Fourth Lab - Experimental Protocol

2.1. Special Materials:

Rulers, plates of Streptococcus viridans, Streptococcus pneumoniae and Streptococcus pyogenes where the sensitivity assay has already been performed.

2.2. Procedure:

Observe results of “P” and “A” disc tests. Record results. Continue study of bacteria of throat as needed.

Observe the demonstration plates of Streptococci for the presence or absence of growth around the optochin discs. Measure the diameter of any zones of inhibition. Be careful not to touch your ruler or your fingers to the plate. If a 6 mm disc is used, a zone of inhibition of at least 14 mm in diameter is considered sensitive, and therefore positive for pneumococci; a diameter between 6 and 14 mm is questionable for pneumococci, and requires more complex testing. For 10 mm optochin discs, a zone of inhibition at least 16 mm in diameter is positive for pneumococci.

3.1. Special Materials:

Rabbit plasma, antibiotic discs, nutrient agar plate, and Staphylococcus aureus as a positive control

3.2. Procedure:

1. Perform a Gram stain and the coagulase slide test on any Staphylococcus aureus candidates growing on your Mannitol salts agar plate, using a sample from the S. aureus demonstration plate as a positive control.

   The Coagulase Test
   Purpose:	To differentiate Staphylococcus aureus from other staphylococci and micrococci.
   Principle:	Detection of presence/absence of coagulase.
   Mechanism:	Coagulase clots plasma
   Reagent:	Rabbit plasma
   Method:	Bound Coagulase/Slide test Put a small amount of water (1 or 2 loopfuls) on a glass slide using the inoculating loop. Pick several colonies of catalase-positive, Gram-positive cocci and make a smooth suspension in the water on the slide. Put a small drop of plasma to the side of the bacterial suspension. Using your inoculating loop, gently mix the plasma with the bacteria. Rock the slide for 45 seconds and look for clumping (Flame the loop!)
   Interpretation:	SLIDE: Positive - macroscopic clumping in 45 seconds or less. Negative - no clumping in 45 seconds.

3.3. Antibiotic Testing

Determine the antibiotic sensitivity of your own or a neighbor's Staphylococcus isolate. Streak the entire surface of a nutrient agar plate with a heavy inoculum of organisms from the previous mannitol salts agar plate - a swab is best for this purpose, but a loop can be used also. Work with a single purified colony. With forceps, carefully place each different type of antibiotic-impregnated disc onto the surface of the inoculated plate. Be careful not to allow the forceps to come in contact with the inoculated surface. Gently tap the top of each disc with a flamed loop to insure good contact with the agar surface. These plates will be incubated at 37°C.

4.1. Special Materials:

3 MacLac plates (plus and minus antibiotics), sterile buffer

4.2. Purpose of Exercise

The purpose of this lab exercise is to detect the incidence of tetracycline (Tetr) and ampicillin (Ampr) resistance in E. coli in the fecal flora of your class. We will use fecal swabs to inoculate MacConkey’s-lactose Agar. MacConkey medium contains bile salts (detergent) that permits growth of a limited class of bacteria able to resist the detergent. Fecal bacteria are able to grow on this medium because they have evolved to live in the presence of bile salts. This medium also contains pH indicator that turns red if lactose if fermented. E. coli isolated from fecal material are typically able to ferment lactose, i.e., are lac+.

4.3. Procedure

1. Use the swab from the culturette to make an initial inoculum on the 3 plates listed below. Note: if the culturette has dried out, you will need to wet it using sterile buffer prior to inoculating the plates. Use your sterile loop to streak from this inoculum to get single colonies.
2. You will inoculate these three media:
   1. Mac Lac tet
   2. Mac Lac amp
   3. Mac Lac

   Mac Lac is MacConkey's agar. Lac indicates it contains the sugar lactose as a carbon and energy source. tet indicates that it contains tetracycline and amp indicates that it contains ampicillin. Antibiotic-resistant E. coli will form colonies on either tet and/or amp medium.