Bacteria Testing – Environmental Microbiology
What is Bacteria?
Bacteria, singular bacterium, are single celled organisms. The cell structure of bacteria is simpler than that of other organisms as there is no nucleus or membrane-bound organelles. Their genetic information is contained in a single loop of DNA. Some bacteria have an extra circle of genetic material called a plasmid. The plasmid often contains genes that give the bacterium some advantage over other bacteria, such as antibiotic resistance.
Importance of Bacteria
There are approximately 10 times as many bacterial cells as there are human cells in the human body. Many people know bacteria only as “germs,” invisible creatures that can invade bodies and cause illness. Few know that many bacteria not only coexist with humans all the time, but do an amazing array of useful things. A lot of these bacteria cells are found lining the digestive system to help maintain a healthy digestive system. Some bacteria live in the soil or on dead plant matter where, along with fungi, they play an important role in the cycling of nutrients. Additionally, bacteria can be used to make vitamins and insulin, and help clean up oil spills.
Some types of bacteria do cause food spoilage, food borne illness and crop damage but others are incredibly useful in the production of fermented foods such as yogurt and soy sauce. Relatively few bacteria are parasites or pathogens that cause disease in animals and plants.
Bacteria are a very large group of microorganisms comprising one of the three domains of living organisms (Archaea, Bacteria, and Eukarya). Bacteria are prokaryotic, unicellular, and either free-living in soil or water or parasites of plants or animals. Bacteria and their microbial cousins the archaea were the earliest forms of life on Earth. Cyanobacteria fossils date back more than 3 billion years. Bacteria are among the earliest forms of life that appeared on Earth billions of years ago. Scientists think that they helped shape and change the young planet’s environment, eventually creating atmospheric oxygen that enabled other more complex life forms to develop. Many believe that more complex cells developed as once free-living bacteria took up residence in other cells, eventually becoming the organelles in modern complex cells. The mitochondria that make energy in human body cells is one example of such an organelle.
Types of Bacteria
There are thousands of species of bacteria, but they can be broken down into five groups based on shape and two groups based on Gram reaction. Bacteria can exist as single cells, in pairs, tetrads, chains or clusters.
The five shapes of bacteria are:
- Spherical shaped, called cocci, like Staphylococcus sp., Streptococcus sp. or Micrococcus sp.
- Rod shaped, called bacilli, like Bacillus sp., Listeria sp., and Escherichia sp.
- Spiral shaped, called spirilla, like Spirillum sp.
- Comma shaped, called Vibrio, like Vibrio sp.
- Corkscrew shaped, called spirochaetes, such as Borrelia sp., Campylobacter sp., and Helicobacter sp.
The two groups based on Gram reaction are:
- Gram-positive, which stain purple due to a thick layer of peptidoglycan in their cell wall
- Gram-negative, which stain pink due to a thinner layer of peptidoglycan and an outer membrane
- Acinetobacter species – Gram-negative bacilli. Causes a variety of diseases, ranging from pneumonia to serious blood or wound infections, and the symptoms vary depending on the disease. Acinetobacter may also “colonize” or live in a patient without causing infection or symptoms, especially in tracheostomy sites or open wounds. Acinetobacter baumannii is becoming more important in hospital infections due to its antibiotic resistance.
- Bacillus anthracis – Gram-positive, endospore forming bacilli. Causes anthrax, a common and deadly disease in cattle and a potential bioweapon against humans. Infection occurs in three forms: Cutaneous (affecting the skin), Inhalational (in the lungs), and Gastrointestinal (in the digestive tract)
- Brucella abortus – Gram-negative bacilli. Causes breeding losses in livestock. Can be transferred from animal to human host.
- Campylobacter species– Gram-negative vibrio. Causes intestinal infections which are generally mild, but can be fatal among very young children, elderly and immunosuppressed individuals. It normally inhabits the intestinal tract of warm-blooded animals such as poultry and cattle, and is frequently detected in foods derived from these animals.
- Carbapenemase-Producing Enterobacteriaceae (CPE) – Gram-negative bacteria. Extremely antibiotic resistant bacteria because they produce the enzyme Carbapenemase which disables the drug molecule. They include common pathogens such as Escherichia coli, Klebsiella pneumoniae, Proteus sp. and Enterobacter sp. These organisms are some of the most common causes of many infections such as urinary tract infections, intra-abdominal infections and bloodstream infections.
- Clostridium botulinum– Gram-positive anaerobic spore forming bacilli. Produce neurotoxins that cause botulism. Botulism is characterized by blurred vision, slurred speech, difficulty swallowing, muscle weakness and dry mouth. In infants, the symptoms can also be lethargy and constipation. Without treatment, botulism can lead to paralysis of the arms, legs and respiratory muscles.
