Pour-plate technique

Agar solidifies at temperatures slightly below 40°C (I04°F) and does not melt until near the boiling point of water. To maintain the liquid state for a sufficient time to carry out the steps involved in plating, liquified agar must be maintained at 44-46°C (III-II5°F) prior to pouring. This is an important concern in that there is the potential for undefined inhibition or cell death resulting from thermal shock. 

Success in using this technique requires that the agar be sufficiently liquid to permit introduction of the sample, dispersion of cells, and plating without cooling to the point of solidifying. Thus, laboratory personnel must work quickly while adhering to aseptic technique. 

Prior to plating, liquified media should be equilibrated to 44-46°C (III-II5°F) inawaterbath (see Supplemental Note I). The water-bath temperature may be monitored by placing a thermometer in a similar-sized test tube filled with water. The baby-bottle technique of touching tubes to one s wrist to estimate temperature is not recommended. 

Prepare a dilution series based on an estimated viable cell number 

Once the final dilution is achieved, vortex and transfer the appropriate volume (usually 1.0 mL or less) to a Petri plate. 

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Several laboratory methods are used to quantify bacteria present in the urine. The most accurate method is the pour-plate technique. This method is unsuitable for a high-volume laboratory because it is expensive and time-consuming. The streak-plate method is an alternative that involves using a calibrated-loop technique to streak a fixed amount of mine on an agar plate. This method is used most commonly in diagnostic laboratories because it is simple to perform and less costly. 

The number of viable cells in the inocula was determined at timed intervals by the pour plate technique with HS medium. Colonies were counted after 5 days of incubation at 30 °C. 

Plates (15 by 100 mm plastic petri dishes) contained a base layer of 20 ml minimal agar (8) with 0.5% glucose. A standard pour-plate technique was used 0.1 ml of the proper dilution and 2 ml of molten 0.6% agar were added to a sterile tube, mixed, and poured over the surface of a base plate. The histidine overlay contained 0.1 ml of 0.1 M L-histidine per 40 ml of agar. Plates were incubated at 37°C for 48 hr, and the ratio of mutants total cells (mutant frequency or MF) was determined. 

Penn-Assay broth. Bacteria were pelleted by centrifugation (1800 X g, 10 min) and opsonized in BBSS containing 10% fresh human serum (0.5 ml/ml pelleted), for 30 min at 37 C. Bacteria were recovered by centrifugation, washed twice with saline and resuspended at a final concentration of 1 x 10 organisms per ml in BBSS as determined by routine pour plate techniques. 

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. 

Grip layer. A layer of coarse aggregate may be bonded to the steel to form a rough layer onto which fresh concrete is subsequently poured. This technique has been the subject of some research involving slab panels, tensile reinforcement being provided in the form of a thin soffit steel plate. However, premature adhesive failures have been reported and an improved bonding technique is considered to be necessary(9). 

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. 

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... 

The determination of carbon and nitrogen utilization profiles can be performed in broth or pour plates seeded with the microorganism of interest. Success using these techniques require that fungi be in pure culture and that carbon and nitrogen sources be free of contamination. To interpret the broth method (Anonymous, 1984), a finely ruled white index card is placed behind the tube and the degree to which haze/turbid-ity hampers visualization is noted. Relative growth may be scored as follows ... 

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]. 

There are several forms of electrophoresis. In slab gel electrophoresis the conducting buffer is retained within a porous gel of agarose or polyacrylamide. Slabs are formed by pouring the gel between two glass plates separated by spacers. Typical thicknesses are 0.25-1 mm. Gel electrophoresis is an important technique in biochemistry, in which it is frequently used for DNA sequencing. Although it is a powerful tool for the qualitative analysis of complex mixtures, it is less useful for quantitative work. 

In this technique the concentration of a drug in an agar plate may (theoretically) be varied infinitely between zero and a given maximum. To perform the test, nutrient agar is melted, the solution under test added, and the mixture poured into a sterile Petri dish and allowed to set in the form of a wedge (Fig. 11.6A). 

The poly (HEM A) sheets were prepared by B. Ratner using a special technique he developed. The HEMA solutions were poured between glass plates, and polymerization was chemically initiated. The chemical and physical properties of this material are very similar to those of radiation-grafted poly (HEMA) insofar as protein adsorption is concerned. Heterogeneous or homogeneous poly (HEMA) films were made by polymerization in solvents in which the poly (HEMA) is insoluble or soluble, respectively the result is a white opaque material in the first case and a transparent material in the second case. The resulting films were washed free of excess monomer and then soaked in the buffer to be used in the fibrinogen adsorption experiment for 10 days at 37 °C prior to the actual experiment. 

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