Streak LAWN Technique Report PDF

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Summary

TREAK AND LAWN PLATE TECHNIQUES LAB REPORT
- to obtain single isolated pure colonies using the two
techniques of Streak and Lawn
Evaluation
- Greatly detailed...

Description

STREAK AND LAWN PLATE TECHNIQUES

INTRODUCTION: Streak Plate Technique: Streak plates consist of the progressive dilution of an inoculum of either yeast or bacteria of the surface of a solidified agar medium that is in a Petri Dish. This technique is used to find out the purity of cultures which are being maintained over time and regular sampling and streaking to show any contamination by other microbes. This method is used for expert practitioners to begin new maintained cultures by using an appropriate isolated colony of an identifiable species with a sterile loop as well as growing the cells in a sterile nutrient broth. Spread/Lawn Plates Technique: The spread/lawn plate technique can be used for quantitative work. If the volume and dilution of the inoculum is known, you can easily determine the viable count of the sample. This is the number of bacteria per cm3. Overall, they result in a heavy or commonly a confluent growth of culture that is spread out evenly on the surface of the growth medium. This indicates that it can be used to test the sensitivity of the growth medium.

AIM AND HYPOTHESIS: The aim and hypothesis of this is to obtain single isolated pure colonies using the two techniques of Streak and Lawn. If more than one shape or colour of the colony on the streak lines are noticeable, it means the culture has more than one type of bacterium or yeast. The aim overall is to ensure that where we streaked using the loop should produce isolated colonies of an organism on the agar plates. For the Lawn technique, we could determine the viable count if the dilution forms 30 and 100 separate countable colonies.

LIST OF APPARATUS: The materials used to perform the experiment was • • • • • • • •

Glass Spreader. 2 Agar Plates Bunsen Burner 70% Ethanol Broth Culture Sterile Pipette Inoculum Agar Inoculating Loop

METHOD

Streak Plate Technique: 1. Open the cap of the bottle which consists of the Inoculum. 2. Hold an Inoculating loop and flame it using a red safety flame. (using right hand) 3. Allow the loop to cool. 4. Lift the bottle or test tube that contains the Inoculum. (using left hand) 5. Remove the cap or cotton wool plug or the test tubes. (using the little finger of right hand) 6. Flame the neck of the bottle or test tube. 7. Place the loop into the culture broth and withdraw. Ensure the Inoculating loop is held as still as possible. 8. Flame the neck of the bottle or test tube again. 9. Replace the cap/cotton wool plug or the test tube using the little finger of right hand. Put the bottle/test tube on the bench or a rack. 10. Then partially lift the Petri dish lid containing the solid medium. 11. Hold the charged loop that is parallel to the surface of the afar. Smear inoculum back and forth across a small area of the medium. 12. Take out the Inoculating loop and close the Petri dish. 13. To prevent any contamination, flame the loop and allow it to cool again. 14. The dish must be rotated through 90 degrees anticlockwise. 15. Once the Inoculating loop is cooled, streak the plate from one area across the surface of the agar in three or four parallel lines. This is to ensure that a small amount of the culture is carried over. 16. Remove the loop and close the petri dish. 17. Again, using the Bunsen Burner to flame the loop and allow to cool. Turn the dish through 90 degrees anticlockwise again and streak from another area of the agar in three or four parallel lines. 18. Once the loop is removed and the petri dish is closed, again flame the loop and allow it to cool. Turn the dish through 90 degrees anticlockwise and streak the loop across the surface of the agar from one area to the next area. 19. Lastly, remove the loop and close the Petri dishes. Flame the loop again. 20. Tape the plate closed and incubate the place in an inverted position. To prepare the Spread/Lawn technique: 1. Loosen the cap of the bottle/test tube that contains the broth culture. 2. Remove a sterile Pasteur pipette from its container and attach a teat using your right hand. 3. Hold the sterile pipette in your right hand and the bottle/ test tube containing the broth culture in your left. 4. Remove the cap/cotton wool plug of the bottle/ test tube with the little finger of your right hand and flame the neck. 5. Squeeze the teat of the pipette, and draw up a small amount of broth. 6. Flame the neck of the bottle/ test tube and replace the cap/ plug.

7. With your left hand, partially lift the lid of a Petri dish containing the solid nutrient medium. 8. Place a few drops of culture onto the surface – about 0.1 cm3/ around 5 drops/ enough to cover a 5 pence piece. 9. Replace the lid of the Petri dish. 10. Place the pipette in a discard jar of disinfectant. 11. Dip a glass spreader into alcohol (70% IDA), flame and allow the alcohol to burn off. 12. Lift the lid of the Petri dish to allow entry of the spreader. 13. Place the spreader on the surface of the inoculated agar. Move the spreader in a topto-bottom or a side- to-side motion to spread the inoculum over the surface of the agar. Make sure the entire agar surface is covered. 14. Replace the lid of the Petri dish. 15. Flame the spreader using alcohol. 16. Let the inoculum dry. This will take some time. 17. If the plate has not been made to assess the population in a serial dilution, it can now be treated further, for example, for testing antimicrobials, before taping and incubation. 18. Tape the plate closed and incubate it in an inverted position.

RESULTS & DISCUSSION: Streak plate techniques are to isolate the pure culture of organisms from mixed population. The inoculum is streaked over the agar surfaces so that it "thins" out the bacteria. Some of the individual bacteria cells are either separated or spaced out. Due to the original sample being diluted by streaking it over successive quadrants, it means the number of organisms decrease. A bacterial lawn plate describes the appearance of bacterial colonies when all individual colonies on a petri-dish agar plate would merge to form a mat of bacteria. This is the technique to plate a liquid sample that consists of the bacteria so that the organisms are easily countable and can isolate. For the streak technique, the results show below that there are discrete colonies in the plate as well as both heavy confluent growth and light growth. Once the results were incubated for 24-48 hours, I was also able to carefully identify that there is more than one colony. The Lawn technique however was also incubated for 24-48 hours and I was able to identify that there was growth but it did not cover the entire plate.

CONCLUSION

The results of this lab did support the initial hypothesis. However, for the Lawn Technique Plate there was no growth in on the sides of the petri dishes. Also, for the Streak Technique there were discrete colonies shown as well as both growth of light and heavy confluent. To evaluate on this, I was able to follow the protocol successfully and ensure that the risk assessment was followed to prevent any contamination, spillage or injuries. However, the problems encountered were that I was unable to produce clear streaks on the agar plate and also ensuring that the entire plate was spread. This means, in order to better my results in the near future, I must make sure I carefully and gently streak the plates as well as using the glass spreader to make sure that the plate is entirely covered....