Third Lab - Experimental Protocol

1. Date and Time

Fall 2015, 2 hours

2. Throat flora exercise

2.1. Special Materials:

“P” and “A” discs, forceps, alcohol, sterile swabs, sterile Phosphate Buffered Saline (PBS), rulers, non-group A strep example, 2 BA plates, plates of Streptococcus viridans, Streptococcus pneumoniae and Streptococcus pyogenes for controls for “P” & “A” tests.

2.2. Procedure

Examine your purification plates from the Second Lab. Make sure you record your observations. Pick 2-3 different types of isolates, Gram stain and record results.
The remaining periods of the Throat Flora laboratory will be used to identify some of the organisms you have isolated from your throat. Use the flow sheet at the end of the notebook to guide your investigations. Some special media and reagents will be available during all subsequent periods, and you should work at your own pace on whichever organisms interest you. Subject to limitations of time and material, the instructors will help you try to identify any unusual isolate that does not fit our key. Remember the following questions as you work - how many different kinds of bacteria can you recognize on the basis of colonial morphology alone? Carry out a Gram stain on each of them. Do the morphologic and staining characteristics confirm the apparent difference in colonial appearance?
Try to make a tentative identification based on the macroscopic and microscopic characteristics of the organisms. Consult with an instructor, if necessary.
One test which is useful for distinguishing among strepococci strains involves the use of discs permeated with optochin (P discs) or bacitracin (A discs).

2.2.1. The Optochin Sensitivity Test - "P" Disc

Optochin susceptibility tests the fragility of the bacterial cell membrane. The optochin disc is used specifically to differentiate between Streptococcus pneumoniae, which is optochin-sensitive, and other α-hemolytic Streptococcus species, which are resistant.

Perform the P disc assay using a swab moistened in PBS to spread α-hemolytic cells from your purified colonies on the BA plates into a 2 centimeter square on a fresh BA plate. Using forceps and sterile technique, place a P disc in the square. Do control P disc assays of Streptococcus viridans and Streptococcus pneumoniae on the same plate. For this experiment, you want to spread a large number of cells in the 2 cm square area, so as to obtain a “confluent lawn” of bacterial growth. Swabbing most of a single colony is sufficient.

2.2.2. The Bacitracin Sensitivity Test - "A" Disc

If you observe β-hemolytic colonies, perform an A disc test. (Some students will not observe β-hemolytic colonies from their throat cultures). Susceptibility to the antibiotic bacitracin, contained in the "A" disc, is a useful presumptive test for differentiation of β-hemolytic group A streptococci (such as Streptococcus pyogenes), which are bacitracin-sensitive, from non-group A, β-hemolytic streptococci, which are resistant.

Perform the test by using the same techniques described in the “P” disc test above. Do control “A” disc assay of Streptococcus pyogenes on the same plate.

Your instructor will prepare a demonstration “A” disc test that will show the different bacitracin sensitivities of group A and non-group A Streptococcus isolates

3. Isolation of Staphylococcus

3.1. Special Materials:

H2O2, mannitol salts agar plate, example of S. aureus and S. epidermidis on mannitol salts agar plate.

3.2. Procedure:

Observe the colony morphology of any isolates on these plates. Using mannitol fermentation and catalase tests, tentatively identify these isolates as Staphylococcus aureus or Staphylococcus epidermidis. If you think you have isolated a Staphylococcus from your throat, perform the catalase test on those colonies from the blood agar plates.
Record Results.

The Catalase Test
Purpose:	To differentiate staphylococci (positive) from streptococci (negative)
Principle:	Detection of the presence/absence of the enzyme catalase.
Mechanism:	In the presence of catalase, hydrogen peroxide is hydrolyzed to water and O2, with release of bubbles of oxygen.
Reagent:	3% Hydrogen Peroxide (H2O2)
Method:	Pick part of a colony of the desired sort and immerse it in a drop of 3% hydrogen peroxide on a microscope slide. Do a negative control, i.e., H2O2 but no added colony material. Look for bubbles.
Interpretation:	Positive - immediate strong bubbling. Negative - delayed or absent bubbling

Note: Blood agar contains catalase (in the blood cells), and will produce delayed, weak bubbling; try not to pick up any of the agar when picking colonies for this test.

Purify one catalase positive colony from your own or a neighbor's plate and streak to single colonies on a fresh mannitol salts agar plate. Incubate at 37°C overnight. Next period you will perform a Gram stain and a coagulase test, and determine antibiotic sensitivity for colonies on this plate.

4. Fecal Flora

4.1. Special Materials:

culturette

4.2. Procedure:

Each student will be given a culturette to obtain a specimen of their own fecal flora. The swab will be inserted into the rectum to obtain a fecal specimen. The swab with specimen is then placed in the culturette and the moisture vial is broken so the organisms will remain viable during transport. Take home a culturette for the fecal sample. Take your sample on the morning of the Fourth Lab for workup that day. Do not take it the night before – the bacteria will not survive well.

BEFORE LEAVING, PUT AWAY EVERYTHING AND CLEAN BENCHES AND THEN YOUR HANDS !!!