LAB EXERCISE 22
MEDICAL MICROBIOLOGY
INNATE IMMUNITY: THE ROLE OF NORMAL FLORA and ANTIMICROBIAL ACTIVITY OF THE SKIN
STUDENT OBJECTIVES
1. Demonstrate and identify the antibacterial characteristics of the skin.
2. Describe the role of normal microbial flora in innate immunity.
3. Describe competitive exclusion.
4. Name various microorganisms commonly found on skin of humans and list identify some common infectious diseases each causes.
5. Compare and contrast innate and adaptive immunity.
6. Identify and describe the first line defenses.
7. Apply knowledge on biochemical testing to deduct conclusions on hypothetical scenarios.
THE LABORATORY ROUTINE
1. Store backpacks and items in the space provided, bringing only necessary items to the lab table.
2 Wash your hands.
3. Put on PPE: lab coats, safety glasses and gloves.
4. Long hair should be tied back.
5. Disinfect your bench top using the ‘APPLY, WIPE, and REAPPLY’ method.
6. Review lab exercise one.
Name: ______
PRE-LAB EXERCISE TWENTY TWO
1. What is the purpose of this laboratory exercise?
2. Describe the principle of competitive exclusion.
3. Explain why mannitol salt agar is a selective medium for isolating normal skin microbiota?
4. List three characteristics of the skin that prevent infection from pathogenic organisms.
INTRODUCTION
An infection is a condition caused by the presence of a disease-causing agent. Such agents are termed pathogens. Our body also reacts to antigens: foreign substances to which our immune system responds. The body’s defense and immune system is divided into two arms: innate (non-specific) immunity and adaptive (specific) immunity.
· Innate immunity: This mechanism is spontaneous and does not involve an active role in the entire immune system. Examples include physical barrier of the skin, mechanical barrier of the mucous membranes, acidity of the stomach, enzymes in the tears and saliva, inflammation, fever, and phagocytosis. Phagocytosis is the process in which a leukocyte engulfs and digests particles or pathogens. Humans are born with this system in place therefore the innate immune system is described as a non-specific response since it is always ready to protect the body against foreign invasion.
· Adaptive immunity: This mechanism requires an active immune response from specific cells and requires a longer duration to remove the pathogens. This mechanism is used when the innate mechanism fails, become overwhelmed, or is bypassed (vaccine needle bypassing the skin into the blood vessel). During this response illness usually occurs because of the longer duration required to promote specific cells. When the adaptive immunity ends, the immune system usually forms memory for that specific pathogen or antigen. Humans are not born with an active adaptive immune system however this specific response to an antigen begins “learning” as soon as the first lines of defense are breached in newborns and infants. As humans age the adaptive system learns and remembers how to best remove or destroy specific antigens this ability to form memory against an antigen is responsible for a fast and efficient secondary response to an antigen. In later years of life the adaptive immune response begins to work inefficiently which is why elderly individuals tend to be more susceptible to certain diseases.
Skin is involved in innate immunity and is considered a first line of defense against foreign invaders such as bacteria, fungi, and pollen. The skin is generally an inhospitable environment for most microbes. The dry layers of keratin-containing cells that make up the epidermis (the outer most layer of the skin) are not easily colonized by most microbes. Sebum, secreted by oil glands, is hydrolyzed to fatty acids that lower the pH of the skin. Sweat glands secrete lysozyme, an enzyme that destroys the cell wall of some bacteria. Salts in perspiration create a hypertonic environment on the skin. However, perspiration and sebum can be used as nutrients by some bacteria, and allow them become established as part of the normal microbiota (flora) of the skin.
Normal skin microbiota are generally resistant to drying and to relatively high salt concentrations. More bacteria are found in moist areas, such as armpits and the side of the nose, than on dry surfaces of the arms or legs. Transient microbes are present on hands and arms due to contact with the environment but typically find the skin a poor place to reproduce. Also, transient microbes you encounter that can survive on the skin usually do not survive since competitive exclusion occurs. Competitive exclusion is a principle that states when two organisms compete for the same critical resources within an environment, one of those organisms will eventually outcompete and displace the other. One of the experiments that will be performed today will demonstrate competitive exclusion.
Most indigenous bacteria on the skin are Gram-positive and salt tolerant. Propionibacterium live in hair follicles and feed on sebum from oil glands. The propionic acid they produce via fermentation maintains the pH of the skin between 3 and 5, which suppresses the growth of most other bacteria. Catalase positive, salt tolerant members of Gram-positive cocci (family Micrococcaceae) are the most frequently encountered bacteria on the skin along with members from the genus Staphylococcus. The catalase test is a useful test to quickly distinguish between Streptococcus which is catalase negative and Staphylococcus which are catalase positive.
