Lab 10

 

Objectives:

Reading:

1. Bacteria of the Skin

1. Tortora, Pages 397; 561

2. Oral and Respiratory Specimens

2. L: pages 46; 67; p: PAGE 97

Normal Flora

Normal flora, also known as normal microbiota, are found on the skin, in the nose and throat, in the conjunctiva of the eyes, in the large intestine, in the lower urethra, and in the vagina.

Once established, normal flora are vital to good health, benefiting the host by preventing the colonization of potentially pathogenic organisms. In today's lab, we will focus specifically on normal skin flora, and we will also learn about human skin pathogens.

The following organisms are considered to be normal flora for human skin:

All are bacteria, except for Pityrosporum and Candida, which are fungi. S. epidermidis, S. aureus, and various diptheroids such as Corynebacterium are commonly found in the conjunctiva. Generally speaking, because of the antimicrobial properties of sweat and oil glands secretions, the numbers of bacteria actually found on the skin are not great. Organisms that do thrive on the skin are generally resistant to both drying and high salt concentrations (halophiles).

Opportunistic microorganisms are those organisms, which given the right circumstances, can become pathogenic. Examples of opportunistic microorganisms of the skin include any of the normal flora of the human body that gain access into the body through broken skin or the mucous membranes. Thus, E. coli, a normal intestinal inhabitant, and S. aureus, normal skin flora, can gain entry and produce disease.

Pathogenic microorganisms are those organisms that will cause disease, regardless of their location. These include Streptococcus pyogenes, viruses (viruses are never considered to be normal flora), certain fungi such as those that cause ringworm (Microsporum, Trichophyton, Epidermophyton), and some parasites (an example is Sarcoptes scabiei, the etiologic agent of scabies).

Today we will examine the colony morphology of the following microorganisms:

Recall that colony morphology is the description of the form of the bacterial colony--its colony configuration, its colony margins, and its colony elevation--on a particular medium. Use the table below to compare and contrast the above bacteria's colony morphologies.

Skin Bacterial Colony Morphology

Bacterial Colony

Colony Configuration

Colony Margins

Colony Elevation

S. aureus

 

 

 

S. epidermidis

 

 

 

Corynebacterium sp.

 

 

 

P. aeruginosa

 

 

 

M. luteus

 

 

 

P. mirabilis

 

 

 

Now, examine the MSA and blood agar plates of your skin sample from last week.

  1. How many different colony types can you distinguish?
  2. How do your isolates compare to the above colony morphologies?
  3. Are your cultures pure or mixed?
  4. If they are mixed, select one colony and streak for isolation on a new plate of MSA and blood agar.
  5. Perform a Gram stain on this colony (see below for instructions).
  6. Perform a catalase, oxidase, and/or coagulase tests on your colony, based on your Gram stain results (see below for instructions).
Gram Stain

Instructions for Gram staining are re-printed below

Materials:
Inoculating loop
Glass slide
Slide holder
Glass marking pen
Bunsen burner
Flint lighter
Bibulous paper

  1. Label your slide, if you plan on keeping it.
  2. Place a small drop of .85% saline on the slide.
  3. Sterilize your inoculating loop and select a colony. Pick it up and mix it with the saline.
  4. Allow the saline/colony solution to dry fully.
  5. Heat fix it.
  6. Gram stain it:
    1. Crystal violet, one minute. Rinse with water.
    2. Iodine, one minute. Rinse with water.
    3. Alcohol, until runoff is clear OR 20 seconds, whichever is shorter. Rinse with water.
    4. Saffranin, one minute. Rinse with water.
  7. Blot dry, using bibulous paper.
  8. Coarse focus on 10x; fine focus, using oil, on 100 x.
Oxidase Test To perform the oxidase test:
  1. Place an oxidase strip on a paper towel.
  2. Using a sterile inoculating needle, scrape part of your bacterial colony onto the oxidase strip.
  3. A blue/dark purple color change is positive.

 

 

 

 

 : The glass slides above have been set on black paper so that the positive, dark purple reaction on the right is more easily disinguished from the negative reaction on the left.

CATALASE TEST

To perform a catalase test,
  1. Place a drop of hydrogen peroxide on a clean glass slide.
  2. Using a sterile inoculating needle, mix a selected colony with the peroxide.
  3. The presence of bubbles of oxygen is indicative of a positive reaction.

