Bacillus Subtilis: Identification And Testing

Wondering just what Bacillus Subtilis is, and how you can use scientific techniques to identify it? Then read on to discover all that you need to know!

Bacillus Subtilis: Identification And Testing

What Is Bacillus Subtilis?

Also known as niger or black bacteria, Bacillus subtilis is a rod-shaped bacterium found in soil and water.

It is commonly associated with food spoilage and disease and is a protease enzyme produced by Bacillus subtilis.

This enzyme has applications in various industries such as pharmaceuticals, biotechnology, food processing, and detergents.

How Can I Test For Bacillus subtilis?

B. subtilis can be identified using a number of biochemical tests, and these include:

Gram Staining

Gram Staining involves the staining of cells with crystal violet, which gives them their characteristic purple coloration.

The Gram stain test will reveal whether the cell wall contains peptidoglycan (a polysaccharide), which is a major component of the bacterial cell wall.

If there are no peptidoglycans present, then the cell walls do not contain any amino acids, and thus cannot be stained.

Gram staining will test for Bacillus subtilis by revealing whether the cell wall contains a peptidoglycan layer, which is the main component of the cell wall.

A positive result indicates that B. subtilis is present.

Catalase Production

The catalase test reveals whether or not the organism produces an enzyme called catalase, which breaks down hydrogen peroxide into oxygen and water.

Catalase production is a common feature of many species of bacteria, but only those that produce this enzyme are able to survive in aerobic environments.

Therefore, if the organism under investigation does not produce catalase, then it should be considered nonviable. Bacillus subtilis is a catalase-positive substance, so this test can be used effectively.

Oxidase Production

The oxidase test reveals whether or not the organism produces an enzyme that catalyzes the reduction of nitrate to nitrite.

Nitrogen fixation is a process where nitrogen gas is converted into ammonia, which is then used by plants.

However, some bacteria also fix nitrogen from the air, and therefore must have the ability to reduce nitrate to nitrite before they can utilize the ammonia for growth.


Motility refers to the ability of organisms to move around within their environment.

In order to perform motility, the organism needs to possess flagella, which are long structures that allow the bacteria to swim through liquids.

Bacillus subtilis is motile because its flagella enable it to move about.

Proteolytic Activity

This test reveals whether or not an organism possesses enzymes that break down proteins.

Proteases are important because they help bacteria digest other organisms, and therefore play a key role in the survival of microorganisms.

Bacillus subtilis is capable of producing proteases and therefore is considered a protease producer.


Hemolysis occurs when red blood cells are broken apart, releasing hemoglobin. This causes the release of iron ions, which can cause damage to surrounding tissues.

ome bacteria secrete substances that lyse erythrocytes, causing the breakdown of the cell membrane and subsequent leakage of hemoglobin.

How Can I Identify Bacillus subtilis?

Bacillus subtilis includes a number of characteristics that help to make it identifiable, and these include:

Rod-Shaped Morphology

Bacillus subtilis has a rod shape, and as such, is easily identified using light microscopy. It is one of the most commonly found members of the genus Bacillus and is often found living in the soil.

Production of Endospores

Endospore formation is a characteristic of bacilli, including Bacillus subtilis, and allows them to remain dormant until conditions become favorable.

When conditions change, the endospores germinate and grow out, forming new colonies.

It Is Gram-Negative

As we have mentioned, gram staining is a technique used to identify different types of bacteria based on their morphology.

Gram-negative bacteria have two membranes, whereas gram-positive bacteria have just one.

Gram-negative bacteria will stain blue with basic dyes like methylene blue, while gram-positive bacteria will stain pink. Bacillus subtilis strains purple, indicating that it is gram-negative.

It Grows at 37°C

Bacillus subtilis grows well at temperatures between 25°C and 45°C. At temperatures below this range, it cannot survive.

What Is Bacillus Subtilis Used For?

Bacillus Subtilis is used in a number of products and activities, and these include:

Food Production

Bacillus subtilis is used in food production due to its ability to produce enzymes that aid digestion. These enzymes include amylases, lipases, and proteases.

Amylases break down starch into sugars, lipases break fat down into glycerol and free fatty acids, and proteases break protein down into amino acids.

Industrial Applications

B.subtilis is also used industrially for the production of antibiotics, biocides, and probiotics. Antibiotics produced by this bacterium include penicillin, tetracycline, chloramphenicol, and streptomycin.

Biocides include formaldehyde and phenols, and probiotics include Lactobacillus acidophilus.

Medical Uses

In addition to being used in food production and industrial applications, B.subtilis can be used in medical applications, including wound healing.

The bacterium produces exopolysaccharide (EPS) polymers, which help to repair damaged tissue.

Other Uses

Bacillus subtilis is also used in other areas, including wastewater treatment, biofuel production, and the control of plant diseases.

Is Bacillus Subtilis Harmful To Humans?

Bacillus subtilis is considered an opportunistic pathogen, meaning that it only becomes harmful to humans when certain factors come together.

The first factor is that the immune system must be suppressed. Suppression of the immune system makes people more susceptible to infections.

This susceptibility increases if they are already ill or injured.

The second factor is that the person’s body temperature must be elevated.

If the body temperature rises above 38°C, then the immune system becomes compromised, and the third factor is that the person must be stressed.

Stress causes the release of adrenaline, cortisol, and other hormones that suppress the immune system.

If any of these three factors occur simultaneously, then B.subtilis may cause illness or even death.

However, if none of these factors occurs, then there is no risk of infection from B.subtilis.

Final Thoughts

Bacillus subtilis is a largely safe bacteria that can have a number of positive applications and purposes, and our handy guide will ensure that you can detect and identify it whenever you need to.

Jennifer Dawkins

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