Materials requested used by our group:

Day 1: Gram Staining

1. Glass Slides
2. Gram Staining Set
3. Inoculating Loop
4. Alcohol Lamp
5. Unknown Broth

Day 2: Plated Media Inoculation

1. Alcohol Lamp
2. Inoculating Loop
3. Unknown Broth
4. 1 Blood Agar Plate
5. 1 MacConkey Agar
6. 1 Mannitol Salt Agar

Day 3: Biochemical Test

1. Coagulase Test
2. Catalase Reagent (Hydrogen Peroxide)
3. Oxidase Reagent (Microdase Disk)
4. Novobiocin Disk
5. Glass Slides and Test Tube
Methods

Day 1: Gram Staining

a. Make a bacterial smear by dipping the loop into the broth and spreading it in the glass
slide then fixing it via flame
b. For staining: Flood the smear with Crystal Violet for 60 sec then wash gently with water
c. Flood the smear with Gram’s Iodine for 60 sec then wash gently
d. Decolorize the smear with alcohol for 10 sec then wash gently
e. Flood the smear with safranin for 60 sec and wash gently
f. Observe microscopically via OIO

Day 2: Inoculation

a. Dip the loop into the bacterial broth aseptically

b. Do multiple streaking method on the plates used (2 BAP, MAC) aseptically.
c. Incubate for 18-24 hrs and Observe

Day 3: Biochemical Tests

Catalase Test

a. Fish a colony from the BAP media then spread it in a glass slide

b. Add a drop of Hydrogen Peroxide to the bacteria

c. Observe (+) = Presence of Bubble Formation
Oxidase Test (Modified Oxidase)

a. Fish a colony from the BAP media and then dissolve it in a water until turbidity is present

b. Add a drop of Microdase Disk aseptically to the tube

c. Observe (+) = Purple Color Change of the Disk

Coagulase Test (Slide Coagulase Test)

a. Drop Rabbit’s Plasma into slide

b. Fish a colony from BAP media then spread in a slide

c. Observe (+) = Presence of Clumping Factor within 10 seconds

d. If (-) it could be confirmed using tube coagulase as confirmatory
Coagulase Test (Tube Coagulase Test)

a. Drop 0.5 ml Rabbit’s Plasma into Test Tube

b. Fish a colony from BAP media then mix in a the tube

c. Observe (+) = Presence of Gel like Clot

Novobiocin Sensitivity Test (Novibiocin Disk)

a. Using BAP streak the colonies via overlapping

b. Then place the novobiocin disk in the assign place

c. Incubate for 24 hours

d. Observe Resistant if >14, Susceptible if <14.1
Interpretation

We based our result in the plated media and the three biochemical tests. Gram Staining is Gram
Positive(+) Cocci, Blood Agar Plate is Non hemolytic, Mannitol Salt Agar is Non Mannitol
Fermenter No yellow pigments of colonies, MacConkey Agar Non Lactose, Catalase Test is
Positive because of Bubble Formation, Modified Oxidase Test is negative because it has no
presence of purple color of the disk, Coagulase Test is Negative, Novobiocin Susceptible Test
Zone inhibition is greater than 16mm.

Microorganism Catalase Coagulase Mannitol Novobiocin

Staphylococcus epidermidis + - - sensitive

Our Unknown microorganism is Staphylococcus epidermidis.