the growth of spoilage organisms and pathogens. In the biotechnology sector, maintaining sterile conditions in laboratories and production facilities is essential for producing safe and pure biological products, highlighting the importance of effective microbial control across a wide range of industries.
Common Bacteria in Food, Soil, and Air
Below is a list of common bacteria categorized by their association with food, soil, and air. These bacteria are identified and differentiated using various biochemical tests such as catalase, coagulase, oxidase, EMB, sorbitol, mannitol, and TSI.
Food-Associated Bacteria
1. Escherichia coli (E. coli)
Tests:
Catalase: Positive
Oxidase: Negative
Coagulase: Negative
EMB: Produces a metallic green sheen (strong lactose fermenter)
Sorbitol: Pathogenic strains like E. coli O157 are sorbitol-negative; others are sorbitol-positive
Mannitol: Typically negative
TSI: Yellow slant/yellow butt with gas production
Associated with: Contaminated water, undercooked beef (especially ground beef), raw vegetables, unpasteurized milk, and dairy products.
Significance: E. coli includes both harmless strains (e.g., those naturally found in the human gut) and pathogenic strains like E. coli O157, which can cause severe foodborne illness, including bloody diarrhea and kidney failure.
2. Staphylococcus aureus
Tests:
Catalase: Positive
Oxidase: Negative
Coagulase: Positive
Mannitol: Positive (turns mannitol salt agar yellow)
Associated with: A wide range of foods including meats, poultry, dairy products (especially unpasteurized milk and cheese), salads (such as egg, tuna, chicken, and potato salads), cream-filled pastries, and sandwiches.
Significance: Staphylococcus aureus is a major cause of food poisoning. It produces enterotoxins that can cause symptoms like nausea, vomiting, stomach cramps, and diarrhea within a few hours of ingestion. The bacteria can contaminate food through improper handling, especially by food handlers who are carriers of the bacteria.
3. Salmonella species
Tests:
Catalase: Positive
Oxidase: Negative
Coagulase: Negative
EMB: Colorless colonies (lactose non-fermenter)
TSI: Red slant/yellow butt with H2S production (black precipitate)
Associated with: Poultry, eggs, raw meat, dairy products, and sometimes raw fruits and vegetables.
Significance: Salmonella is a leading cause of foodborne illness, resulting in salmonellosis. Symptoms include diarrhea, fever, and abdominal cramps. In severe cases, it can lead to hospitalization and even death.
4. Listeria monocytogenes
Tests:
Catalase: Positive
Oxidase: Negative
Associated with: Dairy products, raw vegetables, ready-to-eat meats.
Significance: Causes listeriosis, a serious infection, particularly dangerous for pregnant women, newborns, and immunocompromised individuals.
5. Bacillus species
Tests:
Catalase: Positive
Oxidase: Variable
Coagulase: Negative
Mannitol: Some species positive (turns mannitol salt agar yellow)
Associated with: Rice, pasta, dairy products, spices.
Significance: Produces toxins that can cause foodborne illness.
6. Pseudomonas species
Tests:
Catalase: Positive
Oxidase: Positive
Associated with: Fresh meats, dairy products, vegetables, seafood.
Significance: Known for spoilage, particularly in refrigerated foods, due to their ability to grow at low temperatures and produce off-flavors and odors.
7. Campylobacter species
Tests:
Catalase: Positive
Oxidase: Positive
Associated with: Poultry, raw milk, untreated water.
Significance: A leading cause of bacterial foodborne illness, often linked to undercooked poultry and cross-contamination.
8. Shigella species
Tests:
Catalase: Positive
Oxidase: Negative
Coagulase: Negative
TSI: Red slant/yellow butt with no gas or H2S production
Associated with: Contaminated food and water, particularly in areas with poor sanitation. Common in raw vegetables, salads, and foods handled by infected persons.
Significance: Shigella is highly infectious and causes shigellosis, which can lead to severe diarrhea, fever, and stomach cramps. It is spread through the fecal-oral route, often due to contaminated food or water.
Soil-Associated Bacteria
1. Bacillus species
Tests:
Catalase: Positive
Oxidase: Variable
Coagulase: Negative
Mannitol: Some species positive
Associated with: Decaying organic matter, compost.
2. Clostridium species
Tests:
Catalase: Negative
Oxidase: Negative
Coagulase: Negative
Associated with: Soil, particularly in anaerobic environments.
3. Pseudomonas species
Tests:
Catalase: Positive
Oxidase: Positive
Associated with: Soil, particularly in areas with high organic content.
4. Enterobacter species
Tests:
Catalase: Positive
Oxidase: Negative
EMB: Pink or purple colonies (lactose fermenter)
Associated with: Soil, especially in areas with organic matter.
