A lot of precaution must me exercised during inoculation procedures in order to control the contamination of microbiological cultures, personnel or the surroundings. In order to ensure a smooth operation, make sure that all requirements are available in the immediate vicinity before starting any practical work. The laboratory wares must not be left open for longer duration of time and flaming of the mouth is mandatory in a slightly tilted position before sealing it back with a cotton plug or a screw cap to ensure sterility of the contents and the air column above. Always use sterile pipette and tips for handling cultures or for transfer of sterile content while making sure that the pipette or the tip are not coming in contact with any non-sterile surface. A petri dish must be opened only in between two Bunsen burners or inside a sterile laminar flow hood to prevent any form of contamination.
A wired inoculation loop must be heated until red-hot to ensure that spores are destroyed completely and subsequently cooled before being used in transfer of another culture or before safely putting it away. The loop handle is held like a pen to allow the outer fingers to open or close the cotton plug or screw cap of transfer vessel or culture tubes.
Sterile Pasteur pipettes are used to transfer sterile solutions and microbiological cultures.
Take care to ensure that the pipette bulb is not ill-fitted or the cotton plugs are correctly placed inside the pipette to avoid any leak
A leaking pipette is caused by either a faulty or ill-fitting teat or fibres from the cotton wool plug between the teat and pipette. A dropping (Pasteur) pipette can be converted to delivering measured volumes by attaching it to a non-sterile syringe barrel by rubber tubing. A transparent Pasteur pipette made of LDPE, with clear graduation markings is ideal for easy liquid observation.
Smudging of labels can happen if they are not positioned correctly or labeled with markers and covered with adhesive tapes. If a cotton wool plug accidentally catches fire douse the flame with a dry cloth never blow or dip in water.
An inoculation loop with a smooth circular shape is used for preparing a streak plate. A progressive dilution technique is followed by streaking the plate with a single, pure inoculum over several different areas of the plates to produce individual colonies. A smooth disposable ABS inoculation loop is ideal, however while using a Nichrome loop, a proper flaming technique has to be followed.
Procedure:
A pour plate technique is the one in which a small amount of inoculum from broth culture is added by pipette to the center of a petri dish OR Melted and cooled agar medium in a test tube or bottle is poured into the petri dish containing the inoculum. The petri dish is gently rotated to ensure that the culture and medium are mixed thoroughly and the medium evenly covers the plate. Pour plates allow microorganisms to grow both on the surface and inside the medium. If we know the dilution and volume of the inoculum (around 1 cm3), the viable count of the sample per cm3 can be determined. The dilutions chosen must be appropriate to produce enough ( around 100) countable colonies.
Procedure:
Inoculate the Petri dish
Pour the plate
Using a spreader:
Sterile microbiological spreaders are used to distribute inoculum above the surface of already prepared solid agar medium.
Spread plate:
A spread plate technique often gives confluent growth of culture spread evenly over the surface of the growth medium. The spread plate can be used for quantitative work. If the dilution and volume of the inoculum, usually 0.1 cm3, are known, the viable count of the sample. The dilutions chosen must be appropriate to produce about 100 countable colonies.
Moulds:
Moulds are usually prepared as a spore suspension by ensuring that they are not escaping in to air. While handling moulds, ensure that the inoculum is taken as a small portion of the mycelium with a wire loop or straight wire with the end few millimeters bent at a right angle. When an agar plate with a mould is inoculated at the center, make sure not to accidentally touch the outer surface or allow the spores to fall from the loop or wire. We can achieve this by holding the plate upside down and smartly lifting the base of the dish and inoculating the center of the agar with the wire or loop.
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