- Clostridium difficile– Gram-positive anaerobic spore forming bacilli. Often called C. diff, is a bacterium that can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon. It is difficult to prevent the spread of the illness due to the spores. Illness from C. difficile most commonly affects older adults in hospitals or in long-term care facilities and typically occurs after use of antibiotic medications.
- Clostridium tetani – Gram-positive anaerobic spore forming bacilli. The bacteria enters the body via puncture wounds and releases a powerful toxin causing the illness Tetanus, often called Lockjaw. Symptoms include muscle stiffness starting in the jaw, then the neck and arms, legs and abdomen. This illness is preventable by a vaccination.
- Cyanobacteria – Formerly incorrectly known as blue-green algae. Live in water, where they produce obtain energy through photosynthesis, thus producing large amounts of oxygen in the environment. They can occur in single cells, filaments of cells or colonies. Some blooms produce nerve or liver toxins.
- Escherichia coli (a.k.a. E. coli) – Gram-negative bacilli. Found in the environment, food, and intestines of animals and humans, where it helps digest food and produces Vitamin K. The “bad” strain of E. coli, O157:H7 causes severe foodborne illness.
- Klebsiella species – Gram-negative bacilli. Routinely found in the human nose, mouth, and gastrointestinal tract as normal flora; however, they can also behave as opportunistic human pathogens. Klebsiella pneumoniae can cause pneumonia, meningitis, and urinary tract infections.
- Lactobacillus bulgaricus – Gram-positive bacilli. Helps turn milk into cheese, yogurt, and other dairy products. Additionally, this bacteria helps to prevent food borne illness from other bacteria by neutralizing toxins and killing harmful bacteria by producing its own natural antibiotics.
- Legionella sp. – Gram-negative bacilli. The agent of Legionnaire’s disease and Pontiac fever. It is primarily caused by Legionella pneumophila. Currently there are fifty species of Legionella. Legionella occurs naturally in freshwater environments, including lakes, mud and streams. In building systems, it colonizes cooling towers, evaporative condensers, showers, recreational waters (spas) and fountains.
- Listeria monocytogenes – Gram-positive bacilli. It is the agent of listeriosis, a serious infection caused by eating food contaminated with the bacteria. The disease affects primarily pregnant women, newborns, and adults with weakened immune systems.
- Methicillin Resistant Staphylococcus aureus (MRSA) – Gram-positive cocci. An infection can begin as a minor skin sore and become a serious infections. It is difficult to treat because it is resistant to numerous antibiotics.
- Mycobacterium tuberculosis – Bacilli that are not categorized as Gram-positive or Gram-negative, instead categorized as Acid-Fast. Causes tuberculosis, a major killer from the past that has recently resurged with the advent of AIDS.
- Pseudomonas aeruginosa – Gram-negative bacilli. Infections usually occur in people in the hospital and/or with weakened immune systems. Infections of the blood, pneumonia, and infections following surgery can lead to severe illness and death in these people. Healthy people can also develop this infection when exposed to inadequately chlorinated hot tubs or pools.
- Rhizobium leguminosarum – Gram-negative bacilli. Induces nitrogen-fixing nodules on the roots of legumes such as peas, beans, clover and alfalfa to convert free nitrogen into a form the plant can use.
- Salmonella species – Gram-negative bacilli. Salmonella bacteria typically live in animal and human intestines and are shed through feces. Humans become infected most frequently through contaminated water or food. Symptoms may include diarrhea, fever, and abdominal cramps between 12 and 72 hours after infection. The illness usually lasts 4 to 7 days, and most individuals recover without treatment.
- Staphylococcus(a.k.a. Staph) – Gram-positive cocci. Most are harmless and reside on human skin and mucous membranes of humans. Some species, such as Staphylococcus aureus can cause serious skin infections and food borne illness.
- Streptococcus pneumoniae – Gram-positive cocci. Causes pneumonia, ear infections, sinus infections, and meningitis.
- Streptococcus pyogenes – Gram-positive cocci. One of the most frequent human pathogens. Causes Strep throat, Scarlet Fever, Impetigo and Cellulitis.
- Streptomyces griseus – Gram-positive spore forming bacilli. Found in the soil and produces the powerful antibiotic Streptomycin, which kills both the bacterium that causes the plague and the bacterium that causes tuberculosis.
- Thermus aquaticus – Gram-negative bacilli. A heat-loving bacterium from which scientists extracted the enzyme Taq polymerase which is used in molecular biology to amplify DNA through PCR (polymerase chain reaction).
