Identification of Unknown Bacteria

Brian J. Underhill

Monday/Wednesday Lab; 2:00-4:00 p.m.

I. Unknown bacteria: Echerichia coli

II. Unknown broth number: 143

III: Gram stain and morphology results: Gram negative bacteria; individual bacterium in short rod shapes (bacillus).

IV: Procedures: In order to separate the initial broth mixture into two distinct bacteriological cultures, two fresh agar plates were poured and allowed to cool. An inoculating loop of the broth was then streaked on one, and the plate was incubated. Two distinct cultures appeared: one was reddish-orange in color, the other was whitish. The red colonies (Serratia marcescens) dominated the streak plate, but several small white colonies remained sufficiently isolated to allow them to be separated.

A small sample of the white bacteria (the unknown) was streaked onto the second agar plate in order to create a pure colony. After incubation, the plate revealed only white colonies, indicating that the unknown had been successfully isolated from the original broth mixture. Because this second plate had been allowed to incubate for an extended period of time (a week at room temperature), it was too mature to be used to gram stain. Therefore a sample was transferred to an agar slant in preparation for gram staining.

The new slant was incubated for ~23 hours, then transferred to the lab refrigerator and kept for about 96 hours at 4° C to inhibit cell growth. (Gram staining should be done when the bacteria is 18-24 hours old; refrigeration slowed bacterial growth enough that the gram stain process was effective even after nearly 100 hours – a factoid I found interesting in itself).

Two slides were prepared with samples from the agar slant. The first was a very heavy sample, and the decolorization process took longer than the normal 10-15 seconds. But it was obvious from the purple stain (crystal violet) that was draining from the sample, that the ethyl alcohol was, indeed, washing away the initial stain. After counterstaining, it appeared that the sample was gram negative. Although the microscopic field was packed with bacteria (with few isolated cells), I still concluded the sample was a gram negative bacillus.

To be certain, I fixed the second slide and gram stained it as well. This second slide was a much better sample and exhibited all the characteristics of a gram negative stain (e.g., the alcohol rinse took about 10-15 seconds, isolated bacterium were clearly visible in the viewing field, etc.)

My conclusion that the bacteria was Escherichia coli was based on several factors:

1) The morphology of the bacteria (individual bacillus)

2) The gram reaction of the bacteria (gram negative)

3) Information provided (e.g., that we had already seen the unknown bacteria in the lab)

4) Sketches I had created in my lab book based on previous samples of E. coli