November 8-10

6 Tests were done this week

1) Citrate
2) Indole
3)Nitrate
4)Urea
5) Oxidase
6) Kirby-Bauer Technique


RESULTS
Citrate- Negative test
Indole- Positive for indole because there was a quick appearance of a red layer at the top of the tube.
Nitrate- Positive test
Urea-Negative
Oxidase- Negative
Kirby-Bauer Technique-
Cell Wall
1. Penicillin = NO inhibition took place --> Resistant

2. Vancomycin= 14 mm --> Sensitive


Nucleic Acid
3. Novabiocin= No inhibition took place --> Resistant


Protein 
4. Tetracycline= 25 mm --> Sensitive

5. Erythromycin= 11 mm --> Resistant

6. Chloramphenical= 34 mm --> Sensitive

7. Neomycin= 18 mm --> Sensitive

CITRATE UTILIZATION TEST

Purpose 

To identify if a bacterium can utilize citrate as its sole source of carbon and energy.

Materials

Simmons citrate agar slant tube
Unknown Bacteria "K"

Procedure

I used aseptic technique to inoculate the Simmons citrate agar slant with a loopful of my unknown bacteria . I incubated it for 48 hours in 37 degrees C.
My slant did not turn from green to blue resulting in a negative test. 

Before inoculation

After being incubated. Note this was a negative test because there was no color change.

INDOLE

Purpose 

To determine the ability of some bacteria to split the amino acid tryptophan into indole and pyruvic acid.

Materials

Tryptone broth tube
Kovac's reagent in a dropper bottle 
Disposable gloves
Unknown Bacteria "K"

Procedure

I used aseptic technique to inoculate the tryptone broth tube with my unknown bacteria .
I incubated it for 48 hours in 37 degrees C.
After I incubated it I added 5 drops of Kovac's reagent to the culture

Before adding the Kovac's reagent

After adding Kovac's reagent: This is a positive test

NITRATE

Purpose 

To determine if a bacterium is able to reduce nitrate ions to either nitrite ions or to nitrogen gas. 

Materials

Nitrate broth 
Nitrate reagent A (sulfanilic acid) in a dropper bottle. 
Nitrate reagent B (dimethyl-alpha-naphthylamine) in a dropper bottle 
gloves
Unknown Bacteria "K"

Procedure

I used aseptic technique to inoculate the nitrate broth tube.
I then incubated the broth for 48 hours in 37 degrees C.
After the broth was done being incubated, I added 5 drops of both reagent A and B to the broth. I then gently shook the tube to mix the reagents in with the broth.
A pink color developed within 1 minute which means my bacteria tested positive for nitrate reduction.

Nitrate Broth

Nitrate broth after Incubation

Positive test!

Nitrate Reagents A & B that were used in the Nitrate broth after being incubated

UREA

Purpose 

To determine the ability of a bacterium to hydrolyze urea.

Materials

Urea-containing broth
Unknown Bacteria "K"

Procedure

I used aseptic technique to inoculate the urea broth with a loopfull of bacteria from my unknown culture.
I then incubated it for 48 hours in 37 degrees C although the directions said to incubate it for no more than 24 hours.
There was no change of color. The broth reminded yellow and did not turn pink. Thus my test was negative for Urea

Negative test for urea

Urea broth

OXIDASE

Purpose 

To determine if bacteria have cytochrome oxidase, a participant in electron transport during respiration. 

Materials

Oxidase reagent - N,N,N,N tetramethyl-p-phenylenediamine: the crushable ampule.
Sterile wood stick
piece of filter paper
Agar plate used for the Kirby-Bauer technique. 
Unknown Bacteria "K" in a new slant culture 

Procedure

I first prepared a new agar slant of my unknown bacteria using the procedure done in earlier tests.
I then incubated my new slant for 48 hours in 37 degrees C.
After incubation I used the crush-able ampule and I added a few drops of the oxidase reagent to the bacterial colony growing on my agar plate I used for the Kirby-Bauer technique.
I transferred some of the bacteria from my new slant onto the sterile wood stick and streaked the filter paper with it. A few drops of the oxidase reagent was applied.
Nothing happened which means my test was negative.

New agar slant to be inoculated

After incubation for 48 hours

Negative oxidase test

KIRBY-BAUER TECHNIQUE

Purpose 

To determine the sensitivity of a bacterium to several antibacterial medicines. 

Materials

Mueller-Hinton agar plate, 4 mm thick
antibiotic disk cartridges
forceps
95% ethanol in a beaker
ruler, millimeter
Unknown Bacteria "K" 


The Antibiotic Disks Used


Cell Wall
1. Penicillin
2. Vancomycin

Nucleic Acid
3. Novabiocin

Protein 
4. Tetracycline
5. Erythromycin
6. Chloramphenical
7. Neomycin

Procedure

I used aseptic technique to inoculate the Mueller-Hinton agar plate. I used an inoculating loop contaminated with the unknown bacteria to inoculate the entire surface of the agar with a closely spaced back-and-forth motion. I then inoculated the the agar surface a second and third time in different directions.

On the back side of my agar plate I labeled the areas where all 7 different cartridges would be placed evenly.  Before placing each different cartridge on the plate I dipped the forceps in alcohol, then burned the alcohol off.  I made sure to apply light pressure to the disks, using the forceps, to prevent them from fall off when the plate was to inverted for incubation.

I incubated it for 48 hours in 37 degrees C, making sure the plate was inverted.

After incubation I measured out the diameter in mm of the zone of growth inhibition for each disk. To do this I placed the ruler on the underside of the plate.

Agar plate I streaked with my unknown bacteria

The addition of the 7 different antibiotic disks

Here you can see how the different antibiotics affected my bacteria.

NOTE: Number 1 disk is above the MS and the numbering continues counter-clockwise  with number 7 in the center

.

My diameter results were as follows:
Cell Wall
1. Penicillin = NO inhibition took place --> Resistant
2. Vancomycin= 14 mm --> Sensitive

Nucleic Acid
3. Novabiocin= No inhibition took place --> Resistant

Protein 
4. Tetracycline= 25 mm --> Sensitive
5. Erythromycin= 11 mm --> Resistant
6. Chloramphenical= 34 mm --> Sensitive
7. Neomycin= 18 mm --> Sensitive