Unknown Bacteria Identification

Hashem mohammad zyout Sec:4

ID : 121201
We were asked to determine the type of bacteria from the unknown sample through
the tests that we studied previously
We choose unknown 3 .

At the first we started with growth in 3 types pf agar: Nutrient, Blood,

MacConkey.
For growth the culture we do:

1. Sterilize the place with alcohol

. 2Sterilize the lobby using heat
. 3Take a sample from an unknown tube and wipe it on the surface of the agar
.4 Putting the agar in the incubator for 24 hours

 We did this for all agars

 After that, we made a couple of stain tests, such as a simple
stain to confirm purity and gram stain to determine if our
sample was gram negative or positive.

Simple stain :
1. Take a sample from Nutrient agar and wipe it on the slid by drop of water .
2. Leave it to dry
3. Add Methylene blue stain to heat-fixed slide and leave for 2 minutes.
4. Hold the slide in an angle and rinse gently with water from the squirt bottle.
5. Blot gently with bibulous paper to get rid of excess water.
6. Examine under oil immersion .

Result : we find that our sample is pure of one type gram

stain, and that it was Gram-negative

To make sure we used the gram stain

Gram stain :
1. remove a very small amount of bacteria from the MacConkey
agar
2. Smear the bacteria in the drop of water on your slide
3. Let the slide air dry completely.
4. Heat-fix the slide by running it through the flame 3-4 times 

5. Stain with crystal violet for 1 minute, then rinse gently with water.

6. Treat with iodine mixture for 1 minute, then rinse gently with water.

7. Rinse with ~3-6 drops of 95% alcohol to decolorize, then rinse gently with water.

8. Counterstain with safranin for 30 seconds, then rinse with water.

9. Rinse with water and tap dry. and blot gently with filter paper.
- After confirming our sample had one type of strain of
bacteria, and that it was Gram-negative Bacilli we began
doing the biochemical tests for Gram (–) Bacteria
 KIA
 IMViC.
 Oxidase test–ve (no reaction)

KIA:
- As to the KIA test, or Kliger’s Iron Agar, the results came
back as Acid/Acid with the formation of CO2 gas, which
wasn’t definitive as this result is shared with seven other
types of bacteria.
 IMVIC:

As to the IMViC test, split into Indole, Methyl Red, Voges-

Proskauer and Citrate test, in order the results were:
1- Indole: positive
2- Methyl red: Positive
3- Voges-Proskauer: Negative
4- Citrate: Negative
Which is a common and normal result for the Escherichia coli or E.
Coli

Finally, after inquiring with the Lab Master, I found out that
finding the species of E. Coli is complicated and has its own tests
and was therefore allowed to stop after knowing what Type of
Bacteria it is