Specific Identification of Coliform bacteria present in Cape Fear River Sample
Purpose: To perform tests on known cultured coliforms to identify specific bacteria types that are
present. Last week, you cultured individual coliform colonies from a culture plate onto a
new MacConkey streak plate. New purple/pink colonies should have grown. These
colonies represent coliforms which are present in the original water sample. This week,
you will attempt to grow your bacteria on several different media. Your bacteria’s specific
response to the different media types will act as identifying characteristics that are specific
to a particular coliform type. By using the provided key, you will be able to correctly
identify a particular bacterium that is present in the original water sample.
Note: Your results may vary from the results seen by other groups. Why?
Materials:
1 SIM Agar Tube (Stab)
1 Simmons Citrate Agar Tube (Slant)
1 Christensen Urea Agar Tube (Slant)
1 Inoculating Loop
1 Inoculating Needle
Bunsen Burner
Flame Striker
Labels
Amphyl
Procedure:
1. Collect all of your materials and bring them to your station.
2. Label the tops of: SIM Agar Tube, Simmons Citrate Agar Tube, and Christensen Urea Agar
Tube. Your labels should include date, time, and your initials.
3. Acquire your streak plate cultures from last week. Observe the plate closely and choose a
bacteria colony that you want to test. The colony should be distinctly separate from other
colonies. Use a marker to circle the colony from the bottom of the plate. Also, you should
record any visible characteristics of the colony in your notebook (i.e. color, shape, sheen,
texture, edges, relative size, etc.)
4. Ignite the Bunsen burner to a gentle blue flame to encourage a reasonably sterile environment.
5. Clean the lab bench top with amphyl solution.
6. Wash hands thoroughly with soap and warm water. Dry hands completely.
7. Follow each of the procedures below very carefully!
SIM Media
1. Flame sterilize your needle (should turn red-orange for at least 5 seconds) and ALLOW TO
COOL COMPLETELY – Do Not Set Needle Down!
2. Lift the lid on the culture plate just high enough to insert your needle and gently touch and
lift a small amount of the culture. Pull the needle out without bumping it on the lid. Close
the lid on the culture plate and set aside.
3. Hold the needle in your “writing hand” like a pencil. Pick up the SIM agar tube with your
other hand. With the pinkie of your “writing hand” unscrew the cap on the SIM agar tube –
Do Not Set This Cap Down!
4. Flame the top of the tube for 3-5 seconds to sterilize it.
5. Insert/Stab the needle into the center of the SIM agar all the way to the bottom. Remove the
needle from the tube. Do Not Touch the sides of the tube!
6. Flame the top of the tube for 3-5 seconds again and screw the cap back on the tube.
7. Set the tube back in the rack.
8. Flame the needle to sterilize.
Citrate Media
1. Flame sterilize your loop (should turn red-orange for at least 5 seconds) and ALLOW TO
COOL COMPLETELY – Do Not Set Loop Down!
2. Lift the lid on the culture plate just high enough to insert your loop and gently touch and lift a
small amount of the culture. Pull the loop out without bumping it on the lid. Close the lid on
the culture plate and set aside.
3. Hold the loop in your “writing hand” like a pencil. Pick up the Citrate agar tube with your
other hand. With the pinkie of your “writing hand” unscrew the cap on the Citrate agar tube
– Do Not Set This Cap Down!
4. Flame the top of the tube for 3-5 seconds to sterilize it.
5. Insert the loop to the bottom of the Citrate slant and lightly drag the loop up the surface of
the slant in a “serpentine” manner. Remove the loop from the tube. Do Not Touch the sides
of the tube!
6. Flame the top of the tube for 3-5 seconds again and screw the cap back on the tube.
7. Set the tube back in the rack.
8. Flame the loop to sterilize.
Urease Media
1. Flame sterilize your loop (should turn red-orange for at least 5 seconds) and ALLOW TO
COOL COMPLETELY– Do Not Set Loop Down!
2. Lift the lid on the culture plate just high enough to insert your loop and gently touch and lift a
small amount of the culture. Pull the loop out without bumping it on the lid. Close the lid on
the culture plate and set aside.
3. Hold the loop in your “writing hand” like a pencil. Pick up the Urease agar tube with your
other hand. With the pinkie of your “writing hand” unscrew the cap on the Urease agar tube
– Do Not Set This Cap Down!
4. Flame the top of the tube for 3-5 seconds to sterilize it.
5. Insert the loop to the bottom of the Urease slant and lightly drag the loop up the surface of
the slant in a “serpentine” manner. Remove the loop from the tube. Do Not Touch the sides
of the tube!
6. Flame the top of the tube for 3-5 seconds again and screw the cap back on the tube.
7. Set the tube back in the rack.
8. Flame the loop to sterilize.
Clean-up:
1. Clean your lab bench top with amphyl.
2. Wash your hands thoroughly with soap and warm water.
3. Use the antibacterial hand sanitizer before you leave.
Cultures will be incubated at 37ْ C.
 SIM test can be read within 18-24 hours
 Citrate test can be read within 4 days
 Urease test can be read within 8-48 hours
Use the following sheet for evaluations and identification.
Evaluation Key:
MacConkey Agar Plate
This is a Lactose Test: Does the organism utilize lactose?
+ = purple/reddish colony
- = white or other color colony
SIM Media Stab
This is a Sulfur Test: Does the organism reduce sulfur, and produce hydrogen sulfide gas?
+ = black color
- = yellow color
Indole Test (Add one drop of Kovac’s reagent to the SIM tube)
Does the organism produce indole using tryptophanase?
+ = layer of pink color on the top (allow to react 5-30 minutes)
- = no pink color showing after at least 30 minutes (max. wait time 120 minutes)
** The motility test can be deceptive because movement within the agar may occur but be very difficult to see.
Be lenient when using this characteristic in identifying your bacteria.
Citrate Media:
Does the organism use citrate as its sole source of carbon?
+ = change in color from green to blue at top layer
- = no change in color, all of slant is green
Urease Media:
Does the organism hydrolyze urea?
+ = color change to intense pink/red
- = no color change; slant is pale yellow/buff
Use the above results to identify your specific bacterium with the following table:
Bacteria Genus
Escherichia
Klebsiella
Yersinia
Enterobacter
Citrobacter
Shigella
Salmonella
Proteus
Edwardsiella
Lactose
Sulfur
Indole
Motility
Citrate
Urease
+
+
+
+
+
-
+/+
+
+
+
+
+/+/+/+/+
+
+
+
+
+
+/-
+
+
+
+
+/-
+
+
+
+
-
Slow
Notes:
1. Tests that may have variable results are denoted with +/-. These bacteria can not be definitively identified
with that particular test; hence why we do all of the other tests!
2. If you get test results that are different from anything that you see in the above table, then you have a
bacterium that could not be identified with the tests that we ran. Write this on your data sheet.
3. “Slow” denotes that the reaction may take the entire incubation time before the bacteria gives a positive
result. Refer to your lab for incubation times.