Identification of Unknown Bacteria
Authors: Richard Hendricks, Jessica Prebish; NMU
Broth culture 16 was randomly selected by our group and subjected to qualitative tests for taxonomic identification. The culture did appear homogenous throughout the testing period and is currently retained by Northern Michigan University’s department of Microbiology. We suggest that culture 16 is an example of Escherichia coli.
Techniques used were in accordance with NMU Professor Dr. D. Becker’s lab manual (ISBN 0-390-53911- 2; McGraw Hill). Changes in protocol or interpretation are noted where they were implemented, but strict adherence to the manual prevailed.
Materials and Methods:
Microscope, incubator, and deionizer functioned correctly throughout testing period, with stains, dishes, agars, and test reagents readily available. Lab procedures are considered orthodox and usage thereof is noted chronologically.
We obtained a 24 hour old stock broth culture of the unknown specimen 16. It appeared turbid, reflecting a substantial amount of growth. To determine that the stock culture was pure, we performed a streak plate using loops of the stock broth on a fresh dish of nutrient agar and then incubated it for 48 hours at Standard Temperature and Pressure; 37C, 1 ATM (STP).
A Gram stain was then carried out to differentiate the unknown sample from a broad class to a more specific category of bacteria. The Gram stain distinguishes between Gram-positive and Gram-negative bacteria based on the composition of the cell walls. Gram-positive bacteria appear purple and Gramnegative bacteria appear pink after staining. The first Gram stain produced unsatisfactory results and was then repeated with a clear indication of negativity. Light pink staining was evident on the cells in the field of view (FOV) and a search of the slide revealed uniformity in the sample. From these results we concluded that unknown 16 was Gram-negative in nature, and therefore it could possibly be any of the following bacteria: Alcaligenes faecalis, Escherichia coli, Enterobacter aerogenes, Proteus mirabilis, Proteus vulgaris, or Pseudomonas fluorescens.
We then conducted a negative stain to reveal the shape of the cell and any extracellular features such as the presence of a capsule. The first negative stain was unsuccessful and was therefore repeated. The second stain yielded cocci and coccobacilli shaped cells occurring singly and also arranged in irregular clusters. These appeared somewhat sparsely distributed at 1000x with oil immersion. A simple stain was then performed because previously in lab, the negative stain made it hard to visualize the bacteria. The simple stain was prepared using a sample of bacteria from the 24 hour old broth culture. The appearance of the microbe was again coccus and coccobacillus in shape, yet clusters were grouped in greater numbers, and their concentration was higher for the same FOV. From the results obtained by the negative and simple stain, we assumed that the unknown sample might be Alcaligenes faecalis, due to the shape of the cells and their clustered pattern.
After 48 hours of incubation, the results of the streak plate were particularly successful. This revealed moist, colorless, and opaque streaks of colonies the size of pinpricks in quadrant one, diminishing to one 3 mm round colony in the fourth quadrant that had billowing or irregular edges. All colonies appeared to be the same color, size, and shape. We concluded that the stock broth culture was pure. A Potassium Hydroxyl (KOH) Gram test was then performed to confirm the results of our Gram stain and to assure that our bacteria was Gram negative. The KOH test revealed an extremely fine filament which adhered to a toothpick and stretched three to five mm before breaking from tension. This was first performed with a single loop from the streak plate prepared on 3/15/05 with...