Unknown Bacteria Lab Report

Introduction

The purpose to this lab was to isolate and identify two unknown bacteria from a mixed culture provided to us by our instructor. This study was done by applying all of the methods that have been instructed on thus far in microbiology laboratory class. Each test performed, provided us with some key information about the unknown microbes in question and how the bacteria function.

Materials and Methods

Over a two week period, eight prepared types of test media were provided to identify the assigned unknown mixed cultures. Not all of these tests were performed on every culture, as some were used only for gram positive or gram negative bacteria. The tests performed and what constituted a positive or negative test are as follows:

Lab day 1; today in lab we obtained the unknown mixed culture “041”and one brain-heart infusion agar (BHIA). The first step was the preparation of the medium, the bottom of the BHIA dish was labeled with the bacterium number, initials, and section; then divided into four quadrants. The second step, we used the septic technique to transfer a small amount of culture with a flame-sterilized inoculating loop to the first quadrant, flamed and cooled the loop again then transferred a small amount of the culture from the first quadrant to the second using the quadrant streaking method as illustrated on page 18 of the lab manual, repeating this process until all four quadrants were properly streaked.

Lab day 2; we collected our BHIA medium and began by identifying the morphology and cell-to cell arrangements of the colonies. Two different colonies were observed, the first colony was yellow in color and larger in size and the white colored colony was slightly smaller in size. As instructed, each colony was prepared for gram staining, one slide for the large yellow colony and one for the smaller white colony. After properly gram staining the slides as directed in chapter six of the lab manual, the smears were examined under the microscope. The findings concluded that the smaller white colony stained purple, signifying that this culture was a gram positive bacterium, with the morphology of grape-like clusters such as staphylococcus. The larger yellow colony stained pink with the morphology of rod shaped bacterium, which indicates this bacterium to be gram negative. To isolate the two unknown microorganisms for further testing, another BHIA medium was obtained, the bottom of the dish was divided into two sections and the lawn streaking procedure was performed, as illustrated on page 12 of the lab manual. One side of the BHIA medium was inoculated with the gram positive cocci and the other side with the gram negative unknown bacteria, completing this section of lab by placing the labeled medium into the 350C incubator for 48 hours.

Laboratory day 3; a series of tests that were specific to the unknown gram negative and gram positive cultures were performed. Only two tests were necessary for the gram negative bacterim, which included the Enterotube II and the Indole DrySlide test card. Because immediate results would be obtained from the Dryslide test, this experiment was performed first by using a flame-sterilized and cooled inoculating loop to transfer a small growth from the gram negative colony to a clean reaction area of the test card. After 30 seconds, a pink color in this area was observed, indicating a positive result for indole, the positive result was documented. After properly inoculating the Enterotube II as instructed by our lab instructor, the tube was labeled and placed it in the 350C incubator for 24 hours. There were six different test media that were performed on the gram positive bacteria, starting with the inoculation of the mannitol salt agar (MSA) dish, using a flame-sterilized and cooled transfer loop. Two loop tranfers of our isolated gram positive bacterium were placed onto the surface of the agar, applying the quadrant streaking technique as illustrated on page 18 of the lab manual, labeled our medium and placed it into the 350C incubator for growth and interpretation. On the sheep blood agar (SBA), the flame-sterilized and cooled loop was used to transfer two loops of the gram positive bacteria to the media. Only one-half of the plate was streaked, the inoculating loop was again and a small portion of the culture from the first quadrant was used to streak the second quadrant, repeating this step to inoculate the remaining quadrant. Using ethanol-flamed forceps, one Bacitracin and one Optochin disk were transferred onto the one-half quadrant, spacing the two disks approximately two centimeters apart. In the last step, a flame-sterilized and cooled transfer loop was used to place three stab marks into the second quadrantof the SBA media, labeled the dish and placed into the 350C incubator for 48 hours. The DNase dish was spot inoculated by transferring one loop of the gram positive bacteria with the flame-sterilized and cooled inoculating loop to the center of the plate, the media was labeled and placed it into the 350C incubator for growth and future interpretation. The labeled Enterococcus faecalis (EF) broth tube was inoculated as well as the 6.5%NaCl broth tube by removing the caps from the tubes and placing one loop of gram positive culture into each tube with the flame-sterilized and cooled inoculating loop, mixed the culture in to the broth and replaced the caps. After labeling the bile esculin agar (BEA) slant tube, the cap was removed, one loop of gram positive cocci was placed into the bottom corner of the slanted agar and the culture was streaked upward, towards the top of the agar, the cap was replaced and all three tubes were placed into the 350C incubator for growth and future analysis. For the final test, two drops of hydrogen peroxide (H2O2) were placed on the isolated colony of the BHIA medium, observed immediate formation of bubbles, as O2 was produced indicating a positive test for catalase.

Final lab day four; the test media were collected for explanation and interpretation, the results are as listed:

Gram Negative Enterobacteriaceae

DrySlide test card Positive (+) slide took on a pink color

Enterotube II вЂ"identification system

• Glucose Positive (+) color changed from red to yellow

• Lysine Positive (+) color changed from yellow to purple