I was curious if anyone know of certain techniques used for B. anthracis enrichment. I'm pretty sure it'll grow on any nutrient agar, and it is a faculative anaerobe, but if anyone has any super good information, it would be most appriciated.
also, if anyone has any information on it's sporalation process, i.e. in vitro techniques.
05-07-07, 06:44 PM
That doesn't sound safe to me.
And no, I'm not a bio-terrorist, just a microbiology major looking for additional information for a report I'm writing.
That doesn't sound safe to me.
I'm not isolating or enriching for it, I just need to write about techniques used. If you don't want to write about it on a public forum, you can e-mail it to me, or PM me about it.
05-07-07, 09:31 PM
I don't know anything about it. It must be hard for a student to study in this area these days. I used to know a scientist that worked at Ft. Detrick, MD.
I think you'd have better luck in your college library. Try JSTOR.
try http://www.pubmed.com , search for your topic headline.
People at the lab I worked had researches on B. anthracis. They'd use bicarbonate agar and blood agar to grow the cultures.
They grow smooth colonies in bicarbonate agar and granulous colonies in blood agar:
found you something at the Center for Disease Control and Prevention website.
It's a 22 page long .pdf called "Basic Diagnostic Testing Protocols for level A Laboratories for the Presumptive Identification of Bacillus anthracis"
it's pretty complete
Bacterium Bacillus anthracis is the causing agent of anthrax. B. anthracis is commonly found in low-lying swampy terrain with a warm, loose soil in a temperatures of 27 to 37 degrees Celsius and 60% humidity (O’Donnell, et al., 1981). Sporalation can occur under these conditions and may lead to the establishment of B. anthracis in the micro flora. B. anthracis appears as rods in short chains of 2-4 cells. Typically, the cells are 1x3-5 μm in size. They are Gram-positive endospore forming facultative anaerobic Bacilli. This chemoorganotroph shows the ability to undergo butylene glycol fermentation proven by a positive reaction for the Voges Proskauer test and has complex nutritional requirements (Harwood 1989).
Some biochemical characteristics unique to B. anthracis are the abilities to undergo nitrate reduction via nitrate reductase, the hydrolysis of starch via the activation of hydrolytic enzyme amylase, and the hydrolysis of casein, via the exoenzyme caseinase (Harwood 1989).
Determination of B. anthracis is possible after several differential tests. A motility test will result in the determination that B. anthracis is immotile, a signifying characteristic of B. anthracis. Anaerobic growth is another signifying characteristic that can be used in determining if a culture is indeed B. anthracis. Besides these tests, B. anthracis is very similar to most other species of genus Bacillus (Harwood 1989).
B. anthracis has long had a very important role in microbiology, as it was the first microorganism proven to be pathogenic by Robert Koch in 1877 (Brock 2006). Recent years have brought attention to its ability to be weaponized for biowarfare. Because this bacterium can undergo sporelation, it is ideal for packaging, transfer and release into a population.
Pathogenic B. anthracis produces three proteins; Protective antigen (PA), Lethal Factor (LF) and Edema Factor (EF). When combined, PA and LF form lethal toxin. When PA and EF combine, they form edema toxin. PA is the cell binding B component of these A-B toxins. EF causes edema and LF causes cell growth. No protein factor is toxic by itself, but all three act synergistically (O’Donnell, et al., 1981). The entry of toxin into cells starts with the recognition of a receptor on the cell membrane by PA. Proteolytic cleavage of the PA creates a pore-like structure in the plasma-membrane, which allows for LF and EF proteins to bind to the PA pore. Internalization of the entire structure is followed by all three toxins entering the cytoplasm through receptor-mediated endocytosis. The acidic pH then causes the release of LF and EF proteins into the cell’s cytoplasm. Once in the cytoplasm, LF acts as a protease, and cleaves MAP kinase. This inhibits certain pathways used by this kinase, eventually leading to cell death. EF is an adenylate cyclase, which inhibits immune response, including phagocytosis by macrophages (Groston). Growth of B. anthracis in lymph nodes can cause lymphatic tissues to drain into the lungs and eventually lead to edema and cell death. This can be followed by tissue destruction, shock and death (Barlow, et al., 1981).
