Sterile loop

On agar plates, colonies of M. catarrhalis can be easily identified by their capacity to be moved across the surface of the agar with a sterile loop. 

Withdraw all colonies eventually grew on the ground (Fig. 2) using a sterile loop and resuspend each colony in 200 pi of sterile saline or PBS (wNote 20). 

A. Preculture preparation. A 250-mL Erlenmeyer flask containing 50 mL of mineral salt broth (MSB) (Note 1) with 0.2% L-arginine hydrochloride (L-arginine-HCI) and fitted with a cotton plug is sterilized (Note 2). In a laminar flow hood, a single colony of Pseudomonas putida 39/D (Pp 39/D) (Note 3) is selected from a fully grown plate (Notes 4, 5, 6) and transferred to the solution with a sterile loop taking care not to place the hand over the plate or flask. The flask is then placed in a benchtop orbital incubator shaker at 30°C and 200 rpm for 24 hr. 

An area of four square centimeters is marked off on an ordinary microscopic slide with a wax pencil. The slide is sterilized in a flame, and then 0.05 (%0) c.c. of the milk to be examined is placed on it with an accurately calibrated pipette. An equal amount of sterile nutrient liquefied agar, at 42-45 degrees C. is added and the two drops thoroughly mixed with a sterile loop and carefully spread over the area marked off. The mixture is allowed to harden and then a little plate culture is formed. 

Grow D. discoidmm cells as in qualitative tests and make a suspension in sterile saline of approx 2 x 10 D. discoidmm amoebae/mL using cells scraped from this growth plate. In a separate tube prepare a milky suspension of bacteria (appearance similar to fat-reduced milk) harvested with a sterile loop from a fresh Klebsiella aero eneslzvm (less than 1 week old). 

With the second loop, part of the material is now spread out on a third of the culture medium surface at right angles to the first. Then the dish is turned through a further 20 and part of the material is applied with a third sterile loop to the part of the culture medium surface which has not yet been coated. It is possible in this way to obtain individual colonies, which can then be identified in the so-called "colour series". 

Using a sterile loop, transfer yeast as a single streak onto the agar square. 

Using a sterile loop, transfer a yeast colony onto the agar cube. With sterile forceps, carefully place a sterile cover slip over inoculated agar cube, taking care not to leave an exposed agar surface. 

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