Acetobacter can be found in many places and objects with which we come into contact everyday. By taking samples of these things and growing them in a selective medium (in our case, Hoyer’s medium), we should be able to isolate this genus of bacteria. As there is always the possibility of contamination or another organism being able to grow in the media, various tests are necessary to verify that what we isolate is Acetobacter. Although there was growth in the selective media for other things besides the apples cider, the two organisms we ultimately isolated came from the apples cider as fungi appeared to grow from what we isolated from the soil, and we did not wish to deal with such an obvious source of contamination.
A series of tests were performed on the two isolated organisms to confirm whether or not the organisms were Acetobacter. Although the Hoyer’s tubes were inoculated with several environmental samples, only those tubes that were inoculated with apple cider had growth after 48 hours of incubation. Two distinct organisms were isolated from the apple cider. Thus, our first indication that the isolated organism was Acetobacter came from the fact that the organism came from apple cider, a common source of several species of Acetobacter and that it grew well in the selective media. The two sample colonies were pale, as are many types of Acetobacter. Furthermore, our two isolated strains were ellipsoidal, ranging from almost cocci to rod-shaped in morphology, as are many strains of Acetobacter. The bacteria were motile and Gram-variable, as are many strains of Acetobacter. They grew well in the growth media for the selection and enrichment of Acetobacter. Finally, like all Acetobacter strains, our strains did not form endospores, were obligate aerobes, and tested both positive and oxidase-negative. Based on the data, we can conclude that both organisms we isolated were Acetobacter and that our enrichment protocol was successful in isolating Acetobacter.
When we inoculated samples of the cider into the Hoyer’s media, we found an interesting result--growth of yeast cells. Thus, we should remember that the Hoyer’s media may allow other organisms such as this yeast to grow. Moreover, observations of wet mount slides of this yeast revealed an unidentified organism inside these yeast cells (the organisms inside yeast cells were larger than expected size of an organelle and they exhibited movement). As some strains of Acetobacter are parasitic to yeast, we speculated that this organism could have been that strain. Nonetheless, we did not perform any further tests to pursue this hypothesis.
We changed the protocol slightly. We did not perform any tests for antibiotic
resistance, and we also did not perform the test for mannitol carbohydrate
fermentation. Instead, we added a glucose-fermentation test with oil added
to the top to create an anaerobic environment. When this result was found
to be inconclusive (both aerobic and anaerobic tubes experienced growth,
the change of color associated with an acid, and the presence of bubbles
indicating the evolution of carbon dioxide), we added an oxidative/fermentative
test to test for the ability to ferment.
Acetobacter are found in flowers, fruits, honey bees, alcoholic beverages, vinegar, sugar cane juice, garden soil, and canal water. Although the bacteria do not pose any threat to humans, they do make fruit go bad and can be lethal to yeast. Also, some Acetobacter is capable of nitrogen fixation, which would allows them to live in symbiosis with plants.
We have created a method for fruit farmers, wine-makers, brewers, and producers of vinegar to determine if they have Acetobacter in their products. As Acetobacter may cause such problems as rot in apples and pears, pink disease in pineapples, and discoloration and off-flavor in beer, people investing in these products would naturally be interested to know if Acetobacter is present.
Our protocol is useful for these purposes, as it is easy to follow and
the tests are easily obtainable. Microbiologists everywhere would have
access to these tests or similar substitutes.