5. Proteus species
Tests:
Catalase: Positive
Oxidase: Negative
TSI: Red slant/yellow butt with H2S production (black precipitate)
Associated with: Soil, decaying organic matter.
6. Staphylococcus aureus
Tests: Same as in the food list.
Associated with: Soil contaminated with human or animal waste.
7. Salmonella species
Tests: Same as in the food list.
Associated with: Soil, especially in areas with animal waste.
8. Escherichia coli (E. coli)
Tests: Same as in the food list.
Associated with: Soil contaminated with fecal matter.
Air-Associated Bacteria
1. Staphylococcus aureus
Tests: Same as in the food list.
Associated with: Indoor environments, especially where there is human activity, such as hospitals, homes, and workplaces.
Significance: Staphylococcus aureus can be airborne, especially in areas where infected individuals or carriers are present. It is significant as a potential pathogen and can cause respiratory infections or skin infections if it lands on compromised skin.
2. Staphylococcus epidermidis
Tests:
Catalase: Positive
Oxidase: Negative
Coagulase: Negative
Mannitol: Negative (leaves mannitol salt agar red)
Associated with: Air, especially in hospital environments.
3. Micrococcus species
Tests:
Catalase: Positive
Oxidase: Positive
Associated with: Air in both indoor and outdoor environments, particularly in areas with high dust levels or where there is decaying organic matter.
Significance: Generally considered non-pathogenic, Micrococcus species are commonly found in air and can contribute to the natural microbial flora of indoor environments.
4. Bacillus species
Tests: Same as in the soil list.
Associated with: Air, particularly in agricultural environments where spores can become airborne. Dust particles, soil, and decaying vegetation that become airborne.
Significance: Bacillus spores are hardy and can be easily dispersed through the air. Some species are harmless, while others, like Bacillus cereus, can cause foodborne illness if they contaminate food.
5. Pseudomonas species
Tests: Same as in the food list.
Associated with: Airborne water droplets, soil particles, and decaying plant matter.
Significance: Pseudomonas species are known for their role in biodegradation and can be found in the air, especially near water sources or in moist environments. They are important in breaking down organic pollutants and can sometimes be associated with respiratory infections in immunocompromised individuals.
6. Enterobacter species
Tests: Same as in the soil list.
Associated with: Air, especially in areas with decaying organic matter.
7. Escherichia coli (E. coli)
Tests: Same as in the food list.
Associated with: Rarely airborne, but can be aerosolized in specific conditions.
Lab Experiment
Objective
To understand the environment which lived in and shared with humans by, culturing and identifying microbes from different sources (foods, air, soil, and water) that is in regular contact in society.
Materials
Sterile nutrient broth tubes
Sterile nutrient agar plates
Sterile swabs
Sterile pipettes
Sterile water
Bunsen burner
Inoculating loops
Incubator (37°C)
Glass slides
Cover slips
Gram stain reagents (crystal violet, iodine, ethanol, safranin)
Microscopes
Biochemical test kits (e.g., oxidase, catalase)
Labels and markers
Chosen variable items (foods, soils, water)
Procedure
Sample Collection (Creation of Original Broth)
1. Place approximately 1 gram of each item below in a tube of nutrient broth and incubate at 37°C for 24-48 hours.
o Foods:
Meat (e.g., beef, chicken)
Produce (e.g., lettuce)
Prepared Deli Food (e.g., potato salad, macaroni and cheese)
o Soil:
Potting Soil
Natural Soil (e.g., campus grounds)
Beach Sand
o Water:
Tap Water
River Water
Reverse Osmosis (RO) Water
2. Place open nutrient agar plates in three different locations for 48 hours at (e.g., classroom, hallway, outdoors).
Create a Dilution Series from Original Broth
1. Swab an air plate and inoculate a nutrient broth, incubate at 37°C for 24-28 hours. Repeat for each air plate.
a. This will delay the dilution series for the air samples.
2. For each sample (food, soil, water, air) label 4 microcentrifuge tubes with the following labels;
a. Undiluted, 101, 102, 103
3. Take 1 mL of the broth culture from the original sample and transfer it to an empty microcentrifuge tube labeled undiluted.