- Vancomycin Resistant Enterococci (VRE) – Gram-positive cocci. Enterococci that have developed resistance to many antibiotics such as vancomycin and can cause serious infections, especially in people who are ill or weak. These infections can occur anywhere in the body. Some common sites include the intestines, the urinary tract, and wounds.
- Vibrio sp. – Gram-negative vibrio. Found in saltwater sources and several species of Vibrio are pathogens. Consuming food that is contaminated by Vibrio can result in diarrhea, vomiting and abdominal cramping. Vibrio can also cause serious life-threatening infections with those having a compromised immune system. Strains of Vibrio are known to cause gastroenteritis and septicemia, and Vibrio cholerae is known to cause cholera in humans.
What They Eat
Some bacteria are photosynthetic, which means they can make their own food from sunlight, just like plants. Also like plants, they give off oxygen. Other bacteria absorb food from the material they live on or in. Some of these bacteria can live off unusual “foods” such as iron or sulphur. The microbes that live in the gut absorb nutrients from the digested food.
Where They Are Found
Bacteria are found in every habitat on Earth: soil, rock, oceans and even arctic snow. Some live in or on other organisms including plants and animals including humans.
Bacterial movement refers to the self-propelled movement of bacteria; this is also referred to as motility.
There are several types of bacteria movement. The most common occurs by the use of appendages called flagella. A bacterium can contain a single flagellum, several flagella located at one or both poles of the cell, or many flagella all over the bacterial surface. Flagella can rotate clockwise or counterclockwise. When the motion is counterclockwise, multiple flagella can unite into a flagellar bundle that functions as a propeller. This occurs when the bacterium is moving towards a chemical attractant or away from a repellent. If the flagella turn in the opposite direction, the coordinated motion of the flagella stops, and a bacterium will move in an undirected and random way.
Spirochaete bacteria have flagella that are internal. The bacterium to twist around the axis of the filament. As a result, the bacterium literally screws itself through the fluid.
In a human host, disease causing bacteria can move along the surface of host cells. This movement is due to a bacterial appendage called a pilus. A bacterium can have numerous pili on its surface. These hair-like appendages act to bind to surface receptors and when withdrawn, pull the bacteria along the surface. Movement stops when a suitable area of the host cell surface is reached.
Prevention of Bacterial Illness
- Wash hands before eating, or touching eyes, nose or mouth
- Wash hands after touching anyone who is sneezing, coughing or blowing their nose.
- Don’t share things like towels, lipstick, toys, or anything else that might be contaminated with respiratory germs.
- Don’t share food, utensils or beverage containers with others
- Use disposable cloths or paper towels when possible
- Re-usable cloths should be disinfected after each use
- Wash brushes in a dishwasher regularly or clean with detergent and warm water after each use
- Use two buckets for mopping – one for detergent and the other for rinsing
- Mops and buckets should be cleaned, disinfected and dried after each use
- Keep the U-bend and lavatory bowl clean by flushing after each use
- Use a lavatory cleaner and brush every few days
- Keep the lavatory seat, handle and rim clean by using a disinfectant
- Hygienically clean baths and sinks frequently
- Use disinfectant to clean baths and sinks after having been used by someone who is ill
- Clean shower trays as above for baths and sinks
- If a shower hasn’t been used for a long period, let it run with hot water before using it
- Ensure food preparation surfaces are hygienically clean
- Use separate chopping boards for meat (including fish and poultry) and vegetables
- Ensure proper storage and cooking of all foods
- Wash and dry hands after handling high-risk foods such as raw meat
- Hygienically clean surfaces immediately after use
- Keep tiles and grout in good condition and clean them often
- Hygienically clean or launder the shower curtain frequently
- Clean floors regularly to remove visible dirt with warm water and detergent
- If soiled with vomit, urine or feces, the floor should be cleaned using a disposable cloth and warm water, then disinfected. Make sure the floor is dry before allowing children on it
- Periodically clean carpets and soft furnishings using a suitable product
- Carpets and furnishings can be hygienically cleaned by steam cleaning
- Curtains can be cleaned by laundering or disinfected by steam cleaning
- Keep pet food separate from human food
- Always wash hands after touching animals, their food, toys, cages and litter trays
- Dishes, utensils and tin openers used for pet food should be stored separately
- Clean hard or plastic toys by washing them and storing them once they’re clean and dry
- Some soft toys can be cleaned in the washing machine
- All toys and equipment should be added to a regular cleaning rotation
- Wash hands after handling dirty laundry
- To prevent the spread of germs, all underwear, towels and household linen should be washed at 60°C (140°F) or at 40°C (104°F) with a bleach-based laundry product
- Run the washing machine on empty once a week, either at a high temperature or with a chemical disinfectant to prevent the growth of bacteria
- Don’t leave laundry in the washing machine, as any remaining bacteria can multiply rapidly
- Foot-operated garbage cans are better for hygiene because they reduce the risk of hands picking up germs when they touch the bin lid
- Always wash hands after handling waste material
- Throw garbage away carefully to avoid attracting vermin and insects
All tests for bacteria use a nutritional broth or agar and incubation at a specific temperature to grow
the target organism. Sterile equipment and careful handling techniques are necessary to prevent
contamination of the sample.
