History and Facts

The rod-shaped bacteria was first discovered in 1835 by Christian Gottfried Ehrenberg and given the name Vibrio subtilis. It was later given the more fitting name of Bacillus subtilis in 1872 by Ferdinand Cohn. The non-pathogenic bacteria can be found in soil or in the intestinal tracks of humans and ruminants. It is currently used as a comparative study against other Gram-positive bacteria as wells as a counter measure against some intestinal disorders. It also has the ability to turn into a spore when stressed, allowing it to cope with extreme environments. In doing so, it can even survive in space, and to this day, it holds the NASA record of surviving 6 years in the starry void.

How to Culture

Bacillus subtilis is classified as a BioSafety Level 1, meaning that is considered unlikely to cause any illnesses in healthy adults and poses very minimal risk in the laboratory. However safe, it is still advised to follow standard lab safety procedure using PPE and performing experiments in a room that has a sink and a door separating it from the rest of the building.

B. subtilis may be grown on either nutrient agar pates, slanted agar test tubes or in nutrient broth test tubes. It will usually come in a dehydrated form so it is imperative to rehydrate using nutrient broth before culturing onto an agar plate or slanted agar test tube. To ensure the initial growth in nutrient broth, follow the standard procedures for inoculating into a test tube and incubate at 30°C for at least 24 hours. Afterwards, the culture is viable for inoculating onto the other aforementioned media.

Hence its "Bacillus" genus, B. subtilis is rod-shaped. As can be seen in the colored picture, B. subtilis is a Gram-positive bacteria as it stains purple/violet during the test due to its thick peptidoglycan membrane layers. The greyscale picture shows B. subtilis through an electron microscope and better illustrates the rod-shape.

Positive tests: Gram-stain test, Blood agar test, Salt Mannitol test, Phenol Red with Glucose, Catalase test

Negative tests: MacConkey agar test, gas production from Phenol Red with Glucose test, Sulfur Reduction and Indole Production tests, anaerobic environment test

B. subtilis is a bacillus type bacteria (hence the genus), and is a Gram-positive aerobe. It can grow in a Blood-agar plate though the distinction of whether or not it is beta-hemolytic is unclear as results can vary. B. subtilis cannot grow on a MacConkey agar plate, but it can grow on the Mannitol Salt agar plate, meaning it can survive in environments with a salt concentration higher than 6.5%. In a Phenol Red with glucose broth Durham tube test, B. subtilis is able to perform glucose fermentation but produces no gas. The SIM medium deep agar tests show that B. subtilis is negative in both the Indole Production and Sulfur Reduction tests. Motility is questionable as there is a varying range that is motile with a range that is not motile. Finally, it is catalase positive, meaning it uses catalase in intracellular functions.

However, entering these results into the Gideon database will only yield an 89% chance that the microbe is Bacillus subtilis. Though this is high, there is still a notable chance that the microbe could be identified as another species. There is about a 10% chance, according to the database, that the bacteria may be Bacillus megaterium. In addition, there are very small chances --- less than 1% --- that the microbe could be one of the wide variety that are listed in Gideon based on the test results.

Molecular Diagnostics

Using an in-silico PCR to find a partial sequence of a 16S RNA gene, we can find a full transcript of said gene through a BLAST search. An in-silico PCR was used to amplify the EN1F and EN1R genes from Bacillus subtilis. The former has a melting point at about 58.6°C and a G-C content of about 59%. Meanwhile the latter has a melting point at about 54.4°C and a G-C content of about 52%. AFter acquiring the partial sequences, a nucleotide BLAST search was done on them yielding full sequences. Unfortunately, the full sequences of the genes could not be included as they are much too lengthy to include.

Bacillus subtilis is considered non-pathogenic, meaning it has a very low probability of causing disease in the average healthy adult. To add on, B. subtilis can be naturally found in the gastrointestinal tract of humansHowever, it has been known have an effect in treating some gastrointestinal diseases. It has also been observed to have some benefits in preventing the infection of Salmonella enteritidis.

As far as antibiotics are concerned, B. subtilis has a susceptibility to clindamycin, chloramphenicol, and kanamycin.