October 18 & 20

This week I completed various tests

• Gelatin Hydrolysis test
• Fermentation
• Tripple Sugar Iron Agar test (TSIA)
• Methyl Red Test
• Litmus Milk Reaction

 RESULTS:  

• Gelatin= negative 

• Fermentation= Sugar and Acid were the end products. Gas was produced in both the glucose and lactose vials that were in the test tubes. My bacteria mostly used glucose and lactose. Not the sucrose, although there was some bacterial growth. 

• TSIA = I observed a yellow slant/ yellow butt which = acid slant/ acid butt with gas. There was also a break in the agar. Which means that glucose and lactose &/ or sucrose were fermented.

•  Methyl Red test = Positive test  for mixed-acid fermentation pathway. (my broth turned red)
• VP Test= negative. My broth didn't change colors.

• Litmus Milk reaction= results still unknown. Will not know until 7 days pass.

 Gelatin

Purpose:

To determine the ability of bacteria to hydrolyze gelatin. 

 

Materials needed:

Nutrient gelatin deep tube
unknown bacteria in slant

Procedure:

I labeled the test tube properly and used aseptic technique to inoculate the gelatin by stabbing the gelatin with an inoculating needle with bacteria from my unknown.

I stored the gelatin in the incubator at 37 degrees C for 48 hours then I placed it in the fridge for 15minutes. After removing the gelatin from the fridge I checked it for liquidation and the test came back negative because there was no liquidation.

Gelatin preinoculation

Gelatin after incubation and refrigeration. Note that liquidation did NOT occur. 

Fermentation of Carbohydrates

Purpose:
To determine the ability of some bacteria to ferment a particular carbohydrate. 


Materials:
3 tubes of phenol red- sugar broth with Durham tube

                                   The three sugars used were sucrose, lactose, and glucose. 

Unknown bacteria slant

Procedure:
Labeled the test tubes properly 
Used aseptic technique to inoculate each sugar broth with a loop full of unknown bacteria. 
I incubated the now contaminated broths in 37 degrees C for 48 hours although the instructions said for 24 hours. 
After incubation I checked each test tube for acid production or acid and gas production. 
My Lactose was very pale yellow. The glucose was a brighter shade of yellow and the sucrose stayed red. As seen in the picture below.

Lactose before being contaminated

Glucose before being contaminated

sucrose before being contaminated

Results. From Left to right: Lactose, Sucrose, Glucose.  

                                            Note the gas bubbles in both the lactose and glucose samples. 

 Triple Sugar Iron Agar Test (TSIA)

Purpose:

To differentiate among the gram-negative enteric bacilli as to their ability to ferment glucose, lactose, and sucrose and to produce H2S. 

 

Materials needed:

Triple sugar iron agar slant tube
unknown bacteria in slant
Inoculating needle with a straight wire

Procedure:

I labeled the TSI tube.
Used aseptic technique to remove a small amount of my unknown bacteria with an inoculating needle. I stabbed the bacteria into the TSI agar butt to about three-quarters deep. As I withdrew the needle I streaked in a zigzag pattern along the slant surface.
I incubated the tube at 37 degrees C for 24 hours and noted the changes that occurred.
My tube turned from red to completely yellow. Which means my slant is acidic and the butt had gas produced.

The Agar slant before being inoculated

After 24 hour incubation. Note the color change of the agar from red to yellow.

A better view of the color change that occurred

Methyl Red Test/ VP Test

Purpose:
To determine the ability of some bacteria to ferment glucose via mixed-acid fermentation.  


Materials:
Methyl red (MR-VP) broth tube
Unknown bacteria culture in slant
Barrett's reagent A
Barrett's reagent B
Disposable gloves
Methyl red in a dropper bottle 


Procedure:
Labeled the test tubes properly 
Used aseptic technique to inoculate the MR-VP broth with a loopful of my unknown bacteria. 
After incubating the broth at 37 degrees C for 48 hours I separated the broth into a second test tube (making sure there were equal amounts of contaminated broth in each tube.)
In the first tube I added 5 drops of methyl red. Gently swirled the tube to mix the broth culture and pH indicator.
After swirling I noted that a red color was formed. This redness yields a positive test for the mixed-acid fermentation pathway.

Broth after 48 hour incubation but before splitting it into two test tubes.

MR-VP positive test results

In the second tube I performed the VP test. 

I first put on the gloves before handling the Barrett's reagents.

VP negative test

I added 15 drops of Barrett's reagent A and 5 drops Barrett's reagent B.

After swirling the broth for a few minutes there was no color change which results in a negative test. 

Litmus Milk Reaction

Purpose:

To differentiate among bacteria as to their ability to utilize lactose, protein, and litmus in litmus milk. 

 

Materials needed:

Litmus milk tube

unknown bacteria in slant

Procedure:

I labeled the test tube properly and used aseptic technique to inoculate the litmus milk with a loopful of my unknown bacteria. 

I inoculated the tube at 37 degrees C for 48 hours. examined the tube, and placed it back in the incubator for another 4 days. 

Preinoculation

After 48 hours in the incubator