The GenusBacillus

he genus Bacillus includes over three dozen species, many ofwhich have significance in industrial and medical microbiol-ogy. For example, dried Bacillus subtilis is used as a source of

enzymes in enzyme detergents, and B. polymyxa produces the antibioticpolymyxin. Also, Geobacillus (formerly Bacillus) stearothermophilus isused to test the effectiveness of sterilization, and B. anthracis is the agent ofanthrax, a serious disease of animals—and a health threat to humans as evi-denced by the anthrax letter terrorism in October 2001.

The organisms in this genus are gram-positive, sporeforming rods thatgenerally produce catalase. Most are motile, and many species digest starchand DNA. In this exercise, Bacillus species will be isolated from the soil andtheir presence confirmed by noting typical characteristics of the organ-isms. Initially, the other soilborne aerobic organisms will be destroyedwith heat so as to select out Bacillus spores.

pecial Materials

• Nutrient agar plates• Starch agar plates• Iodine solution• Hydrogen peroxide (3%)• Tubes of motility test agar• Malachite green• Safranin• Heated water bath• Soil samples and test tubes• Gram stain reagents

rocedure

1. Heat a water bath to approximately 80° C. Place a generous pinch of rich soilin several milliliters of water in a test tube, and immerse the tube in the hotwater for approximately 10 minutes, then remove it and let the liquid cool.This procedure will destroy most organisms other than bacterial endo-spores and will stimulate the spores to germinate.

2. Select or prepare one nutrient agar plate and one starch agar plate.Label each on the bottom side with your name, the date, the name of themedium, and the designation “soil sample.’’

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PURPOSE: to isolate andidentify Bacillus species from soil samples.

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3. Aseptically obtain a loopful of the cooled soil sample, and streak the nutri-ent agar plate for isolated colonies according to the streak-plate technique,as explained in Exercise 2A. Repeat with the starch agar plate, and incubateboth plates in the inverted position at 37° C. Not more than 24 hours of incu-bation will be necessary. Refrigerate the plates until the next laboratory peri-od.

4. Observe the plates for uneven, white, spreading colonies, which maycontain Bacillus species. Test for catalase production by placing dropsof hydrogen peroxide onto the colonies on nutrient agar and noting theformation of bubbles of oxygen gas. Starch digestion, a characteristic ofmany Bacillus species, may be ascertained by adding iodine to coloniesgrowing on the starch agar and noting whether clear areas form aroundcertain colonies, indicating the starch has been hydrolyzed. Representa-tions of the plates may be entered in the Results section. Samples ofcolonies thought to contain Bacillus species should be isolated on nutrientagar slants and stained according to the method described in Exercise 7Ato determine whether endospores are present. Gram stains also may beprepared.

5. When Bacillus species have been identified by the above tests, motility maybe determined by inoculating motility test agar with samples, according tothe procedure described in Exercise 7C.

6. Since Bacillus species often produce antibiotics, the isolates may also betested for this ability in the following way: Inoculate a nutrient agar platewith a single streak across the center of the plate (see Figure 34.1). A testorganism supplied by the instructor is then inoculated at a right angle to theoriginal streak, and the plate may be incubated at 37° C for 24 to 48 hours.

7. Observe the motility test agar for evidence of motility, and enter this infor-mation in the Results section. Examine the antibiotic plate and notewhether the growth of the test organism has been inhibited in the region ofthe Bacillus species. Growth inhibition provides evidence of antibiotic pro-duction. Representations of the test results should be prepared. Drawappropriate observations from the results obtained in this exercise.

uestions

1. Summarize the biochemical characteristics of members of the genusBacillus.

2. What might result if the soil sample were not heated prior to the attemptto isolate Bacillus species?

3. Why are microscopic observations of particular value in the identifica-tion of Bacillus species?

4. Given the choice, might it be more advisable to use catalase productionor starch digestion as an identifying test for Bacillus species? Why?

5. Name several instances in which members of the genus Bacillus makepractical contributions to human welfare.

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!When streaking the plate,avoid exposure to the air,since Bacillus spores can easily enter the plate andintroduce errors.

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T H E G E N U S B A C I L L U S 21 193

Name

Date Section

Exercise Results

The Genus Bacillus

Bacillus Species from Soil

21

Stained Smears of Bacillus Species

Source: ______________________ ______________________

Magnif.: ______________________ ______________________

Nutrient Agar Plate Starch Agar Plate

Source:

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194 21 T H E G E N U S B A C I L L U S

Observations and Conclusions:

Motility Test Antibiotic Production

Source:

Magnif.:

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