inoculating a culture plate in bacteriology

Streaking culture plates in Bactaeriology

1. STREAKING CULTURE PLATES IN BACTE R IO L OGY D r . T . V . Rao MD 5/7/2016 1

2. Think Before Inoculation 2 Before inoculation, important information is written on the bottom of the plates, close to the rim 1date of inoculation 2.temperature of incubation 3.duration of incubation 4.microorganism inoculated

3. What is streaking 3 The most common method of inoculating an agar plate is streaking. Streak plates 1.With this method, a small amount of sample is placed on the side of the agar plate (either with a swab, or as a drop from an inoculating loop if the sample is a liquid). 2.A sterile loop (flamed until red hot, then cooled by touching the agar away from the inoculated sample) is then used to spread the bacteria out in one dir e ction fro m th e initial site of inocu l ation. Thi s is done b y movin g th e loop from side to side, passing through the initial site

4. What is streaking 4 The loop is then sterilised (by flaming) again and the first streaks are then spread out themselves. .This is repeated 2-3 times, moving around the agar plate. What should happen is that single bacterial cells get isolated by the streaking, and when the plate is incubated, forming discrete colonies that will have started from just one bacterium each.

5. The essential step in inoculating culture plates 5 There are several essential precautions that must be taken during inoculation procedures to control the opportunities for the contamination of cultures, people or the environment

6. Prompt action with Optimal utility 6 Operations must not be started until all requirements are within immediate reach and must be completed as quickly as possible

7. Expose the inoculum in test tubes and containers fo r m inimal Time 7 Inoculum is grown in test tubes and must be open for the minimum amount of time possible and while they are open all work must be done close to the Bunsen burner flame where air currents are drawn upwards.

8. Th e Neck of T est tub e cont a ining inoculum to be heated briefly 8 On being opened, the neck of a test tube or bottle must be immediately warmed by flaming so that any air movement is outwards and the vessel held as near as possible to the horizontal

9. Exposure to Environment should limited to minimum 9 During manipulations involving a Petri dish, exposure of the sterile inner surfaces to contamination from the air must be limited to the absolute minimum

10. Work with absolute sterility 10 The parts of sterile pipettes that will be put into cultures or sterile vessels must not be touched or allowed to come in contact with other non-sterile surfaces, e.g. clothing, the surface of the working area, the outside of test tubes/bottles

11. Work Using a wire loop 11 Wire loops are sterilised using red heat in a Bunsen flame before and af t er us e . Th e y mus t b e heat e d t o r e d hot to make sure that any contaminating bacterial spores are d e str o yed. Th e handle of the wir e l oop is held close to the top, as you would a pen, at an angle that is almost vert ica l . Thi s leave s th e littl e fing e r free to take hold of the cotton wool plug/screw cap of a test tube/bottle

12. Flaming procedure 12 The flaming procedure is designed to heat the end of the loop gradually because after use it will contain culture, which may ‘splutter’ on rapid heating with the possibility of releasing small particles of culture and aerosol formation

13. Handling of the Inoculating loop 13 Position the handle end of the wire in the light blue cone of the flame . Thi s is t h e cool area of the flame

14. Perfect the art handling the Inoculating loop 14 Draw the rest of the wire upwards slowly up into the hottest region of the flame, (immediately above the light blue cone). . Hold there until it is red hot.

15. Heat and cool the Loop 15 Ensure the full length of the wire receives adequate . heating. Allow to cool then use immedi a tel y .

16. Handle the Inoculating loop 16 Do not put the loop down or wave it around.. Re-sterilise the loop immediately after use .

17. Be careful some times the Loop may not contain inoculum must redraw the inoculum 17 If a loop does not hold any liquid the loop has not made a comp l e t e circle. T o cor r ect t h e problem, first ensure that the loop has been sterilised and then reshape the loop with forceps. Do not use your fingers because of the possibility of puncturing the skin .

18. Working with bacteria and yeast Streak plate The loop is used for preparing a streak plate. This involves the progressive dilution of an inoculum of bacteria or yeast over the surface of solidified agar medium in a Petri dish in such a way that colonies grow well s e 5 p / 7 / 2 a 1 r 6 ated fro m each o t he r .

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20. The aim of the procedure is to obtain single isolated pure colonies Loosen the cap of the bottle containing the inoculum. Hold the loop in the . right hand. Flame the loop and allow to cool. 20

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22. Handling matters in getting the right inoculum Lift the bottle/test tube containing the inoculum with the left hand. . Remove the cap/cotton wool plug of the bottle/test tube with the little finger of the right hand. . Flame the neck of the bottle/test tube. 22

23. Handling loop and Inoculating the material matters Insert the loop into the culture broth and withdraw. At all times, hold the loop as still as possible. . Flame neck of the bottle/test tube. 23

24. Do not Practice the way 24

25. Practice for Perfection Replace the cap/cotton wool plug on the bottle/test tube using the little finger. Place bottle/test tube on bench. . Partially lift the lid of the Petri dish containing the solid medium. 25

26. P r actice , P r actice b est w a y t o P erfecti o n Hold the charged loop parallel with the surface of the agar; smear the inoculum backwards and forwards across a small area of the medium (see streaked area . 26

27. Follow the Instructions for safe keeping of Petri dish Remove the loop and close the Petri dish. . Flame the loop and allow it to cool. Turn the dish through 90° anticlockwise. 27

28. Streak the Plates in a defined Manner With the cooled loop streak the plate from area ‘ across the surface of the agar in three or four parallel lines . Make sure that a small amount of culture is carried over. 28

29. Streak the Plates in a defined Manner Remove the loop and close the Petri dish. . Flame the loop and a l lo w t o cool. T urn t h e dish through 90° anticlockwise again and streak from ’ across the surface of the agar in three or four parallel lines . 29

30. Carry with further inoculations Remove the loop and close the Petri dish. Flame the loo p and a llo w t o cool. T urn the dish through 90°anticlockwise and streak loop across the surface of the agar from ‘ into the centre of the plate 30

31. Complete the work Close the petri dish Remove the loop and close the Petri dish. Flame the loop. . Seal and incubate the plate in an inverted posit i on. The r e a r e alternative methods for preparing a streak. 31

32. References 32 Basic Practical Microbiology© 2006 Society for General Microbiology ISBN 0 95368 383 4 Google resources from images