Identification of unknown #7

Karen Williams

November 15, 2014

Introduction

The purpose of this exercise is to identify two bacterial unknowns. By identifying the

bacteria , we can detail information on weather or not the bacterium is pathogenic or harmful as well

as those that may be beneficial to us in some way. This is an important aspect in research.

Bacterium plays an important role in environment , they can manufacture their own food by

photosynthesis and some can derive nutrition from organic substances. Bacterium in our body are

beneficial due to digesting food – spoilage. Tools that aid in accurate bacterial identification include

microscopes for morphology , staining , biochemical testing and serological testing as well as various

types of cultural media.

Methods and Materials

In identifying unknown bacteria #7 the procedures that were followed. On a labeled slide

a small drop of water was placed using an inoculating loop. Next the unknown bacteria was added

to water and mixed. The drop was then spread out in a thin layer and left to air dry. After the smear

was fully air dried, the slide was passed through the upper part of a flame two times, using a slide

holder. This heat fixed the slide for better staining. The next step was the Gram stain. The gram

stain is differential stain in which a decolorizing step occurs between the applications of two basic

stains. Finally the slide was thoroughly dried , it was viewed under microscope at 100x oil

immersion. Two slides that had isolated colony, under the microscope showed gram positive and

gram negative. A TSA, Trip tic Soy Agar using the quadrant streak method. Isolation and sterilization

steps were taken. The plate was incubated at 37 degrees for 24-48 hours. After this period plate was

examined for growth and color.

Following procedure for the Gram positive unknown #7, Phenylethyl Alcohol Agar (PEA)

was used, which is a selective medium that encourages growth of Gram positive organisms while

inhibiting the growth of Gram negative.

Catalase test was performed. This test detects an organisms ability to produce catalase,

which cleanses hydrogen peroxide produced in the electron transport chain during respiration.

Performing this test a drop of 3% H2O2 on a glass slide, using plastic loop, transfer bacterial growth

from the agar and mix into H2O2. Once you see bubbling, that will indicate the presence of catalase.

The catalase test was positive.

Mannitol Salt Agar (MSA), test is selective and differential, the last test for the Gram

positive unknown performed . It is high salt concentration, inhibits the growth of most bacteria.

It contains the sugar, Mannitol and allows pheno red for the pH indicator. Staphylococcus aureus

will ferment the mannitol and form yellow in the reddish agar, due to the phenol red becoming

yellow in the presence of fermentation acids.

The Gram negative unknown #7, first test is the Eosin Y Methylene Blue (EMB),

this agar was used as a selective medium and differential medium. It inhibits the growth of

Gram positive bacteria, allow the growth of Gram negative bacteria. Encourages growth of fecal

coliforms and differentiate them. Also contains lactose ans sucrose which serve as fermentable

carbohydrates sources. EMB incubation 37 degrees, for 24 to 48 hours. A strong acid

production colonies are purple to black, with or without metallic green sheen. The bacteria

was positive for lactose.

The next test performed was