Macconkey Agar Lab Report

Unknown #1 Vomit
Serratia marcescens & Bacillus cereus

Biol 251 Microbiology

5/14/2009

Introduction
The purpose of this study is to differentiate and identify two unknown organisms provided by the instructor in a nutrient broth. It is only known that the two organisms are from vomit; one is gram-positive and the other is gram-negative. It is necessary to first separate the two organisms by inoculating a nutrient agar plate using the streak-plate method. The initial streak-plate procedure was performed and placed in the incubator at 37◦C for 24-48 hours. Upon observing the growth on this plate, it is fairly obvious that one of the organisms is Serratia marcescens. However,
Both organisms were inoculated onto opposite sides of the PEA plate and incubated at 37◦C for 18-24 hours. Observation shows that for #1, there is no growth and no change in the appearance of the medium, while #2 does have growth, but with no change in the appearance of the medium. This is further indication that #2 is gram-positive, while #1 is most likely gram-negative.
MacConkey agar: MacConkey agar is both a differential and selective medium used to select for gram-negative bacteria. It also differentiates lactose-fermenting bacteria from non-lactose-fermenting bacteria. Only #1 was used to inoculate this medium. After inoculation, the plate was incubated at 37◦C for 18-24 hours. Observation shows there is a bright red growth but no change in the appearance of the medium. This indicates no lactose fermentation, however since there is growth, this means the bacterium in gram-negative.
Mannitol salt agar: This medium was inoculated with both specimens and incubated at 37◦C for 18-24 hours. There being no growth and no change in appearance of the medium for either organism, this indicates there was no mannitol fermentation and they do not tolerate
The methyl red test is used to identify enteric bacteria based on glucose metabolism. The Voges-Proskauer test is used to determine if an enteric bacterium produces acidic or neutral end products. First, the medium was inoculated with isolate #1, and incubated at 37◦C for 48 hours. After incubation, 2.5 ml of the medium was transferred to another tube to be used for the Voges-Proskauer test. For the methyl red test, five drops of pH indicator methyl red is added to one of the tubes of medium. After being gently mixed by rolling the tube between the palms of the hands, the results are observed. The MR-VP broth has changed to a yellow-orange color. This is a negative reaction and indicates that pyruvic acid was metabolized to neutral end products. For the Voges-Proskauer test, 10 drops of Barritt’s reagent A is added to the second tube of medium. The culture was shaken, then immediately 10 drops of Barritt’s reagent B was added and it was shaken again. For the next 15 minutes, the culture was shaken every 3-4 minutes. Observations were made at 15 minutes. There was no change in the medium which indicates a negative reaction and also indicates that #1 does not ferment glucose. This is another contradiction of expected results and is contradicted in the next