Spread-plate technique

Compared with the pour plate described above, this procedure utilizes previously sterilized bent glass rods (spreaders or hockey sticks) to distribute a defined volume of sample evenly over the surface of the solidified agar medium. Compared with the pour-plate method, thermal shock is not a problem. However, complete transfer and separation of individual cells to yield separate countable colonies may be a concern. Also, excess moisture present on the agar s surface may result in unexpected colony spread 

Prepare agar plates as described in the section B.4.2. Allow agar to fully solidify prior to proceeding. 

While rotating the plate by hand, gently distribute the sample over the agar s surface using the sterilized glass spreader ( hockey stick ). Many laboratories utilize turntables for this purpose. Replace Petri plate cover and discard spreader into disinfectant. 

After sufficient time has elapsed for the liquid to fully absorb into agar (15-30 min), incubate in the inverted position. 

After the appropriate period of incubation, count developed colonies. Multiply colony number by the reciprocal of the dilution and report. Remember, where 1.0 mL of inoculum was used, a further dilution is created and must be taken into account (see section C.2 and C.3.1). 

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Checking the spread plate technique according to ISO 7932 (1987) using Mannitol Egg Yolk Polymyxin agar (MEYP). 

In Tables 1 and 2 the confidence limits (geometric mean CB,j fp/mr ) for the most frequently used combinations of capsules and replicates are given for MEYP and PEMBA respectively. For the spread plate technique on SBA only indicative values based on 4 sets of data are given in Chapter 8 of the certification report. 

Spread plate technique according to the German Federal Food Law method no L 00.00 - 25 (1992) using Pyruvate Egg yolk Mannitol Bromothymol blue Agar (PEMBA). Spread plate technique according to the Nordic Committee on Food Analysis method no 67, second edition (1982) using Sheep Blood Agar (SBA). 

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. 

The spread plate technique may be automated by the use of a piece of apparatus known as the spiral plater. An agar plate is rotated on an Archimedean spiral whilst being inoculated. The volume of the sample decreases (and is therefore effectively diluted) as the spiral moves towards the outer edge of the plate. A specialised counting grid relating the area of plate to the sample volume enables colonies in the appropriate sector to be counted. An electronic colony counter travelling in the same Archimedean screw may be used. 

Spread-Plate Technique The water sample (0.1-0.5 mL) is simply spread uniformly on the surface of agar and then incubated. The incubation temperature and duration for bacterial growth depend on the bacteria of interest. This method is simpler than the pour-plate technique described below as the plates can be prepared in advance. With this method, the morphology of the colonies can be easily distinguished [77]. The colonies can also be transferred with relative ease. Also, the bacterial cells in the samples are not heat-shocked due to exposure to media at 40°C contrary to the jx)ur-plate technique. 

An account of the changes that have occurred in the HPC methodology in the United States since 1905 can be foimd in Ref. [116]. Many types of media are available for use in determining heterotrophic coimts of flora (Table 4.6) and the three techniques that can be used to determine HPC in water are pour-plate and spread-plate techniques and membrane filtration technique [2]. 

Microorganisms were enumerated by the spread plate technique (Lorch et al., 1995) using tripticasein soy agar (TSA Becton Dickinson, Mexico) for bacteria, and potato dextrose agar (PDA Becton Dickinson, Mexico) with chloramphenicol (Sigma), 100 mg L, for fungi. 

Two classic techniques for separation and enumeration of microbial populations utilize pour plates or spread plates. Both suffer from logistical and interpretational difficulties. It is generally necessary to plate multiple dilutions (in duplicate) of the same sample in order to arrive at plates that are countable and statistically valid. In the case of pour plates, embedded colonies may be difficult to recover and transfer. Further, where either method is used for enumeration purposes, it is assumed that each developed colony arose from a single cell. As previously noted, in the case of yeasts this may not be a completely valid assumption. 

Spread plate method A technique used to prepare pure cultures by placing a diluted sample of cells on the surface of an agar plate and then spreading the sample evenly over the surface. 

Three methods may be used for the enumeration membrane filtration, plate count, and most probable number (MPN) method. The advantages of the membrane filter method are its low limit of detection (LOD) of < 1 CFU/g or mL and the efficient separation of the micro-organisms from components of the product, particularly antimicrobial agents. For the pour-plate method, the sample is generally 1 10 dissolved in the diluent, and 1 mL of the dilution is mixed with the agar. This corresponds to a LOD of 10 CFU/g or mL. The LOD is sometimes higher (e.g. 100 CFU/g or mL) if the product needs to be further diluted due to microbial inhibition, or lower in case of products with low microbial acceptance criteria. If the spread plate count technique is used the LOD is a factor of ten higher (>100 CFU/g or mL), because only 0.1 mL of the... 

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). 

Streichen, Streichbeschichten, Streichverfahren (Auftrageveifahren) spread-plate method/technique Spatelplattenverfahren spreading... 

Technique of thin-layer chromatography. Preparation of the plate. In thin-layer chromatography a variety of coating materials is available, but silica gel is most frequently used. A slurry of the adsorbent (silica gel, cellulose powder, etc.) is spread uniformly over the plate by means of one of the commercial forms of spreader, the recommended thickness of adsorbent layer being 150-250 m. After air-drying overnight, or oven-drying at 80-90 °C for about 30 minutes, it is ready for use. 

FIGURE 1 In the powder diffraction technique, a sample is spread on a flat plate and exposed to a beam of monochromatic (single-frequency) x-rays. The diffraction pattern (inset) is recorded by moving the detector to different angles. 

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