Microbiology Lab

Exercise 9: Pure Culture Techniques

Ex 9: Pure Culture Techniques – T-streak for isolation

• Students will perform a t-streak for isolation from an assigned mixed broth labeled either “Mix A” or “Mix B".
• Procedure

1. Prepare your work area
2. Gather supplies
• Petri dish
• Inoculating loop
• Sharpie
• Bunsen burner and striker
• Assigned broth mixed culture
3. Perform a t-streak
• Label the bottom of your Petri dish (fig. 1)
• Aseptically collect a sample of your assigned bacteria
• From a broth, collect 1 loopful
• From a solid media, collect a SMALL amount of bacteria by just touching the loop to the media where the bacteria are growing
• Beginning in top third of plate, streak back & forth, moving toward the top of the “T” (fig. 2)
• Flame the loop and let it cool
• Touch the loop to the left 1/3 of the Petri dish
• Streak up TWICE into the top 1/3 of the plate
• Streak up and down the left 1/3 of the plate moving toward the “T” (fig. 3)
• Flame the loop and let it cool
• Touch the loop to the right 1/3 of the Petri dish
• Streak left TWICE into the left 1/3 of the plate
• Streak left and right moving toward the top of the “T” (fig. 4)
• Flame the loop

§ Put the Petri dish into the incubator

Practice Gram staining

• Students will Gram stain 2 bacterial cultures
• One from a broth mixture of a Gram + and a Gram – bacteria
• What is the morphology & arrangement of the Gram + bacteria?
• What is the morphology & arrangement of the Gram – bacteria?
• One from an agar slang
• Is the culture Gram + or Gram -?
• What is the morphology & arrangement of the bacteria?

Review for the Midterm

• Lab safety & protocol
• Brightfield microscopy
• Bacterial morphology & arrangement
• Smear preparation
• Simple staining
• Negative staining
• Gram staining
• T-streak for isolation (pure culture techniques)