Proteus Vulgaris Lab Report

Identification of Proteus vulgaris from an Unknown Sample
Lakhram Bhisham
March 31, 2016
01:447:390 General Microbiology
TA: Jennifer Goff

ABSTRACT
This report delineates how unknown #405 was identified as Proteus vulgaris out of a possible seven species of Enterobacteriaceae by applying various tests that are able to distinguish between members of the family. Inevitably, the results for many tests are identical across multiple species, which is expected due to the organisms’ evolutionary relatedness. However, some tests are specific to select organisms and contribute to the basis on which these species are differentiated. Of the seven possible organisms that unknown #405 could have been, only P. vulgaris tested positive for phenylalanine
Physiological Reactions and Motility Test Results. All of the possible unknowns were of the family Enterobacteriaceae, and thus, performed similarly on a number of tests. All organisms evaluated were facultative anaerobes, showing densest growth at the top of the fluid thioglycolate medium with lessening growth moving down the tube. This oxygen requirement is consistent with the conditions of the intestines, where enteric bacteria are found. Facultative anaerobes generate superoxide ions whenever they respire aerobically and use oxygen as a terminal electron acceptor. The superoxide ion, which is extremely reactive, is converted to H2O2 by the enzyme superoxide dismutase following the equation 2O2- + 2H+ → H2O2 + O2. Catalase must then break down H2O2, which is also a reactive oxygen species, according to the equation 2H2O2 → 2H2O + O2. All enteric bacteria in this experiment tested positive for catalase activity, showing bubbling when added to H2O2, indicating that O2 (and water) were being released. Although enteric bacteria use oxygen as a terminal electron acceptor, tests for oxidase activity indicated that they possess terminal oxidases other than cytochrome c oxidase. That is, the oxidase reagent did not change from pink to purple when it came in contact with any of the organisms, indicating a negative result. Instead of oxygen, some facultative anaerobes employ nitrate as a terminal electron receptor; the nitrate broth was used to test for nitrate to nitrite
Both media are selective and favor the growth of gram-negative bacteria. EMB contains eosin and methylene blue while SS contains bile salts and brilliant green dye, all of which inhibit the growth of gram-positive bacteria. As the name suggests, SS agar should theoretically only allow the growth of Salmonella and Shigella. In fact, many gram-negative bacteria cannot grow on SS agar. However, some enteric bacteria are able to grow on this medium, due to shared characteristics with Salmonella and Shigella, though their proliferation is limited. Both EMB and SS agars are also differential as they contain lactose, which causes indicator dyes in the media to change color in response to increased acidity when it is fermented. On an EMB plate, the colony color of a lactose fermenter might range in appearance from intensely pink to purple/black to a metallic green depending on the degree of lactose fermentation. On an SS plate, lactose fermenters will appear pink. In addition, SS agar contains thiosulfate, which if reduced to hydrogen sulfide by an organism, will react with ferric iron in the medium to produce a black center in the bacterial colonies. Kligler’s iron agar (KIA) slant also served as an indicator for hydrogen sulfide production, but from the degradation of cysteine, not thiosulfate. If synthesized, the H2S reacted with ferrous salts in the