After graduating from college, you obtain a job with the Department of Homeland

Question

After graduating from college, you obtain a job with the Department of Homeland Security to generate devices for detecting the biothreat agent, Bacillus anthracis, the causative agent of anthrax. One morning, your entire town is quarantined to their homes because Bacillus anthracis has been detected within the town borders, leading to suspicion that the town has been maliciously attacked with a bioweapon.

As an anthrax expert you understand that the spore form of B. anthracis is much more likely to be transmitted to humans than the vegetative form of B. anthracis. A major goal therefore, in assessing the risk of the B. anthracis released in your town, is to determine the percentage of the released B. anthracis that is spores.

Unfortunately, you do not have in your apartment a microscope, or any of the molecular reagents or equipment to tell the difference between spores and vegetative bacilli. However, you do have Petri dishes filled with bacterial growth agar, as well as some liquid growth medium from your child’s 2nd grade science project. You also happen to have a liquid pipetter and pipette tips at your home. You know that both B. anthracis spores and living vegetative B. anthracis bacteria will grow equally well on the agar plates.

Based on this information, describe a simple overall experimental approach that you could perform in the kitchen of your apartment to determine the percentage of the released B. anthracis that is spores. Be sure to describe how the data from your experimental approach can be used to calculate the percentage of spores in your sample. (Only the writing within the allotted space will be considered).

Explanation / Answer

Ans: Here based on the question Anthrax spores and vegetative cells may be distributed in soil or circulating in air. Here we can have two approaches. Approach1: Take some amount of soil (using some old level measuring tonic cap or disposable plastic spoons of equal size, because for different places you should use fresh spoons to avoid cross contamination and ending up with wrong data) from different potentially contaminated places. Simple sure shot methodology is dissolve these samples separately in known amount of water (say X grams soil in 100ml sterilised water, measure water using kitchen measuring mug) and incubate 5-10min with constant shaking. Assuming all the vegetative cells and spores gets dissolved in the water. Leave for five more minutes to settle the soil. Now you may take 2x25ml of water from 100ml solution one 25ml goes to pasteurisation at 72°C for 15 seconds to kill most of the vegetative cells and spores only should survive, actually spores gets activated at this temperature and germinate faster. From this take one ml and spread on the agar solid media plate, incubate overnight in the yogurt making incubator rack at 37°C and measure the colonies (say you have counted 50 colonies=X). The other 25 ml sample, from which you take 1ml and spread on the agar solid media plate, incubate overnight in the yogurt making incubator rack at 37°C and measure the colonies (say you have counted 100 colonies=Y). This means in plate Y spores + vegetative cells = 100 = 100%

Now from the above information we can calculate percentage of spores in the sample. Y(100)-X(50) =50 spores i.e., in terms of percentage 50%. You can back calculate to say Xg of soil in 100ml and how many spores and vegetative cells in a given sites environmental sample.  

Approach2: Take a kitchen filter paper of 1square foot and apply sealing wax or vaseline or any greasy material uniformely and expose to air 2-3 metres above ground place in a potentially contaminated site for uniform time, like this you may collect samples from several sites, entire 1square foot paper you may use to dissolve in a known amount of sterilised water or cut and take 1 square inch paper and do the further growth and colony enumerating procedures as per the above given protocols

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