13 - Isolate an individual species of bacteria

What is the aseptic technique used for?

• To avoid contamination of the sample from outside substances (e.g. microorganisms)

• Important to get reliable & repeatable data

What is streak plating?

• A technique in which bacteria are spread out on a nutrient agar plate so that distinct individual colonies can be seen

• These colonies can then be grown on clean agar plates to produce non-contaminated samples of one species of bacteria

What can the colonies from streak plating be identified as?

A particular species of bacteria via indicators (e.g. size, colour & texture of colony)

What is streak plating an alternative to?

Pour or spread plating

Outline the aseptic technique

• Wipe down surfaces with antibacterial cleaner before & after experiment

• Use a Bunsen burner so convection currents draw microbes away from the culture

• Flame the wire loop before using to transfer bacteria

• Flame the neck of any bottles before use to prevent bacteria entering the vessel (air moves out, so unwanted organisms don’t move in)

• Keep all vessels containing bacteria open for minimum amount of time

• Close all windows & doors to limit air currents

What equipment is needed for this practical?

• Bench disinfectant

• Paper towels

• Bunsen burner

• Inoculating loop

• Mixed culture of bacteria

• Three nutrient agar plates

• Adhesive plates

What is the method for this practical?

1. Loosen the cap of the mixed culture tube

2. Sterilise the inoculating loop in a Bunsen burner flame & allow to cool

3. Flame the neck of the culture tube, dip the loop into the culture, then re-flame the neck & close the tube

4. Slightly open the Petri dish lid & streak the loop lightly across one section of the agar

5. Rotate the plate 90°, re-streak from the previous section into a new area (3-4 streaks). Repeat to dilute the bacteria (final streaks should not overlap the first)

6. Flame the loop again

7. Tape the Petri dish lid loosely (to prevent anoxic conditions) & incubate for 24 hours

8. Next day, observe & sketch colonies

9. Use a sterile loop to pick a white colony & streak it onto a fresh agar plate

10. Do the same with a yellow colony on another fresh plate

11. Tape the lids & incubate again for 24 hours

12. Observe & sketch final results

What are white colonies likely to be?

Salmonella

What are yellow colonies likely to be?

Staphylococcus 

What is the risk, safety precaution & in emergency for the hazard: disinfectant

Risk:

• flammable

Safety precaution:

• keep away from naked flame

In emergency:

• put out fire & seek assistance

What is the risk, safety precaution & in emergency for the hazard: biohazard

Risk:

• contamination & infection

Safety precaution:

• use disinfectant & wash hands with soap after dissection

• don’t incubate at human body temperature

• don’t open agar plate post incubation

In emergency:

• seek assistance

What is the risk, safety precaution & in emergency for the hazard: naked flame

Risk:

• fire hazard & burns

Safety precaution:

• keep away from flammable materials

• tie up long hair

• keep away from edge of desk

In emergency:

• put out fire & seek assistance

• run burns under cold water immediately