Micro-Unknown Research

Student McStudent BIOL 2420 – 3U2

Unknown Report Activity Log #2

Date Activity Media/ Reagent

Results/Observations/ Comments

10/18

Received broth of unknown bacteria # _2_. At 3:00 p.m, the unknown was streaked for isolation on Tryptic soy agar (TSA). This streak plate (#1) was incubated at 37oC for 48 hrs at 4:05. At 3:15, the unknown was streaked again for isolation on tryptic soy agar. This streak plate (#2) was incubated at 25oC for 48 hrs.

TSA Cultural characteristic of the broth culture: Sediment- accumulation in the bottom of the tube

10/20

At 2:55, the plates from 10/18 were observed. At 3:05, one isolated colony was removed from streak plate #2, streaked for isolation, and a stock plate was made on tryptic soy agar. This stock plate was incubated at 37oC for 48 hrs at 4:05 p.m. At 3:15, a Gram Stain was performed on a clean slide from an isolated colony from streak plate #2. Streak plate #1 (from 10/18) was relabeled as the reserve plate and was stored at 25oC at 4:05. The slide with the Gram Stain and streak plate #2 were both thrown away at the end of class in the biohazard bag (at 4:00).

TSA

Slide

Crystal Violet

Gram’s Iodine

Acetone/Alcohol

Safrinin

Cultural characteristics of the colonies:  Form: at 37° C= circular &

at 25°C= punctiform  Size: at 37° C= 1.5 mm &

at 25° C= 0.5 mm  Margin: Entire  Texture: Butyrous-

buttery/smooth  Elevation: Flat/ Slightly

raised  Surface Appearance:

Glossy and smooth- also opaque

 Pigment: +/- (both)

Results of Gram Stain: Gram Positive and Cocci (Staphylococcus)

10/25

At 2:55, the stock plate from 10/20 was observed and an isolated colony was used at 3:00 for the Catalase test. In this test, the substrate hydrogen peroxide, H2O2, was used to determine the liberation of O2 gas. The slide was discarded at 3:10 in the biohazard bag. At 3:15, an isolated colony was removed from the stock plate and streaked for isolation on the Blood Agar Plate (BAP).

TSA

H2O2

Slide

BAP

Catalase Results: (+) Bubbles were formed from the unknown after H2O2 was added.

Unknown #2

10/25 Cont.

This plate was incubated at 37oC for 24 hrs at 4:05 p.m. At 3:25, another isolated colony was taken from the stock plate and inoculated into Nitrate broth. This broth was incubated at 37oC for 48 hrs at 4:05 p.m. At 3:35, Phenol Red Broth was inoculated with the unknown. This is four different broths that are Glucose, Sucrose, Mannitol, and Lactose. The broths were incubated at 37oC for 24 hrs at 4:05 p.m.

Nitrate Broth

Phenol Red Broth (Glucose, Sucrose, Mannitol, Lactose)

No more results/observations for the day.

10/26

Professor removed both the Blood Agar Plate and Phenol Reb Broth from the incubator and placed them in the fridge at 4:05 (24 hours later).

BAP

Phenol Red Broth N/A

10/27

At 2:55, the Blood Agar Plate from 10/25 was observed. At 3:00, this blood agar plate was disposed of in the biohazard bag. At 3:05, the Phenol Red Broth, from 10/25, was observed for growth and changes. At 3:15 the broths were put into the biohazard waste bag. At 3:20, the Nitrate Broth from 10/25 was observed for growth. Noticing growth was present, five drops of the reagents sulfanilic acid and α- naphthylamine, were added to the tube. After turning red, it was placed in the biohazard waste bag at 3:35. At 3:40, an isolated colony from the stock plate was used to streak for isolation in the Mannitol Salt Agar (MSA), which was incubated at 37oC for 48 hrs at 4:05 p.m. At 3:50, an isolated colony from the stock plate (from 10/20) was used to produce another stock plate. This TSA, stock plate #2, was incubated at 37oC for 48 hrs at 4:05 p.m. The first stock plate (from 10/20) was thrown away at 4:00, in the biohazard waste bag.

BAP

sulfanilic acid and α-naphthylamine

Nitrate Broth

Phenol Red Broth

TSA

MSA

TSA

Hemolysis Test Results: strongly Beta: The stab marks had clear zones around them. The rest of the blood agar had a yellow tint.

Nitrate Reduction Results: A red precipitate formed

Phenol Red Broth Results: All four broths changed into a yellow color. No gas was visible.

Unknown #2

Date Activity Media/ Reagent

Results/Observations/ Comments

11/1

At 2:55, the Mannitol Salt Agar from 10/27 was observed. At 3:00, this Mannitol Salt Agar was disposed of in the biohazard bag. At 3:05, a clean slide was used to hold a small amount of rabbit plasma for the Coagulase Test. The tryptic soy stock plate #2 from 10/27, was then used to transfer a small amount of the unknown into the rabbit plasma on the slide. After stirring the plasma and unknown together and noticing clots, the slide was disposed of in the biohazard bag at 3:20.

MSA

TSA

Slide

Rabbit Plasma

Mannitol Salt Agar Results: Changed into a very bright, yellow color and lots of growth was visible. Growth was in all quadrants.

Coagulase Test: Clotting was visible.