Staphylococcus aureus is species of bacteria commonly part of the normal microbiota of the skin and is also considered an opportunistic pathogen it is the second most commonly isolated bacteria in hospitals and often associated with nosocomial (hospital acquired) infections. Unlike many other species in the genus Staphylococcus, S. aureus is able to ferment the sugar mannitol. Staphylococcus aureus also produces an enzyme called coagulase that clots the fibrin in blood. The production of this enzyme is highly correlated with pathogenicity and distinguishes S. aureus from other species of Staphylococcus. Testing for mannitol fermentation or for the presence of coagulase are biochemical tests that can be used to identify S. aureus from other staph species.
Although many different bacterial genera (and yeasts) live on human skin, we utilize a selective and differential medium to attempt to isolate staphylococci in this exercise. You will also expose two microorganisms to the environment on your skin. Then you will try to determine which of the two is a typical inhabitant based on how they survive the antimicrobial activity of the skin.
MANNITOL SALT AGAR PLATE
Use the swab for about half of the plate then switch to a loop to get isolated colonies. Be sure to flame in between streaks. The final plate should look something like below.
WEEK ONE – ANTIMICROBIAL ACTIVITY OF THE SKIN
Materials per 2 students: Tryptic Soy Agar (TSA) plate (1), Sterile cotton swabs (5), Sterile saline, Mixed broth culture of Bacillus subtilis and Micrococcus luteus
Procedure:
1. Divide the bottom of a TSA plate into 5 sections and label each section with one of the following numbers: 0, 15, 30, 45, and 60.
- Select four areas on the back of your hand, making sure each area is free from cuts and abrasions. Mark the spots on your hand using a pen or marker with the same numbers (except “0”) as above (or use your knuckles as markers).
- Moisten one sterile cotton swab in the mixed broth culture, then slightly swab each marked area on your hand with the culture. Do not wash your hands for the duration of this test. Using the same swab, streak the 0 sector on your plate to show the mix of bacteria at time zero. Biohazard the swab.
- After fifteen minutes, moisten a fresh swab with sterile saline and swab over the area marked 15 on your hand “picking up” the organisms left on your skin. Streak the sector of the agar plate marked 15. Discard swab in the biohazard bag.
- Repeat the procedure in step 4 above with the area 30 at 30 minutes, 45 at 45 minutes, and 60 at 60 minutes.
- After the hour long test, wash your hands well with soap and water.
- Invert and place the labeled plate in the container provided for incubation.
WEEK ONE-ISOLATION OF SKIN MICROBIOTA
Materials per student: Mannitol Salt Agar (MSA) plate (1), Sterile cotton swab (1) , Sterile saline
Procedure:
1. Wet the swab with saline, and push against the wall of the test tube to express excess saline. Swab any surface of your skin, the sides of your nose, behind your ear, inside your arm, would be ideal areas for this exercise.
2. Swab one-half of the mannitol salt plate with the swab.
3. Discard the swab in the biohazard bag.
4. Using a sterile loop, streak back and forth into the swabbed area a few times, then streak to obtain isolated colonies according to the diagram on the previous page.
5. Place your inverted plate in the tub for incubation at 35°C until the next lab period.
WEEK TWO ANTIBACTERIAL ACTIVITY OF THE SKIN
Examine the TSA plate inoculated last period and estimate the relative amount of growth of Micrococcus luteus (yellow colonies) and Bacillus subtilis (off-white colonies). Record your results.
WEEK TWO ISOLATION OF SKIN MICROBIOTA
Materials:
Your incubated MSA plate 3% hydrogen peroxide
Procedure:
1. Examine the isolated colonies. Members of the Micrococaceae usually form large opaque colonies. Record the appearance of 3 colonies and any evidence of mannitol fermentation (yellow halos) in your lab manual.
2. Test for catalase production by carefully adding 1 drop of H2O2 onto the 3 isolated colonies.
Name ______
EXERCISE TWENTY TWO LAB REPORT
Antibacterial Activity of the Skin
Sketch your plate and label the two organisms in the original culture.
1. Explain what happened to the each bacterial species over the hour:
2. Which organism became “extinct” on your skin?
Isolation of Skin Microbiota
Source of inoculum: ______
Mannitol Salt Agar Plate: Describe 3 colony types if possible you may have to look at one of your lab partners plates:
Number assigned to Colony (colony ID)1 / 2 / 3
Colony description
Pigment of colony
Mannitol fermentation
Yes/No
Catalase reaction +/-
3. What is a lysozyme? How does it destroy certain bacteria?
4. List at least 3 biochemical tests that could be performed to positively identify the organisms on your body. Hint: you can use tests we have performed in other labs.
5. An unknown organism is growing in small colonies on an Mannitol Salt agar plate (MSA plate). The media is red to begin with and it is still red following the incubation period. What can you conclude from the results of this growth pattern?
6. You inoculate an MSA plate with an unknown bacterium. The media is red to begin with and it is still red following the incubation period. Following the incubation period there is no growth. What can you conclude from the results?
7. An unknown organism is growing in small colonies on an Mannitol Salt agar plate (MSA plate). The media is red to begin with and it is now yellow following the incubation period.
a. What can you conclude from the results of this growth pattern?
- You then perform a coagulase test and the result is positive. What can you conclude about the identity of this organism?
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