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Bacterial Hydrolysis of Casein

Carolina Labsheets™

In this lab, students culture two bacteria on skim milk agar. The protein casein gives milk its white color. Some bacteria secrete protease enzymes that can hydrolyze casein. When these bacteria are grown on skim milk agar a clear area develops around the colonies, indicating that casein has been hydrolyzed into its component amino acids.

This activity assumes that students have previously worked with bacteria and are familiar with sterile technique. It also assumes that they know how to streak plates. If this is not the case, we recommend that students complete the activities described in our “Introduction to Sterile Technique” LabSheet before beginning this activity.

Student Lab Sheet

Needed Materials*

Bacillus subtilis plate culture (154922)

Escherichia coli plate culture (155067)

Skim Milk Agar, Prepared Media Bottle (155763A)

petri dishes, pack of 20 (741250)

Bunsen burners or alcohol lamps

inoculating loops


disinfectant (e.g., 70% ethanol, Lysol®, bleach solution)

wax pencils

Optional Materials
Tube cultures (154921 and 155065) can be substituted for the plate cultures. If using tube cultures, students must flame the mouth of the culture tube before inserting the inoculating loop and again after withdrawing it. Each workstation also will need a test tube rack for holding the tube cultures. See our Techniques for Studying Bacteria and Fungi manual for more detail on the proper handling of tube cultures.


This activity requires that students work with bacterial cultures and open flames. They should use sterile technique at all times and wear appropriate safety equipment. All work surfaces should be wiped down with disinfectant before and after the lab. All students should wash their hands before and after the lab. Cultures remaining at the end of the lab should be destroyed by autoclaving or by being flooded with disinfectant overnight before proper disposal.

Ensure that students understand and adhere to safe laboratory practices when performing any activity in the classroom or lab. Demonstrate the protocol for correctly using the instruments and materials necessary to complete the activities, and emphasize the importance of proper usage. Use personal protective equipment such as safety glasses or goggles, gloves, and aprons when appropriate. Model proper laboratory safety practices for your students and require them to adhere to all laboratory safety rules.


Students can work individually or in pairs.

On the day before the lab, pour the skim milk agar plates. Consult the instructional video “How to Melt and Pour Agar Plates,” searchable in the "Activities" section of our Web site, www.carolina.com, or follow the instructions accompanying your bottled media. One 125-mL bottle of media will pour 5 plates.

Set up workstations for inoculating the plates. Each station should have the following materials:

bacterial cultures
Bunsen burner or alcohol lamp
inoculating loop

Set up separate workstations for the skim milk agar plates, each equipped with wax pencils for marking the plates.

Incubate Bacillus subtilis at 30°C and Escherichia coli at 37°C. If you must incubate them in the same incubator, incubate at the lower temperature. If you do not have an incubator, the plates can be incubated at room temperature. Examine them for growth at 24-hour intervals. The plates should be ready for use 2 or 3 days after inoculation.

Optional: The lab can be a stand-alone activity or it can be done in conjunction with our “Bacterial Growth on MacConkey Agar,” “Bacterial Hydrolysis of Starch,” and “Bacterial Hydrolysis of Lipids” LabSheets to give a broader view of the biochemical diversity found within bacteria. The following kits explore the topic in even greater depth: Bacterial Fermentation Kit (154710), Bacterial Biochemical Identification Kit (154715), and Enteric Biochemical Detective Kit (154717).

Answer Key to Questions Asked on the Student LabSheet

Record your observations of the growth and appearance of the two bacteria on skim milk agar.

Bacillus subtilis: Heavy growth surrounded by a clear zone
Escherichia coli: Heavy growth; color is white, off-white, or gray

Which, if either of the bacteria, secretes protease? How do you know?
Bacillus subtilis secretes protease. There is a clear area indicating breakdown of the protein casein.

Student Lab Sheet

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