Spread plate procedure

One test deficiency to be aware of is inadequate dis-persement of a cream or ointment on microbial test plates. Firms may claim to follow USP procedures, yet in actual practice they may not disperse product over the test plate, resulting in inhibited growth as a result of concentrated preservative in the nondispersed inoculate. The spread technique is critical, and the firm should document that the personnel performing the technique have been adequately trained and are capable of performing the task. Validation of the spread-plate technique is particularly important when the product has a potential antimicrobial affect. 

Ion plating was used by Yoshida and co-workers [183] to make carbon fiber reinforced Al. A typical ion plating procedure is to spread the carbon fiber with an air knife, feed into a vacuum chamber at about 2 Pa in Ar, apply a potential of 0.5 kV to form an Ar plasma. The Al wire fed is into a crucible where it is evaporated onto the moving fiber. The coated fiber formed by ion plating can be processed by hot pressing/diffusion bonding. 

This type of transfer is slightly more complicated than the transfer of a colony from solid to liquid media hut is as important as these techniques are used to enumerate microorganisms. To perform these procedures, graduated pipettes are used to transfer specihc volumes to dilution blanks or to pour/spread plates (Section 14.3). Where reusable glass pipettes are used, these should be washed and placed into a metal canister for autoclaving and subsequent drying in an oven prior to use. Alternatively, sterilized disposable pipettes can be used for transfers (Section 18.7). 

Spread plates are used to enumerate aerobic or heat-sensitive microorganisms and can be prepared a few days prior to use. This procedure utilizes bent glass rods ( hockey sticks ) to distribute a defined volume of liquid evenly over the surface of the solidified agar medium (Fig. 14.4). Normally, the maximum volume that can be placed on a spread plate is 0.1 mL, unlike pour plates, which can receive up to 1 mL. The method suffers from difficulties in complete transfer and separation of individual cells needed to yield separate countable colonies. Excess moisture present on the agar surface can also result in unexpected colony spread and uncountable plates. 

Procedure Spread 25 grams of lead monoxide in a thin layer on an iron or aluminum plate 2-4 mm. thick. Either use the variety of lead oxide which has not been fused and is known under the name of massicot, or use lead carbonate, which on being heated yields a very pure and finely divided lead monoxide. Heat the lead oxide over a ring burner so adjusted that the flames do not quite touch the metal plate. The plate must be kept just below a perceptible red heat. Continue the heating for 6 hours or more and turn over the powder frequently with an iron spatula. When the change is complete, the product is dark brown when hot, a bright scarlet-red when partly cooled, and a somewhat less brilliant red when entirely cold. 

PEO/Li salt films were prepared by dissolving ultrapurified PEO (12 g) and the purified lithium salt, e.g., LiC104 (0.81 g), in 120 mL of acetonitrile (AN), to obtain a PEO/Li+ molar ratio of ca. 36 1. A small aliquot of the PEO/Li salt solution was placed on a glass plate and then spread with a film applicator (Gardner AR-5312) adjusted to ca. 0.76 mm gap. After evaporation, the films thus formed were allowed to dry in a temperature-controlled vacuum desiccator installed inside the glove box at ca. 25°C for 6 h. The temperature was then gradually raised while under vacuum to 100°C over a period of about a day, held at that value for 48 h and then allowed to cool to room temperature. This overall procedure yielded smooth PEO/Li salt films 50 to 65 pm thick, which were stored in sealed polyethylene bags. 

In paper chromatography we use filter paper, marketed for this purpose. It comes usually in the form of a 2-5 cm-wide tape, from which a strip of the necessary length can easily be cut. The more modern technique of thin layer chromatography (TLC), makes use of thin sheets of aluminium oxide, silica-gel, cellulose or some other material, supported by a metal sheet or a polymer. Chromatographic thin layers can be prepared in the laboratory from commercially available adsorbents. A thick suspension of these is made with water (usually a 2 1 w/w mixture of water adsorbent is made up) and this is then spread on a metal plate with a suitable spreader device. Techniques vary from device to device, and the instructions of the manufacturer should be followed whenever thin layer plates are to be prepared. Ready-made thin layer sheets are also available commercially. These contain the active material spread on a plastic support. Thin-layer chromatographic materials, especially ready-made plates, are much more expensive than chromatographic paper, but normally offer faster and sharper separations than the paper. The procedures described in Section VI.20 can be carried out both on a slow chromatographic paper (e.g. Whatman No. 1) or on a cellulose thin layer (e.g. Whatman cellulose). 

Microbial Test Methods The selected microbial test methods determine specific sampling and analytical procedures. When the product has a potential antimicrobial effect and/or preservative, the spread technique on microbial test plates must be validated. In addition, the personnel performing the analytical techniques have to be qualified and adequately trained for this purpose [6],... 

Various binders have been used to give mechanical stability to the layer spread on the suppon plate. Basic requirements are that the binder should not interfere with solute-sorbent interactions, with elution, and detection procedures. At the same time, these binders have to provide compact and adherent layers together with the sorbent. There are various binders that have been applied to prepare the stationary phase for planar chromatography (Table 10.7). 

---