13--3 Protocol for medically important microbes

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Period 1

Materials

Cultures of the following grown in BHI Broth tubes or Agar slants:

Figure 13-6 Cultures

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Strains to be used in the experiments of seciton 13-3

place table in figure

Streptococcus mitis Staphylococcus aureus
Enterococcus faecalis Staphylococcus epidermidis
Enterococcus durans Neisseria sicca

5 plates of Blood Agar

3 plates of Trypticase Soy Agar (TSA)

4 plates of Vogel-Johnson Agar

4 sterile cotton swabs

Candle jar

Special Safety Precaution: Take care with the Staphylococcus aureus and all cultures form the nose an throat. Treat these cultures as pathogens.

Figure 13-7 Grams stains, set 1

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Typical Gram stains if Streptococcus mitis, Staphylococcus aureus and Enterococcus faecalis.

Gram stain of 3 strains in table

Figure 13-8 Grams stains, set 2

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Gram stains of Staphylococcus epidermidis, Enterococcus durans, Neisseria sicca

Gram stain of next 2 strains in table

  1. Each member of the pair should select three of the organisms and prepare gram stains of them, preferably on the same slide. Take care that you do not use excess water in rinsing off the stains and reagents, as doing so may lead to overdecolorization. Leave the slide on the microscope for your partner to examine. Note the pleomorphism of S. mitis.
  2. Prepare streak plates for isolated colonies as follows:
    • Streak S. mitis, E. faecalis and E. durans onto plates of Blood Agar and the other three cultures onto TSA. (Note: If the cultures are provided on agar slants, take the inoculum from the suspension of cells in the liquid at the base of the slant.) On the Blood Agar plates, make a few small vertical jabs with the edge of your loop into the medium where you streaked the first phase, as indicated by your instructor.
    • Bring the plates to the stage for one-day incubation at 37°C. All of the plates can be placed in the candle jar.
    • The additional carbon dioxide provided by the candle jar enhances the growth of some of these organisms. The candle jar is not a method for anaerobic incubation, as the burning candle will not consume all of the oxygen in the jar.

  3. Streak the two species of Staphylococcus for isolated colonies onto plates of Vogel-Johnson Agar. Incubate at 37°C until the next period. Note the explanation of this medium on page 14
  4. Optional (non-required) exercises involving isolation of organisms from the human body:
    • Prepare a throat culture on Blood Agar for isolation of Streptococcus and Neisseria as follows: Swab the tonsil area with a sterile cotton swab; a sterile tongue depressor can be used if available. Rub the swab onto a small area of the surface of a Blood Agar plate. With a loop, streak from this inoculum for isolated colonies. Place the plate in the candle jar for one-day incubation at 37°C.
    • Isolation of Staphylococcus aureus from the nasal cavity may be attempted: With great care, sample one of the nostrils with a sterile swab. Using the procedure in step 4a, streak a plate of Vogel-Johnson Agar for isolated colonies. Incubate at 37°C until the next period.

Period 2

Materials

2-4 tubes of Brain Heart Infusion (BHI) Broth

Dropper bottles of 3% hydrogen peroxide and oxidase reagent

Empty petri dishes for the slide catalase test

Demonstrations of various streptococci (step 3)

Figure 13-9 Growth on blood agar

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Colonies of S. mitis, E. faecalis and E. durans on blood agar.

Growth of strains on Blood agar

Figure 13-10 Growth on TSA

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Colonies of S. aureus, S. epidermidis, N. sicca on TSA.

Growth of strains on TSA

Figure 13-11 Throat culture

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An example of the growth of colonies from a throat swab.

Throat culture on Blood agar

Figure 13-12 Nasal culture

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Colonies of a nasal culture grown on Vogel-Johnson agar.

Nasal culture on V-J agar

  1. Examine the Blood Agar and TSA plates of the six cultures prepared in Period 1:
    • Describe the colonial characteristics (including pigmentation) of each organism.
    • Note the hemolytic reactions of the Streptococcus and Enterococcus species. Observe around the isolated colonies and also the slices made into the medium.
    • Perform the slide catalase test (Grab a bit of growth and place it on the slide. Place the slide inside a petri dish. Add a few drops of 3% H2O2 and observe for the evolution of bubbles) for each organism, taking care to observe the reaction through the lid of a petri dish as per the instructions. What would happen if we added the hydrogen peroxide directly to the colonies growing on Blood Agar? Discard the slide into the disinfectant and the petri dish into the usual place.
    • Perform the oxidase test directly on the plates.
    • Having obtained the Gram, catalase and oxidase reactions for each organism, what two ways can you differentiate between the three genera, such that each way involves only two of these tests?
  2. Examine the Vogel-Johnson Agar plates of the two species of Staphylococcus:
    • Note the differences, if any, between the two species regarding colony size, tellurite reduction (black color in colony) and mannitol fermentation (pH indicator turns yellow).
    • In a selective isolation procedure for Staphylococcus aureus from a clinical specimen or other sample (as in the nasal culture below), one would ignore the mannitol reaction and pick black colonies for further testing. If the gram reaction, morphology, arrangement and catalase reaction indicate a probable Staphylococcus, then the coagulase test is done.

    • To set up for the coagulase test to be run next period, inoculate each organism into a tube of BHI Broth and incubate at 37°C for 1-2 days.
  3. Examine the demonstrations of the various streptococci of clinical interest:
    • Streptococcus pyogenes on Blood Agar: Note the ? hemolysis characteristic of this species. A throat culture yielding a large number of small, ?-hemolytic colonies may indicate strep throat.
    • Streptococcus pneumoniae on Blood Agar: Note the ? hemolysis and the sensitivity to the antibiotic optochin, as discussed in the introduction.
    • Streptococcus salivarius on high-sucrose medium (Heart Infusion Agar+10% sucrose): Note the gumdrop appearance of the colonies. For more information about S. salivarius and the role its slime production plays in tooth decay, read the chapter on host-microbe interactions in the microbiology textbook.
  4. Examine your throat culture on Blood Agar as time permits. (Plates can be saved for later in the refrigerator.)
    • The smallest colonies (usually ?-hemolytic) are oral streptococci. Pick one or more of these colonies and confirm the probability of Streptococcus by the gram stain and the catalase test.
    • Place a dropperful of oxidase reagent onto the isolated colonies. Any colony which turns blue within seconds is probably Neisseria. A confirmatory Gram stain can be made.
  5. Examine your nasal culture on Vogel-Johnson Agar for the presence of relatively large, black colonies (see step 2). From a representative colony, prepare a gram stain and perform a slide catalase test. If a probable Staphylococcus is indicated, inoculate the colony into BHI Broth and incubate with the tubes prepared in step 2b.

Period 3

Materials

Small capped test tubes (1 per BHI Broth culture)

Tube of EDTA-treated rabbit plasma (0.5 ml per BHI Broth culture)

Micropipettes and sterile tips

Figure 13-13 Coagulase test

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A movie of typical reactions in the coagulase test. The positive control (A) is S. aureus, the negative control (B) is S. epidermidis. The third reaction (C) is a typical reaction from a nasal isolate.

coagulase test. Provide both positive and negative controls

  1. Perform the coagulase test on each tube of BHI Broth by adding 0.5 ml of culture to a tube of rabbit plasma. Incubate in the 37°C water bath or incubator tray. Results may be read after several hours of incubation. Be sure to compare the reaction of the unknown microbe to the positive and negative controls provided.
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