Day 3 - Odds and Ends: Miscellaneous Tests

We performed some miscellaneous tests to discover some different properties of our bacteria. 

TSI (Triple Sugar Iron) Agar Test:
This test helps differentiate between different types of gram-negative bacilli, by testing the bacteria's ability to ferment glucose, lactose, and sucrose and to produce H2S.

Materials:
TSI agar slant tube
Pure culture of our bacteria on an agar slant
Inoculating needle

Steps:
1.  Label TSI tube with our group number and test type.

2.  Using aseptic technique, inoculate the TSI agar slant from our bacteria's agar slant.  For this inoculation, we stabbed the bacteria into the TSI agar slant and then as we withdrew the needle, we streaked side to side along the surface of the agar. 

TSIA Test

3.  Incubate the tube at 35 degrees for 24 hours.

4.  After incubation, examine the tube for color changes, gas appearance, and black precipitate. 

5.  Discard the tube properly.



Results:


Discussion:  We have a positive result.  Our bacteria produced acid in the slant and butt of the tube, along with gas.  Based on this, our bacteria uses glucose, sucrose, and lactose.


Urea Hydrolysis Test:
This test determines whether our bacteria can hydrolyze urea.  If so, the urea will be hydrolyzed into carbon dioxide and ammonia, which will raise the pH of the medium.  So a pH indicator will enable us to figure out if our bacteria hydrolyzes urea.


Urea

Materials:
Urea-containing broth
Pure culture of our bacteria on an agar slant

Steps:
1.  Label the urea tube with our group number and type of test.

2.  Use aseptic technique to inoculate the urea broth.

3.  Incubate the urea tube at 35 degrees for 24 hours.

4.  After incubation, examine the tube for color change.  A bright pink color indicates a positive result.

5.  Discard tube properly.

Results:


Discussion:  We had a negative result, so our bacteria cannot hydrolyze urea into carbon dioxide and ammonia.  We incubated the culture longer to verify these results.


Litmus Milk Reactions: 
Since bacteria differ in their abilities to digest lactose, protein, and litmus in litmus milk, we will use this to help determine the identity of our bacteria.  There are 4 possible results: 1) acid production through fermentation of lactose (indicated by lighter-colored medium), 2) acid curd (red at top) with litmus reduction, 3) peptonization with alkaline reaction, 4) alkaline reaction, and 5) no reaction.





Litmus Milk

Materials:
Litmus milk tube
Pure culture of our bacteria on an agar slant

Steps:
1.  Label the litmus milk tube with our group number and type of test.

2.  Use aseptic technique to inoculate the litmus milk tube from our bacterial agar slant.

3.  Incubate the inoculated tube at 35 degrees for up to 7 days, checking every 24 hours.

4.  Discard the tube properly.


Results:


Discussion:  We have a positive result!  Our tube medium was a lighter color and soft curds formed.  This means that our bacteria ferments lactose into lactic acid (hence the color change) and forms curds in the process.



Motility Testing:
This test indicates whether our bacteria are motile.





 

Motility

Materials:
Tube with motility test medium
Pure culture of our bacteria in broth culture

Steps:
1.  Label the tube with our group number and type of test.

2.  Use aseptic technique to dip inoculating needle into our bacterial broth culture and stab our bacteria into the center of the motility test medium.

3.  Incubate at 35 degrees for 24-48 hours.

4.  After incubation, examine the motility tube.  If bacterial growth spreads out from the stab, it is a positive result for motility.

5.  Discard the tube properly. 


Results:


The uniform and cloudy appearance is due to our bacteria moving out from the center.
The original deep stab is not visible.

Our positive result (left) with another group's negative result (right)

Discussion:  Our bacteria likes to move!  It is definitely motile.  This verifies the hanging-drop slide we saw under the microscope, in which we saw our bacteria swimming.