Isolating Bacteria from a Mixture

Carolina Labsheets™

In this lab, students are introduced to a basic technique used in the study of bacteria: isolating a single species of bacterium from a mixture. Environmental surfaces are often sampled and the samples streaked on plates to isolate bacteria. In this activity, students practice the technique by using a mixture of bacteria of known composition.

Needed Materials

Bacteria broth culture

Mixed Suspension of Introductory Bacteria (154760 broth culture)

Liquid culture containing Bacillus subtilis (rods), Micrococcus luteus (spheres), and Rhodospirillum rubrum (spirals)

or

Mixed Suspension of Pigmented Bacteria (154765 broth culture)

Liquid culture containing Serratia marcescens (rods) and Micrococcus luteus (spheres)

Bunsen burners or alcohol lamps

nutrient agar plates (821862)

inoculating loops

disinfectant (70% ethanol, Lysol®, 10% household bleach, etc.)

wax pencils

Optional Materials

If you do not have burners or lamps to flame sterilize inoculating loops, you can substitute sterile, disposable inoculating loops (703037).

Safety

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.

This activity requires that students work with bacterial cultures and open flames. Have students use sterile technique at all times and wear appropriate safety equipment. Have them wipe down all work surfaces with disinfectant before and after the lab and wash their hands before and after the lab. Disinfect cultures and any contaminated items remaining at the end of the lab by autoclaving or by flooding with disinfectant overnight before proper disposal.

Procedures

If you are not using prepared media plates, you will need to pour plates on the day before the lab. Use either prepared bottle medium (776360) or, if you have an autoclave, dehydrated medium (789640). One unit of 776360 will pour four to five 100-mm plates.

Isolation Streaking

Each student will need access to a Mixed Suspension culture and the following:

• burner
• inoculating loop
• nutrient agar plate
• wax pencil

Streaked plates can be incubated at room temperature. Check the plates daily for signs of growth. Rhodospirillum grows slowest and may take up to 5 or more days to develop usable colonies. The other bacteria will likely show growth in 2 days. If so, prepare slides of Bacillus and Micrococcus first and wait for Rhodospirillum to develop.

Examining the Isolation Plate and Culturing of Isolated Colonies

• isolation streaked plate with visible bacterial colonies
• burner
• inoculating loop
• nutrient agar plate
• wax pencil

Examining the Pure Culture Plate

Pure plate culture produced in the preceding activity

Optional: The plates of pure cultures produced in this activity can be stained for viewing. See our Introduction to Prokaryotes: Bacteria Labsheet for details.

Answer Key to Questions Asked on the Student LabSheet

1. Do you see any indication that more than one species of bacteria is growing on your plate? If so, explain.
   Yes. There are colonies of different color growing on the plate. [Colonies of Bacillus subtilis are white, Micrococcus luteus are yellow, and Rhodospirillum rubrum are pink. Serratia marcescens is pink to red.] Students may comment on other qualities, such as shape or texture of the colony.
2. According to your observations, how did the zigzag pattern of streaking the plate help isolate different colonies of bacteria?
   Each time a streak was made, the loop picked up fewer bacterial cells. Eventually, so few cells were picked up that individual bacteria cells were left on the streak. Each cell divided to produce a colony of a single species.
3. How does the growth on the pure culture plate differ from what you obtained on the isolation plate?
   There were colonies of different colors on the isolation plate. All the colonies on the pure culture plate are the same.
4. Were you successful in establishing a pure culture of one bacterial species on the plate? Explain your answer.
   Yes, all the bacterial growth on the plate is uniform. There are no colonies of differing colors. [Some students may argue that they need to perform additional tests before they can answer this question. This is an acceptable answer.]