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Microorganisms from the genus Bacillus are a diverse groups of gram-positive, variable-sized rods. Colonies can vary tremendously from species to species often forming very unusual morphologies. They are grouped together taxonomically due to their ability to form endospores. Endospores are resting structures that are resistant to heat, radiation, dryness, harsh chemicals and extremes in pH. This is truly a state of suspended animation since the spore can survive for many thousands of years and then germinate, if favorable conditions are detected.

Endospore formation (sporulation) is triggered when environmental conditions become poor due to depletion of an essential nutrient. The developmental process that produces a spore takes several hours and involves profound changes in the cellular morphology, biochemistry and physiology of the cell. Sporulation is tightly regulated with close cooperation between two sister cells, one cell eventually being packaged as a endospore, the other devoting most of its resources to building the spore and then lysing. This process serves as a simple tractable model system for understanding the regulation of development and is of interest to developmental biologists as well as microbiologists. You can learn more about Bacillus and endospores in the microbiology textbook.

Nutrition

Most Bacillus species are chemoheterotrophs, but their metabolism is very diverse. B. cereus and B, subtilus carry out a 2,3 butanediol fermentation similar to some Enterobacteriaceae, except these species also produce glycerol as an end product. B. schlegelii is able to grow chemolithoautotrophically in a minimal salts medium using CO₂ as a carbon source, H₂ as an energy source and oxygen as a terminal electron acceptor.

Some species are pathogenic to other organisms. Bacillus anthracis is the causative agent of anthrax - historically a serious threat to live stock and humans. It has recently hit the public the public consciousness due to the percieved threat of bioterrorism. Bacillus was first described by Robert Koch while working on this disease. The use of antibiotics and a vaccine against the disease has eliminated it in most industrialized countries. Several other bacilli (including B. thuringiensis, B. popilliae, and B. larvae) are pathogenic to insects. These microbes produce a crystalline protein during sporulation that is toxic to certain species of insect. They are of interest to scientists as biocontrol agents since some of the susceptible insects are agricultural pests.

Thermophillic Bacillus species have also been described and industry is interested in them for the production of heat stable enzymes. Many Bacillus species will excrete extracellular enzymes that degrade polysaccharides, proteins and lipids. These thermophillic enzymes are used to process raw materials into commercially valuable products. One example is the conversion of starch to glucose units by heat stable amylases.

Endospore formation has allowed Bacillus species to disperse themselves widely in the environment and it is possible to isolate them from almost any source. In this experiment we will isolate spores from soil.

A soil sample is suspended in saline and heated at 80°C. This treatment destroys all vegetative cells leaving mostly heat resistant spores. This is an effective pretreatment that is so selective for endospore-forming bacteria, a non-selective rich medium can be used in the next step. Mesophillic and thermophillic Bacillus will be enriched for by incubating plates at 30°C and 55°C respectively. The experiment will culminate with students comparing the heat tolerance of endospores from a mesophile and endospores from a thermophile.

Period 1

Materials

Sample that may contain endospores (brought in by you)

balance capable of weighing 1 g

4 tubes 0.85% saline (9 ml)

80 °C water bath

6 plates of Nutrient Agar + 50 mg/L MnCl₂ • 4H₂O (NAM)

55°C incubator

1. If your sample is a solid, weigh out 1 gram and place it into one tube of 0.85% saline. Vortex for 1 min. to dislodge the endospores
2. If your sample is a liquid, pipette 1 ml into a tube of 0.85% saline and mix for 10 sec..
3. Place your tube in the 80°C water bath for 10 minutes. Make sure the water level of the bath is at least equal to the water level in your saline suspension. What does this treatment do?
4. Serially dilute your sample to 10^-4 using the remaining saline dilution blanks.
5. Pipette 0.1 ml of the 10^-2, 10^-3, and 10^-4 dilutions onto separate plates of NAM. Perform this in duplicate.
6. Incubate one set of plates at 30°C and the other set at 55°C for 2 days. (It may be wise for your instructor to take the plates out of the 55°C incubator after 1 day of incubation and refrigerate them.)

Period 2

Materials

2 plates of NAM

Plate of bacillus @ 30

Plate of Bacillus @ 55

1. Examine your plates for the presence of colonies. Streak one isolate from the 30°C plates onto a plate of NAM. Also pick one isolate from the 55°C plates and streak for isolated colonies on a plate of NAM.
2. Incubate the 30°C plate for 2-5 days at 30 °C and the 55°C isolate at 55°C for 1-2 days.

Period 3

Materials

2 NAM slants

2 Starch Agar plates

2 tubes of Voges-Proskauer Broth

2 tubes of Thioglycollate agar

2 Citrate Agar slants

Malachite Green for endospore stain

2nd Plate of bacillus @ 30

2nd Plate of Bacillus @ 55

Wet mount of Bacillus (have both)

1. Pick an isolate from the plate incubated at 30°C and one from the 55°C plate. Perform a Gram stain and endospore stain on each. Be sure to note the location of any endospores, their shape (round vs. ellipsoid), if the spores swell the sides of the bacteria (swollen sporangium), and the size of the cells (large vs. small). For cell size, it may be a good idea to compare a neighbors isolate to yours to get an idea of what is small and what is large.

(Note: Some endospore forming bacteria will not sporulate under the conditions we are using. If you are unable to observe endospores, redo the endospore stain next period using a bit of growth from the NAM slant. Many Bacillus species will sporulate under these conditions)

2. After you have verified that your isolates are potentially Bacillus (Gram positive, endospore forming rods that grow in the presence of oxygen) stock each culture on a NAM slant.
3. We will be identifying the 55° isolate to the species level using the media listed above. Inoculate the starch agar, Vogues-Proskauer broth, thioglycollate agar, and citrate agar with your 55°C isolate. Incubate at 55°C. Your instructor will remove all tubes from the 55 °C incubator after two days incubation.
4. Also inoculate your 30°C isolate into the above media and incubate at 30°C for 2-5 days.

Your assignment for next period is to design an experiment to compare the heat resistance of endospores from a mesophile to endospores from a thermophile. You have available the materials listed below in Period 4. Before you come in to lab, discuss with your lab partner the design of the experiment and agree on a prediction of the results.

Period 4

Materials

Bottle of sterile saline (50 ml)

Basket of sterile test tubes

Plates of NAM (You are limited to 14 plates)

100°C water bath

Voges-Proskauer reagents

Timer

Reactions in starch agar

Reacitons in VP broth

Reactions in Thioglycollate agar

Reactions in simmons citrate agar

1. Run your planned experiment to test your hypothesis.
2. Also read the tests of the medium inoculated in the last period for the 55°C and 30°C isolates. Consult the following table to determine the species of your thermophillic Bacillus strain.

Bacillus strains. make figure of below.

Table 10-1. Differentiating properties of thermophillic Bacillus species

Thermophillic species	Sporangium Swollen	Spores Round (Not Ellipsoid)	Degradation of Starch	Voges-Proskauer	Citrate	Anaerobic Growth
B. acidocaldarius	+	-	+	-	-	-
B. circulans	+	-	d	-	d	d
B. badius	-	-	+	-	-	-
B. licheniformus	-	-	+	+	+	+
B. schlegelii	+	+	-	-	-	-
B. stearothermophilus	+	-	+	-	d	-

+ =90% of isolates are positive
-=90% of isolates are negative
d=11-89% of isolates are positive

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