4. Transfer 0.1 mL from the undiluted tube and transfer it to the 10-1 tube containing 0.9 mL of sterile water to create a 1:10 dilution.
a. Mix the solution thoroughly to ensure even distribution of bacteria.
b. Subsequent Dilutions:
i. Take 0.1 mL from the first dilution (101) and transfer it to a second dilution blank containing 0.9 mL of sterile water. This creates a 1:100 dilution (or 102).
c. Repeat this process as needed to achieve further dilutions, such as 103 (1:1000), 10-4 (1:10,000), etc.
d. Serial dilution process
i. Original Broth (undiluted): 1 mL (total volume) = Undiluted
ii. 1st Dilution: 0.1 mL from the undiluted sample + 0.9 mL sterile water = 1:10 (101)
iii. 2nd Dilution: 0.1 mL from the 1:10 dilution + 0.9 mL sterile water = 1:100 (102)
iv. 3rd Dilution: 0.1 mL from the 1:100 dilution + 0.9 mL sterile water = 1:1000 (103)
5. Plating the Dilutions
a. After creating your dilution series, plate a small, consistent volume (0.1 mL) from each dilution onto separate nutrient agar plates. This will allow the bacteria to grow and form colonies.
b. Label each plate with the corresponding dilution factor to keep track of which plate corresponds to which dilution.
c. Using a sterile pipette, transfer 0.1 mL from each dilution onto the surface of the labeled nutrient agar plates.
d. Spread the inoculum evenly over the surface of the agar using a sterile spreader.
6. Incubate the lawn plates at 37°C for 24-48 hours.
Colony Counting
1. After incubating your agar dilution plates, you'll see individual bacterial colonies that have grown from the bacteria in your sample. Each colony originated from a single bacterium or a group of bacteria, so the number of colonies represents the number of viable bacteria that were in the portion of the sample you plated.
2. Count the colonies on plates that have between 30 to 300 colonies. Plates with fewer than 30 colonies may not be statistically reliable, and those with more than 300 colonies can be difficult to count accurately due to overcrowding.
3. Calculate the CFU per mL
o Use the following formula:
§ CFU/mL = (Number of colonies counted x (Dilution factor)) / (Volume plated)
§ Number of colonies – This is the total number of colonies on the individual dilution series plate.
§ Volume Plated – This is the volume of the diluted sample that you spread onto the agar plate. It’s usually 0.1 mL.
§ Dilution Factor – The dilution factor is the extent to which the original sample was diluted. For example, if you took 1 mL of your original sample and mixed it with 9 mL of sterile water, you created a 1:10 (or 101) dilution. If you repeated this process, the next dilution would be 1:100 (or 102), and so on.
o Example Calculation:
§ Suppose you counted 150 colonies on a plate where you plated 0.1 mL of a 1:1000 dilution.
§ The calculation would be:
§ CFU/mL = 150 × (103) / (0.1)
§ CFU/mL = 150,000 / 0.1
§ CFU/mL = 1,500,000
§ This means there are approximately 1.5 million viable bacteria per milliliter in the original sample.
Streaking from Original Broth
After incubation of the original broth, observe the growth.
Name, date, and Label 12 agar tubes of each type, MacConkey, Sorbitol, Mannitol.
Using a sterile inoculating loop, perform streaking on selective media tube slant from each broth culture and air sample.
Incubate the streaked plates at 37°C for 24-48 hours.
Staining of Colonies
After incubation of the media tubes, prepare a smear from colonies grown on the media to a glass slide.
Perform Gram staining for each for each culture that is grown.
Observe the stained slides under a microscope to determine Gram reaction and morphology.
Perform an endospore stain for each culture that is grown.
Observe the stained slides under a microscope to determine endospore formation, which may mean that the bacteria is in the bacillus genus (B. cereus).
Additional Testing of Colonies
After incubation of the media tubes inoculate a TSI test for each colony to help confirm potential pathogenic bacteria, such as Shigella, E. coli, Salmonella.
After incubation of the media tubes perform simple biochemical tests (oxidase, catalase, coagulase) on colonies formed. Coagulase positive may mean a Staphylococcus like S. aureus. Catalase positive could be Shigella, E. coli, or Salmonella.
Data Recording
Record the variables of the Environmental Sampling
2. Record observations of colony morphology, Gram stain results, and endospore.
3. Record observations of biochemical test results.
4. Record colony types, colony count, and CFU
5. Compare the findings to reference materials to tentatively identify the bacteria.
Review Questions
1. What is the primary purpose of creating a dilution series when working with bacterial samples?
2. Why is it important to incubate nutrient agar plates at 37°C for 24-48 hours after plating the dilution series?
3. How do you calculate the Colony-Forming Units (CFU) per mL from a dilution series? Provide the formula.
4. What factors can influence bacterial growth in environmental samples?
5. Describe the procedure for performing a Gram stain on isolated bacterial colonies.
6. What are some common biochemical tests used to identify bacteria in environmental samples, and what do they test for?
7. Why is it necessary to obtain isolated colonies from a bacterial sample before performing Gram staining and biochemical tests?
8. Explain the role of environmental sampling in public health and how it helps prevent disease outbreaks.