Did you isolate Azotobacter from your enrichment experiment? Briefly outline the evidence in support of your conclusion.
2 days after initial inoculation from the collected soil sample, the WINOG1 plate displayed substantial growth of large white colonies, with a mucoid appearance - Glistening mucoid colonies are a typical Azotobacter macroscopic observation - As the WINOG medium is a nitrogen free medium, substantial growth on the plate implies the presence of nitrogen fixing bacteria; and a subsequent subculture confirmed this (and eliminated the possibility of trace nitrates being carried from the soil) as substantial growth was also displayed on the new plate, WINOG2.
A hanging drop was performed on a singular colony extracted from the WINOG2 plate, which showed bacteria displaying motility - Most, but not all Azotobacter are motile.
Observing the WINOG2 singular colony under the microscope showed spherical cells, roughly 2µm in diameter, which stained inconclusively (gram staining variability). Most Azotobacter strains are gram negative, although it is known that Azotobacter can display gram variability in its staining.
Having observed a motile spherical bacteria, which displayed gram variability in its staining, enriched from soil, with a mucoid colony appearance - I feel confident that a species of Azotobacter has been enriched in this experiment, but not enough evidence was collected for a species identification.
Enrichment culture experiment to isolate Bacillus anthracis
Bacillus anthracis is a gram positive, aerobic soil bacterium, and is the causative infectious agent of the disease anthrax \cite{Edwards_2005}. B. anthracis is an obligate pathogen, and is currently believed to only be able to reproduce inside its host, which in the wild typically consists of herbivorous ungulates \cite{Smith2000}. B. anthracis is well known for its resilience to stressful physiological conditions, like many Bacillus species, B. anthracis can form endospores under extreme stress; and within its host B. anthracis is able to evade phagocytosis by means of a poly-D-glutamic acid capsule \cite{Koehler_2009}.
Ecologically, B. anthracis prefers to live in humus rich, alkaline soils, ideally fortified with high Ca2+ concentrations \cite{Hugh_Jones_2009}, as it's been shown that Ca2+cations play an important role in both the maintenance of endospore dormancy, as well as in germination \cite{Dragon1995}. The soil for this experiment will be taken from farmland in Sonora, TX, a site with soil that fits the above conditions, as well as the site of two anthrax outbreaks since 2000 and a site of interest in Blackburn et al, an approximate geographic distribution of B. anthracis over the continental US \cite{Blackburn2007}. The sample will taken between the months of November and February, as previous experiments have shown minor, but significant differences in B. anthracis endospore concentration in soil between seasons \cite{Lindeque1994}.
To isolate B. anthracis, we will first suspend the soil sample in deionised water at a 1:2 (weight/volume [w/v]) ratio and vigorously shake it for 5 minutes. The sample will then be physiologically shocked, to trigger sporulation, by either submerging the sample in a 70°C water bath for 30 minutes, or by ethanol shock, by adding 99% pure ethanol to the sample, at a 1:1 w/v ratio and leaving the sample at room temperature for 30 minutes. After the shock, the sample will be serially diluted into 0.1, 0.01 and 0.001 dilutions, and then individually streaked onto PLET medium plates, which has been used historically used to isolate B. anthracis from soil \cite{Knisley1966}. The medium is complex, and contains digests of animal tissue as the main organic carbon and nitrogen source (as B. anthracis is an obligate pathogen and will not grow without the digests) as well as numerous selective agents; chiefly, polymyxin (an antibiotic produced by Paenibacillus polymyxa, which disrupts gram negative cell membranes \cite{WILKINSON_1964}), lysozyme (which hydrolyses the 1,4-beta-linkages in peptidoglycan), EDTA and thallous acetate (which in combination select against B. cereus \cite{2015}). It's important that sodium chloride is also added to maintain the osmotic equilibirum. The full breakdown of the media is listed below, and has a pH of 7.4±0.3 at 25°C.