Carlos Franco                                                                                                                            Return to Biology Menu

Summer 2001

Living Environment


Isolating bacteria from a mix culture



1.      Students will isolate three species of bacteria using the plate streak method

2.      Students will implement sterile microbiology techniques.

3.      Students will produce pure cultures of each microorganism at the end of this exercise

Duration of activity: (3) days

            Day 1: Streaking plates with mix culture (30 min)

Day 2: Inoculating nutrient broth tubes with colonies in streaked plates (20 min)

            Day 3: Observe plates and record/ discuss results (30 min)


Science Standards:


Standard 5: Students will apply technological knowledge and skills to design, construct, use and evaluate products and systems to satisfy human and environmental needs

 Standard 2: Students will access, generate, process, and transfer information using appropriate technologies.




Culture of Escherichia coli


(1)Inoculating loop per group

Culture of Serratia marcescens

(8) Nutrient agar plates per group

Culture of Micrococus lutea

(6) 3ml/nutrient broth tubes


(1) Bunsen burner per group


(1)Incubator @37 °C (optional)

(1) Pack of colored pencils



Before commencing this activity prepare the following:

1.Inoculate 5ml of nutrient broth with Escherichia coli, Serratia marcescens, and Micrococus lutea. Place in incubator @ 37°C  for 24 hours or 48 hours at room temperature;

2. Prepare nutrient agar plates (18ml/plate) and nutrient broth (3ml/tube) 24 hours before activity .  Store plates and tubes in a refrigerator until ready to use.


Standards addressed:

1.      Interpretation and analysis of data

2.      Hands on activity

3.      Cooperative learning


Q. Have student brainstorm why is it important to separate the components in a mixture.  What can we learn by separating the components of a mixture?

 Have students write out the methods they would use to separate the components of a mixture of sand, iron filing, and oil.

Possible keywords in responses: density, magnetism, filtration; funnel, etc.


Procedure: Day 1

  1. Obtain from your teacher the following materials:
    1. (1) Inoculating loop
    2. (1) Bunsen burner
    3. (2) Agar plates keep plates close to avoid contamination
    4. (1) Mix culture of bacteria
  2. With a marker label the bottom of the agar plates: plate #1 and plate #2 respectively, your group name, and the today’s date.      
  3. Turn on your Bunsen burner and adjust your flame until it is blue.
  4.  Place the inoculating loop over the flame until the loop turns bright orange.
  5. Remove loop from flame and wait about ten seconds and dip the loop only into the mix culture.
  6. Take the loop and make four streaks exactly as shown in the following diagram:


Make sure that when you are making the streaks they intersect at each corner as illustrated by this diagram.


While streaking make sure you minimize talking to avoid contamination.  As soon as you finish cover the plate immediately.






  1. Incubate @ 37°C for twenty-four hours or leave at room temperature for forty-eight hours

Homework #1:

Write descriptive paragraph for each specie of bacteria in the mix: Escherichia coli, Serratia marcescens, and Micrococus lutea. Describe their physical characteristics as well as the conditions they need to grow best.

Hint: Use a search engine on the Internet, i.e.. Infoseek, Lycos, etc. and search for each specie at time. 


Procedure: Day 2 

  1. Observe your plates and draw what has grown using color pencils.  You will note that there are three types of colonies present in the agar, each color corresponds to a different species of bacteria.
  2. In which streak are most of the bacterial colonies growing? Explain
  3. Obtain three nutrient agar plates and label them white, red, and yellow.
  4. Turn on the Bunsen burner, adjust the flame, and sterilize the loop and described earlier.
  5. Choose a white colony from streak #4 and after loop has cooled-off (10 Sec) tap your chosen white colony and streak plate label WHITE as shown in diagram below
  6. Sterilize your loop
  7. Choose a red colony from streak #4 and after loop has cooled-off (10 Sec) tap your chosen white colony and streak plate label RED.
  8. Sterilize your loop
  9. Choose a yellow colony from streak #4 and after loop has cooled-off (10 Sec) tap your chosen yellow colony and streak plate label YELLOW.
  10.  Incubate @ 37°C for twenty-four hours or leave at room temperature for forty-eight hours
  11. Sterilize your loop






Streak plates as illustrated in this diagram





Homework #2

Describe the streak- cross method as a method of separating bacteria from a mix culture.  How does streak -cross method work?

Hint: Internet search engines


Procedure: Day 3

  1. Observe your plates and answer the following questions based on your understanding of this activity as well as the completed Internet assignments
    1. Were all the colonies in each plate the same color?  If they were not give a possible explanation for your observation.
    2. Why were you asked to choose your colonies from streak #4 and not from rest of the streaks?
    3. Why was it important that each streak intercepted as seen in figure #1?
    4. Based on your Internet search what color colonies does each bacteria express in nutrient agar?
    5. Sterile technique is important in microbiology.  Explain why this was so in this activity.
    6. If you were given a mixture composed of bacterium that produced same color colonies, would the method you implemented in this activity help you separate this mixture? Explain Hint: Homework#1