The Lab Exercises have been created over many years as a team effort by the Molecular Biology and Microbiology faculty.
1. Date and Time
Fall 2004, 2 hours
2. Throat flora exercise
2.1. Special Materials:
Rulers, plates of Streptococcus
viridans, Streptococcus pneumoniae and Streptococcus
pyogenes where the sensitivity assay has already been performed.
Observe results of "P" and "A"
disc tests. Record results. Continue study of bacteria of throat as
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
3. Isolation of
3.1. Special Materials:
Rabbit plasma, antibiotic discs, nutrient agar
plate, and Staphylococcus aureus as a positive control
- 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.
Staphylococcus aureus from other staphylococci and
||Detection of presence/absence of
||Coagulase clots plasma
||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
- Rock the slide for 45 seconds and look for
clumping (Flame the loop!)
||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
4. Microbiology fecal isolates.
4.1. Special Materials:
3 MacLac plates (plus and minus antibiotics),
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+.
- 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
- You will inoculate these three media:
- Mac Lac tet
- Mac Lac amp
- 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
BEFORE LEAVING, PUT AWAY EVERYTHING AND CLEAN
BENCHES AND THEN YOUR HANDS !!!