05/01/2023 Dr.T.V.Rao MD 1

STREAKING CULTURE PLATES IN BACTERIOLOGY

Dr.T.V.Rao MD

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Think Before Inoculation •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

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What is streaking •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 direction from the initial site of inoculation. This is done by moving the loop from side to side, passing through the initial site

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What is streaking •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.

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The essential step in inoculating culture plates

•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

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Prompt action with Optimal utility

•Operations must not be started until all requirements are within immediate reach and must be completed as quickly as possible

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Expose the inoculum in test tubes and containers for

minimal Time • 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.

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The Neck of Test tube containing inoculum to be

heated briefly •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

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Exposure to Environment should limited to minimum•During

manipulations involving a Petri dish, exposure of the sterile inner surfaces to contamination from the air must be limited to the absolute minimum

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Work with absolute sterility •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

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Work Using a wire loop •Wire loops are sterilised using red heat in a Bunsen flame before and after use. They must be heated to red hot to make sure that any contaminating bacterial spores are destroyed. The handle of the wire loop is held close to the top, as you would a pen, at an angle that is almost vertical. This leaves the little finger free to take hold of the cotton wool plug/screw cap of a test tube/bottle

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Flaming procedure •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

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Handling of the Inoculating loop

• Position the handle end of the wire in the light blue cone of the flame. This is the cool area of the flame

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Perfect the art handling the Inoculating loop

• 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.

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Heat and cool the Loop • Ensure the full length of the wire receives adequate heating. . Allow to cool then use immediately.

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

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Be careful some times the Loop may not contain inoculum must redraw the inoculum

•If a loop does not hold any liquid the loop has not made a complete circle. To correct the 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.

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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 separated from each other.

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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.

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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.

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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.

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Do not Practice the way

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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.

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Practice , Practice best way to Perfection

• 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 .

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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.

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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.

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Streak the Plates in a defined Manner

• Remove the loop and close the Petri dish. . Flame the loop and allow to cool. Turn the dish through 90° anticlockwise again and streak from ’ across the surface of the agar in three or four parallel lines .

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Carry with further inoculations

•Remove the loop and close the Petri dish. . Flame the loop and allow to cool. Turn the dish through 90°anticlockwise and streak loop across the surface of the agar from ‘ into the centre of the plate

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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 position. There are alternative methods for preparing a streak.

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References •Basic Practical Microbiology© 2006 Society for General Microbiology ISBN 0 95368 383 4

•Google resources from images

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•Program Created by Dr.T.V.Rao MD for the benefit of Medical Microbiologists

for learning basic principles in inoculating the culture plates, as a

Mission for Universal education of skills in Microbiology

•Email•doctortvrao@gmail.com