5. Environmental Microbiology (Part I) Introduction Microorganisms have colonized every single environment on the planet from the ice caps to volcanoes and deserts. They are found floating in the air on dust particles, in fresh and salt water, on the surface of objects, clothes, tables, floors, and in soil. They live in close associations with other living organisms: protozoans, fungi, plants and animals. They constitute the normal flora of the body and are essential to good health. In hospitals, however, where susceptible patients must be protected from hospital-acquired (nosocomial) infections, the concentration and the distribution of microorganisms are a matter of great importance. Frequent monitoring of the environment is one of the responsibilities of the hospital epidemiologist who may be a microbiologist, nurse, or physician. Pathogens may be spread if they contaminate surfaces and medical devices. Fungal molds and spores can be toxic or cause severe allergic reactions. The successful growth of microorganisms in the lab depends on the recreation of the conditions in which they normally grow. Temperature, nutrients, pH, and other physical and chemical conditions must meet the requirements of the organisms. Only a small percentage of known microbes can be grown in the lab. This is known as culture bias. Identification of microorganisms starts with the description of the physical appearance of the colonies formed on agar. Colorful colonies are frequently isolated from the environment where the organisms are exposed to potentially damaging sun rays. Pigments such as carotenoids and melanin protect cells against the damages of intense light. Other pigments play a role in photosynthesis. A few compounds are colored because they contain metals or conjugated double bonds. In this case, color does not reflect a physiological role. Large colonies with irregular borders signal that the organism is probably motile. Slimy colonies are often formed when capsules are present. Bacteria are not the only microorganisms that will grow on the Petri dishes. Mold and yeasts are also abundant in the environment. In this lab, several types of environments are sampled for the presence microorganisms. Air, selected areas in the college, and body tissues will be tested. The Petri plates will be incubated at temperatures close to the temperatures in which the microorganisms are routinely found. Next session, we will compare the results as a class.
Relationship to Class Instruction: Chapters 3, 4 and 17.
Purpose of Laboratory 
Isolate microorganisms from a wide variety of sources and describe their colonial morphology.
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Results will be analyzed in lab 6.
Material and Methods Tryptic Soy Agar (TSA) Petri plates, 2 plates per group Blood Agar Petri plates are used for samples from throat or nose only; 1 per group Sterile cotton swabs Sterile tubes to collect liquid samples Sterile distilled water tubes to moisten swabs
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Hazardous Waste Container
Procedure Special Safety Precautions Discard all biohazard material appropriately: Petri dishes in the biohazard bag; swabs in the biohazard bag
Air Exposure 1. All Petri plates are labeled with initials of lab partners, today’s date, session, and experiment (air or environment.)
intervals: 0 min. (control), 30 min., 60min., 90min., 120min., 180min. Results from the whole class will be recorded to compare extent of microbial contamination versus time of exposure.
2. Pick 2 TSA Petri plates per lab group.. One set of TSA plates, labeled Air, is used to monitor the contamination of plates as a function of length of exposure to air. Different times of exposure are written on the plates. Each group will leave the plate open to the air for the time interval written on the bottom of the plate. Plates will be exposed to air for the following time
3. Open the plate for the length of time written on your plate. Record both the time the plate was open and the time the plate was covered in lab notebook. 4. All plates will be exposed at the back of the classroom in the same location to minimize variability.
Dry Surfaces: e.g. Floor 5. Label the second TSA agar plates as indicated above. Divide plate into 4 quadrants by drawing with a Sharpie 2 intersecting lines on the bottom of the plate. Mark one sections Finger. Choose the environment to be tested for the 2nd, 3rd and 4th sections. Pick a sample to swab and mark the quadrant with your choice. Suggestions: lab bench, doorknobs, eyewash, etc…Mark your environment on the plate and record it in you notebook.
7. After taking a sample as described above, gently rub the swab over the agar in a zigzag motion. The instructor will demonstrate. Remember to work fast and leave the plate open only long enough to inoculate the medium.
6. To test a dry surface, place a swab into a tube of sterile water to moisten. As you withdraw the swab, press it against the inner wall of the tube and express excess fluid
9. Finger: gently touch your section with the thumb from your dominant hand.
8. Example: culture off the floor (swab a 10mm square area, ½″). Inoculate the 2nd quadrant of the TSA agar plate. Discard the swab in the hazardous waste. Repeat for all dry locations of your choice
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Moist Surfaces 1. Use a dry swab.
3. Moisten a dry swab, squeeze out extra liquid and inoculate a plate.
2. Collect liquid: eyewash, water fountain or faucet water in an empty sterile tube.
Throat and Nose 1. Draw a line on the bottom of the plate to divide it into 2 sections. Each section will be used for a swab from one partner.
4. For a throat specimen: open your mouth wide, lower the tongue and gently touch far back in the tonsil area with a dry sterile swab. Inoculate blood agar plate. DON’T POKE YOURSELF. A small sample is sufficient.
2. Follow demonstration and wash your hands before and after sampling.
5. For a nose specimen: Touch the back of the interior of the nostril area and inoculate blood agar plate.
3. All swabs are biohazards and should be treated accordingly.
Incubation 1. Incubate plates after inoculation according to the environments that were tested.
3. Blood Agar plates are incubated at 35°C for 24-36 hours. 4. We will observe and discuss your results in an open lab forum next class.
2. TSA are incubated at room temperature until the next session. Use the labeled drawer in the cabinets at the back of the lab.
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