How Do We Culture Bacterial Colonies and Prepare Them for Identification?


Pierre Orbe

Talent Unlimited High School, Manhattan


Summer Research Program for Science Teachers

August 2007



This lesson is broken up into 5 parts and time is required for the cultures to grow before you can isolate and stain them. Parts I and II can be done in the same day within a 45 minute Lab period if students have been given a pre- lab session on how to do these steps the day before.


Aim: How do we Culture Bacterial colonies and prepare them for identification?


Part I: Obtaining Bacterial Samples


Lab Materials: 



  1. Determine beforehand where you'd like to sample for bacterial cultures. Make sure you get permission, and discuss what kinds of bacteria you might expect to find in each location.
  2. At each location, put on a pair of gloves. Dip a sterile swab into the dirt, water, or other substance to be sampled. Lightly rub the end of the swab onto a sterile agar plate like you're painting a line onto the surface of the agar. This will give you a mixed culture of microbes.
  3. Carefully dip the same swab into your water-filled tube - this will give you a backup in case your first plate culture doesn't work out.
  4. Mark your plate with the date, location, and your initials to keep track of your culture.


Part II: Isolating a Pure Bacterial Culture


Lab Materials:



  1. From the line streaked onto the surface of your plate (the mixed culture of microbes), you can try one of two techniques to separate them:
    1. Use a second sterile cotton swab or a sterile toothpick to touch a portion of the line on your plate and gently streak it across the plate in a zigzag pattern. Or:
    2. Back at the lab, take a sterile inoculating loop (loop is made sterile by passing it through flame from Bunsen burner then cooled) and dip the loop into the culture of microbes. Then streak this in a pattern over the surface of the agar plate. The inoculating loop is sterilized following each streak series. As the pattern is traced, bacteria are rubbed off the loop onto the medium. The last cells to be rubbed off the loop should be far enough apart to grow into isolated colonies.

    2.   Streaked plates are then incubated overnight at 37 degrees Celsius. Selected colonies can then be picked up from these plates with a sterile inoculating loop and transferred to separate agar plates or culture tubes (such as typtic soy agar slants or tubes filled with Luria broth) to form a pure culture. Once you have obtained a pure bacterial culture, you are ready to stain your microorganism.



Part III: Staining Bacterial Colonies


Lab Materials:



  1. After pure culture is obtained, microorganism is fixed to a microscope slide using an inoculating loop as follows: flame loop then allow it to cool, dip loop into pure culture medium then smear it onto microscope slide.
  2. Allow smear to air dry. Smears can also be heat fixed by carefully passing the slide through flame.
  3. Cover the smear with crystal violet dye for 30 seconds. Because the purple stain imparts its color to all cells, it is called a primary stain.
  4. Rinse the purple dye off with distilled water then cover the smear with Gram's iodine for 10 seconds.
  5. Rinse gently with distilled water. Decolorize the smear with alcohol.
  6. Rinse with distilled water then cover the smear with safranin for 30 seconds. Rinse the smear with distilled water and blot the slide dry.
  7. Observe slide under a microscope. Bacteria that are gram negative will appear pink while those that are gram positive will appear purplish.


Part IV: Identifying Bacterial Species

This kit is one that I plan to try this year in order to help run some tests to identify what the bacterial colonies might be.  Otherwise there are different resources for identifying and categorizing bacteria based on size and color, but there are so many species this will become difficult.



Enterotube II, a marker-sized device that contains an inoculating wire running through 12 separate agar-filled chambers that perform 15 biochemical tests. Manufactured by Becton Dickinson. For more information about prices, availability, and local distributors, call 1-800-638-8663 ext. 2.



National Science Standards:

Teaching Standard A: Teachers of science plan an inquiry-based science program for their students. In doing this, teachers


Content Standard C: As a result of their activities in grades 9-12, all students should develop understanding of


NS 9- 12.5 Science And Technology


NS 9- 12.6 Personal And Social Perspectives