Methods for Bacteria Testing
- Presence/absence (P/A) — The sample is added to a bottle containing broth and incubated. A color change and/or fluorescence indicates the presence of the target bacteria. Often used for E. coli, Coliforms and Enterococcus.
- Most Probable Number (MPN) — The sample is diluted and added to a series of tubes containing nutrient broth. The tubes are incubated and then examined for the presence of turbidity and/or gas. Often used for E. coli, Fecal Coliforms and Total Coliforms.
- Membrane Filtration (MF) — The sample is filtered and the filter is placed on a petri dish containing growth media. After incubation, the filter is examined for the growth of the target organism. Often used for E. coli, Fecal Coliforms and Total Coliforms.
- Plate count agar — The sample is mixed with an agar in a large petri dish and incubated. After incubation, the agar is examined for bacteria colonies. This test is usually used for total bacteria count.
Presumptive and Confirmation Tests
Two tests are necessary for most methods, a presumptive test and a confirmation test.
- Presumptive test — Uses growth media that facilitates the growth of the target organism. A positive result is an indication of the target organism but can include a false positive result. The P/A, MPN and MF methods are presumptive tests.
- Confirmation test — Uses media that is more selective for the target organism and sometimes uses a selective incubation temperature.
Ensuring Accurate Results for Microbiological Tests
To make sure the results are reliable, samples must be collected and preserved appropriately. Good laboratory technique is essential for accurate microbiological tests. The use of high-quality laboratory equipment and prepared media save time and minimize errors. Additionally, analysts must be highly trained and Quality Assurance procedures are essential.
How to Collect Bulk Samples for Bacteria Testing
- Wear suitable gloves
- Cut a small piece (about 4 square inches) of the suspect material (e.g., carpet, drywall, wallpaper, wood); taking care not to disturb the bacteria
- Place the sample inside a clean plastic bag
- Close the bag and label the sample appropriately
- Fill out the chain of custody form and send it together with the samples to the lab
How to Collect Swab Samples for Bacteria Testing
- Dry swabs are recommended for wet surfaces and wet swabs for dry surface
- Wear suitable gloves
- Remove swab from tube (If using swabs with a wetting agent, drain most of it on the sides of the tube before sampling)
- Swab the test surface by rolling the swab lightly back and forth. For quantification of the amount of bacteria on the test surface, swab a known surface area (for example, 100 square centimeters)
- After swabbing, insert the swab in the tube – Firmly close cap and label the sample appropriately
- Label the swab appropriately
- Fill out the chain of custody form and send it together with the samples to the lab
How to Collect Air Samples for Culturable Bacteria Testing
- Select suitable agar media for sampling, typically TSA.
- Various sampling pumps can be used, such as SAS or Anderson A6.
- Remove the lid of the plate and place in the sampling device.
- Ensure the pump has been calibrated to the appropriate flow rate
- Run the pump for the length of time to achieve the desired total sample volume.
- Label the bottom of the plates with appropriate information.
- Place the samples in a cooler/box ensuring the samples are not in contact with ice packs (to avoid having the samples frozen).
- Fill out the chain of custody form and send it together with the samples to the lab for incubation and identification of the resulting bacteria.
Key Points To Remember When Sampling:
- Use a permanent marker to label the samples.
- Complete a chain of custody with the relevant information.
- For air samples, record the flow rate and sampling time or the total air volume collected on the form.
- Secure the samples and the chain of custody form in a shipping container.
- For samples that do not require culturing, refrigeration is usually not needed when submitting the samples to the laboratory for analysis.
- Samples must be sent via Overnight Shipping and not sent for weekend delivery.
- When collecting samples, write down and include with the sample(s) the following information:
Name of collector Email address Company Date sample taken Mailing Address Date sample relinquished Telephone Number The type of analysis requested Turn-around time* *Note, same day analysis is not available for bacteria