Joanna Burel
04/20/15
Dr. Leonard
Prassanna Rao
Microbiology BIO 2010
Section 03

Isolation and identification of unknown bacteria

ABSTRACT
A mixture of two unknown bacteria was obtained and handed out in microbiology lab. It contained one gram positive and one gram negative bacteria. The gram positive rod bacteria had small round yellowish morphology able to grow at 37 degrees Celsius. It was found to be a facultative anaerobe, oxidase positive, fermenting glucose and MR, VP, indole and catalase tests negative. It was found not to grow on DA, EMB and XLD agar and to grow on PAA. These were signs that the bacteria may be a species of bacillus based on Bergey’s manual. The second bacteria was a motile, gram negative rod which was able to grow at 37 degrees Celsius. The bacteria produced larger cloudy colonies. It was found to be an aerobic motile bacteria. It was able to grow on XLD and EMB, DA agar but not MSA. The microorganism was oxidase, catalase and MR positive and VP, indole negative. It was most likely salmonella typhimurium based on the table of Bergey’s manual.

INTRODUCTION
Microbiology is the study of tiny organisms too small to be seen with the naked eye and requires the use of a microscope. These tiny organisms are called microorganisms and include bacteria, virus, fungus and protozoa. Microorganisms play a major role in many areas of everyday life. One important reason to study them is because they are often a cause of diseases. Microorganisms can live and grow in different conditions. The main goal of this lab was to identify two different bacteria from a list of 24 bacteria (Bergey’s manual) depending on these living conditions and their morphology. In order to do this we performed a number of biochemical tests such as differential and selective tests on the separated forms of the bacteria to obtain independent results. The two bacteria were called A and B. In this project, bacteria A and bacteria B were separated from the mixture using the streak plate isolation. This method consists of an agar plate providing nutrients for growth of the bacteria. After isolation, the two different cultures appear distinctively. Pure culture can then be harvested and used for the rest of the experiment to perform the different tests. Streak plate is also helpful to determine general aspect of bacteria like color of colonies, shape, size and arrangement. The pure cultures can be grown in slants containing agar which allow them to multiply and create a stock. Then, gram staining is a major step in identification of unknown. It helps scientists determining gram-positive and gram-negative bacteria and visualizing their shape. When bacteria appear purple on gram staining, they are gram-positive. Their thick peptidoglycan retain the crystal violet stain. When bacteria appear pink or red, they are gram-negative. Their thin peptidoglycan allows decolorizer to wash crystal violet stain and the safranin colors them in pinkish/red. Microorganisms have an ability to grow and develop with or without oxygen. This ability is an important hint in identification. Agar deep stabs are used to determine this ability. Aerobic species would grow on top of the agar whereas facultative anaerobic or anaerobic would grow deep in the agar. Selective media ensure growth of specific species of bacteria and inhibit growth of the others. This is why uses of selective media is important to identify unknowns. Selectivity depends on components of the media and living conditions of microorganisms. Four different selective media were used, Phenylethyl Alcohol Agar (PAA), Desoxycholate Agar (DA), Mannitol Salts Agar (MSA), Xylose Lysine Desoxycholate Agar (XLD), and Eosin Methylene Blue Agar (EMB). PAA media is selective for gram-positive cocci as streptococcus and staphylococcus. These organisms are not inhibited by phenylethyl alcohol. It inhibits gram negative bacteria. MSA is composed of Sodium Chloride in high concentration. It then selects bacteria that do not undergo dehydration. This media kills most of listed bacteria except staphylococcus. Plus, color of the plate after inoculation informs about which species it is. A yellow growth represents an organism that can produce acid whereas a red growth isn’t. DA is composed of desoxycholate citrate which inhibits the growth of gram positive bacteria. A change of color informs about pH. A red color signifies a pH of 6.8 and below and a yellow color signifies a pH of 8.0 and up. XLD agar selects for shigella and salmonella species. The agar contains sodium desoxycholate that inhibits the growth of other bacteria. Color of the growth helps identify further, a pink growth is a shigella species whereas a pink/red growth is a salmonella species producing ferric ammonium citrate precipitate an...

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