Naturally a soil-dwelling bacterium, B. anthracis sporalates under extreme environmental conditions, and then can be ingested by herbivores like cattle, goats and sheep. When these animals are infected, transmission to humans can occur via ingestion of their uncooked meats, inhalation of spores on the coats or introduction of cells or spores into the skin. Transmission to humans can occur in three separate ways.
The first pathway of infection, known as cutaneous anthrax, can occur when the bacterium or its spores enters a cut on the skin of the host. This can occur during the handling of contaminated wools, hides or other products from infected animals. Initially, skin infection is noted by an insect-bite like itchy bump. The bump will develop into a fluid-filled vesicle within 1-2 days and then rupture to form an eschar, usually 1-3 cm in diameter, with a characteristic black necrotic center. B. anthracis can induce pronounced edema by the release of edema toxin. Deaths are rare from this sort of infection, and have a mortality rate of about 20% for untreated victims (Lindsay et al., 2001).
Gastrointestinal anthrax is caused by the consumption of contaminated byproducts from infected organisms. Symptoms included acute inflammation of the intestinal tract, nausea, loss of appetite, vomiting and fever. These can be followed by vomiting of blood and severe diarrhea. While the mortality rate is not known for certain, it has been estimated to be between 25-60% (Lindsay et al., 2001).
Pulmonary anthrax, or Inhalation anthrax, if left untreated is known to have a 100% mortality rate (Balows et al., 1981). Pulmonary anthrax is caused by the inhalation of B. anthracis spores, and is the agent of the 2001 bio-terrorism attack in the United States (Brock 2006). After inhalation, the spores incubate for 1-6 days before disease symptoms are seen. Initial symptoms include fever, malaise and fatigue (Lindsay et al., 2001). These are commonly followed by severe respiratory distress with dyspnea, diaphoresis, stridor and cyanosis. Shock and death usually occur 24-36 hours after respiratory distress. The infections can be viewed using a chest X-ray, revealing a widened mediastinum with pleural effusions (Lindsay et al., 2001).
Antibiotic therapy is commonly used as treatment for B. anthracis infections. Penicillin, ciprofloxacin and doxycycline are common treatment options. As always, early treatment initiation should be executed (Brock 2006).
The U.S Department of Defense has licensed a human vaccine for those working in high-risk environments such as agriculture. This is a cell-free filtrate that contains protective antigen and alum. It has a success rate of 93% with cutaneous anthrax (Lindsay et al., 2001).
Recent research has been conducted to further classify B. anthracis as a species, using modern insertion-PCR techniques to map tRNA genes of B. anthracis, B. cereus and B. thuringiensis. This research is important because all three of these species have similar virulence mechanisms (Daffonchio et al., 2006). Further understanding of these mechanisms can lead to breakthroughs in anti-bioweapon technologies.
Isolation of B. anthracis is relatively simple. It is very similar to most Bacillus species, and requires no specific enrichment techniques (a common TSA plate would be sufficient). Special care should be given to sterile-technique and proper safety precautions should be used when handling, as it can be extremely harmful. Handling and analysis of B. anthracis should be performed with a certified Class II biological safety cabinet (Lindsay et al., 2001). Decontamination should be successful with commercial bleach solutions containing 5.25% hypochlorite (Lindsay et al., 2001).
Isolating B. anthracis can be done by inoculating a Tryptic Soy Agar (TSA) plate with a soil sample. If using samples from an infected host, several techniques are used pertaining to the kind of infection. For cutaneous anthrax, during the eschar stage, rotating swabs beneath the edge of the eschar without removing the eschar, then inoculating 5% Sheep blood agar plates (SBA) with the swabs. Gastrointestinal anthrax cultures can be obtained via a stool sample from the infected host, and isolated on the same SBA plates. Blood cultures can produce the organism, if obtained before the initiation of antibiotic treatment (Lindsay et al., 2001).
Incubation should occur at about 37 degrees Celsius, under ambient conditions. Growth has been observed within 8 hours of inoculation. Isolated colonies are 2-5 cm in diameter, and are irregularly round with a ground-glass appearance (Lindsay et al., 2001).
That's what I turned in so you guys can read.