The colonies on the slide on the top, above, clearly show evidence of catalase production because of the bubbles of oxygen produced after hydrogen peroxide was added. No bubbles of oxygen were produced by the colonies on the slide below. These organisms are catalase negative.

Coagulase

  • This test distinguishes between Staphylococcus aureus and Staphylococcus epidermidis.
  • Recall that S. aureus is a pathogen and S. epidermidis is considered to be normal skin flora
  • Thus, it is performed on Gram positive, catalase positive cocci.
  • It detects the ability of S. aureus to clot plasma.
  • It involves incubating the bacteria with plasma that has been treated to prevent normal clotting from occurring (at 37o C.).
  • If S. aureus is present, the plasma will clot within 2-4 hours (although, some may take as long as 24 hours).
  • If S. aureus is not present, the plasma will remain clot-free.
  • Positive and negative controls, using a known S. aureus (for the positive) and a known S. epidermidis (for the negative) are required.
  • This test is specific to S. aureus.
  • Thus, just as growth on MSA that causes the medium to turn yellow (due to fermentation of the mannitol) is specific to S. aureus, so is a coagulase positive test of a Gram positive, catalase positive coccus specific for S. aureus.
Instructions for performance of coagulase test will be provided in class.

 

 

 

 

 

 

 

 

The tube on the left, above, contains freely flowing plasma, indicative of a coagulase negative result. The tube on the right contains clotted plasma, indicative of a coagulase positive result.

Oral and Respiratory Samples

Although potential pathogens may be found in the upper respiratory tract, normal flora suppress their growth by competing for nutrients and by producing inhibitory substances. This is not true of the lower respiratory tract, which is essentially sterile.

  1. Using a sterile toothpick, take a sample of plaque or calculus from your teeth.
  2. Place the entire toothpick into a tube of nutrient broth that contains glass beads.
  3. Vortex carefully; the glass beads will break up the sample.
  4. Now streak two blood agar plates.
    • One will be incubated aerobically
    • One will be incubated anaerobically
  5. Using a sterile swab and tongue depressor, collect a respiratory sample from the back of your lab partner's throat.
  6. Roll the swab on blood agar.
  7. Streak for isolation.
  8. Now switch, and repeat for yourself.
  9. We will examine these at next week's lab.

Take Out Food for the Brain:

The most widespread disease in the world is about to be tamed. In the February issue of Infection and Immunity, University of Florida dentist and researcher Jeffrey Hillman explains how tooth decay, primarily caused by Streptococcus mutans, will soon be relegated to medical history books, if upcoming clinical trials prove to be successful. The accepted theory of tooth decay describes how S. mutans, a naturally occurring bacterium of the mouth (normal flora) breaks down sugars, and in the process, produces lactic acid. Over time, lactic acid destroys tooth enamel. This destructive process eventually results in tooth decay.

Researchers at the University of Florida have removed the gene that is responsible for lactic acid production from one strain of S. mutans. (Recall that each gene is responsible for the production of a protein. In this case, the eliminated gene was responsible for the production of the enzyme that catalyzed the production of lactic acid.) The new strain is called an "effector" strain, and not only does it not produce lactic acid, but it LOVES high-sugar diets and it rapidly overwhelms other strains of S. mutans, resulting in an almost pure population of the effector strain.

This ability to successfully colonize by this new strain will be tested in upcoming clinical trials. In these studies, the number of applications required to change the normal flora population of the human mouth will be determined. According to Hillman, if all goes well, it is expected that

...the ideal application would be to treat infants when their first teeth appear. Infants normally acquire S. mutans via contaminated saliva from their mother or primary caregiver. The child would simply visit their dentist for a squirt of solution on their teeth. The approach also is designed for use in older children and adults.
The genetically engineered strain is not known to cause disease or pre-dispose to disease. Nor is it expected to eliminate the need for brushing teeth. The effector strain does not remove unsightly rotting food and plaque!

How do you feel about using genetically altered forms of bacteria to prevent or eliminate tooth decay? You tell me.

Take Home Thought

Designer jean$...designer gene$. What price tag do caries-preventers carry?








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