Chapter 24: Human Microbiota

The human body is host to many different microorganisms. This includes those microbes that are permanently present, called the resident microbiota, and a number of transient (temporary) microbes. The normal (resident) microbiota is unique to each individual, although some bacteria are found on every human. Each part of the human body has its own microbiota, dtermined in large part by the physical and chemical environment of the body there. 

Skin. Human skin is, for the most part, aerobic, dry, salty, and acidic, although there are exceptions. Underarm moisture and anaerobic pores are two examples. It should be no surprise, then, that the most common resident skin bacteria are those that can tolerate dry salty conditions. Virtually all resident skin bacteria are Gram-positive (remember the salt tolerance of Staphylococcus?). Virtually all are aerobic (Micrococcus) or facultatively anaerobic (Staphylococcus), although a few anaerobes inhabit skin pores (Propionibacterium) and some aerotolerant Streptococci can be found as well. Skin bacteria can easily be cultured on TSA and most often distinguished by simple staining and colony characteristics.  

Throat. The moist, warm and somewhat less aerobic environment of the throat harbors numerous streptococci, diphtheroids (irregular Gram-positive rods), and staphylococci. Because the mucous lining is a better source of nutrients, such as iron, fastidious microbes often flourish there. Occasionally, Gram-negative bacteria such as Neisseria or Haemophilus may be found as well. The most common throat culture in use is the blood agar plate because of its ability to differentiate among various streptococci. Many bacteria produce compounds called hemolysins which break down red blood cells. The bacteria thus gain access to iron, a scarce mineral for them. The pattern of hemolysis (red blood cell lysis on the agar plate) distinguishes bacteria. Streptococci that cause a clearing in the agar are termed beta-hemolytic, those that partially break down red blood cells (leaving a green halo) are alpha-hemolytic, and those that cannot break down red blood cells are gamma-hemolytic. Since some staphylococci also lyse red blood cells, a catalase test can be done to distinguish catalase-positive Staphylococcus from catalase-negative Streptococcus.

Nose. The nasal cavity is a transition environment between the skin and the respiratory tract, and therefore is inhabited by microbes that you would find on skin, such as Staphylococcus, as well as those that need a moister environment, including Haemophilus (Gram-negative) and Streptococcus. Of particular interest to health care is that the nose is a common site for colonization by Staphylococcus aureus (much more so than skin or throat), and testing for S. aureus colonization usually is done by nasal swab. 

GI tract. The predominant residents of the GI tract are anaerobic bacteria such as Bacterioides and Clostridium, aerotolerant Enterococcus spp., and numerous facultatively anaerobic enterobacteria, including E. coli. Most microbes in the large intestine carry out fermentation of fiber and other left-over nutrients, and a significant proportion of feces consists of microbial cells. Although the anaerobes are difficult to culture, most enteric samples are very successful at growing the enterobacteria. Initially, a swab of the enteric tract is cultured on a selective/differential medium (Eosin Methylene Blue Agar = EMB). Only Gram-negative bacteria grow well on this medium. Lactose-fermenting Gram-negatives (Escherichia, Klebsiella, Citrobacter, Enterobacter) form colonies with a purple center and/or a green metallic sheen. Lactose-negative bacteria (Proteus, Salmonella, Vibrio, Providencia) form white or pink colonies. These colonies are usually tested further using SIM media, citrate slants, and Triple Sugar Iron (TSI) slants to further distinguish them.

Typical microorganisms

Skin
Throat
Nose
Enteric
 
Procedure
Each lab group will get:
Throat swab (1 student + 1 assistant)

Using a sterile long cotton swab, swab the back of one student's throat. Gently rub the cotton tip over the entire surface of the blood agar plate. Discard used swab in the beaker with disinfectant on the front lab bench.

Enteric swab (1 student)

Take the EMB plate and a regular cotton swab to the restroom. Swab the skin near the anus and gently rub the cotton tip over the entire surface of the EMB plate. Discard used swab in the beaker with disinfectant on the front lab bench.

Nasal swab (1 student)

Using a sterile cotton swab, gently swab the inside of one nostril - at least 1 inch up the inside of the nostril. Use the swab to inoculate a MSA plate by swabbing the entire MSA surface.

Skin swab (1 student)

Dip a regular cotton swab into saline solution, then vigorously rub a 1-inch square on your forearm. Next, gently rub the cotton tip over the entire surface of the TSA plate. Discard used swab in the beaker with disinfectant on the front lab bench. 

Second lab period

  1. Examine your TSA (skin) plate. Describe 4 different colonies (color, size, etc). Simple-stain all 4 bacteria. Conduct a catalase test on each colony. Tentatively identify.
  2. Examine your nasal swab MSA plate. Describe the color - pink/red/orange/yellow. Select 1 colony (choose a colony on yellow agar if you have one). Simple-stain this colony with Crystal violet. Any colonies that you tentatively identify as Staphylococcus spp. should be tested with a coagulase test

        Mannitol Salt Agar (MSA)

    1. Selective for Gram-positives (salt tolerant)
    2. Differential for S. aureus (Mannitol fermentation)

    ·  S. aureus ferments mannitol = yellow quadrant

    ·  S. epidermidis does not = pink/purple quadrant

  3. Examine the blood agar plate for areas of hemolysis. Identify the hemolysis patterns - which are seen on your plate? Which are most common? Select two colonies from different hemolysis patterns. Describe the colonies (color, size). Perform a catalase test on these two colonies. Note: This catalase test must be performed on a glass slide or the inside of the Petri plate lid since red blood cells in the agar are catalase-positive.
  4. Examine the EMB plates for growth (Bonus point) and for differences in color. Choose one lactose-positive (purple/green) and one lactose-negative (pink/white) colony and perform a citrate, SIM, and TSI test on each. Label and bring up front to be incubated.
Third lab period

  1.  Coagulase test (if done): evaluate as + or -

  2. Triple Sugar Iron agar (TSI)
    1. Differential for fermentative enterobacteria (contains Glu, Lac, Suc)
    2. Data recorded as results for SLANT/BUTT:

·  Slant: A = acid (yellow), N = neutral (orange), K = basic (magenta)

·  Butt: A = acid (yellow), N = neutral (orange), K = basic (magenta), G = gas bubble trapped within the agar

·  Example: yellow butt and purple slant is recorded as K/A

 

  1. Citrate, SIM test
    1. Evaluate as we have done this semester
    2. For indole test add Kovacs Reagent (hood)

Data Sheet & Review Questions

 

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