Pour-plate

Roll tubes may be used as an alternative to plates. In these the sample is added to a tube containing molten agar which is then rotated rapidly on a mechanical roller until the agar sets. The tubes are incubated and counted. This method uses less agar than traditional pour plates and is widely used in the dairy industry. 

See Fig. 14.1 for setting up a dilution series using 9mL dilution blanks. Serially dilute the juice or wine, thoroughly vortexing each dilution blank before transferring 1 mL into the next blank. 

Place 0.1 or l.OmL from each dilution blank into a sterile Petri dish. 

Immediately after autoclaving, place the appropriate agar medium in a water bath at 45°C/113 F to 50 C/122 F. Once the temperature has equilibrated, aseptically add approximately 25 mL of the agar to each plate (smaller Petri plates will use less agar medium). 

Carefully mix each plate using a figure eight pattern. Allow the plates to cool and solidify undisturbed, usually for at least an hour. 

Invert the plates (upside down) and incubate at the appropriate temperature and time. 

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Estimation The above medium is reinforced with lOg/i of thiocyanate, sulphur is omitted and it is prepared as pour plates by the addition of 3% agar. Organisms other than Thiobacilli will grow from spread samples, but the Thiobacilli are easily distinguished by sulphur haloes (see Fig. 2.19). 

Plate on 24-well format LB agar supplemented with antibiotic and, if appropriate, X-Gal and IPTG (dilute a 20% X-Gal, in dimethyl formamide, stock 1 1000, dilute the IPTG 500 mM stock 1 500 in warm agar before pouring). Plate 10 pi of cells, shake plates well to spread the cell suspension, and allow at least 10-15 min for the plates... 

Repeat this for each test organism. For positive control use sterile DW instead of sanitizer solution. For negative control use sterile strips. Swab each strip thoroughly with a premoistened Ca alginate swab and place in Lecithin broth (or other suitable neutralizer). Vortex for 30 sec, then perform the usual pour plate method. 

The method selected shall be capable of isolating the numbers and types of organisms that have been estimated significant relative to system control and product impact for each individual system. The recommended method is membrane filtration pour plate and most probable number may be used per requirements. 

YM5YM + 0.5% acetic acid (add just before pouring plate). 

Fig. 10.2 Various monitoring devices (1 to r) corrosion/fouling/ biofouling mesh coupons, LPRM monitor, corrosion rack, deposit monitor, membrane filter on poured plate, and dip-slide... 

Viable Plate Count A viable cell is defined as one that is able to divide and form a colony. There are two ways of performing a plate count—the spread plate method and pour plate method. With the spread plate method, a volume of no larger than 0.1 mL is spread over the agar surface. With the pour plate method, the sample is mixed with melted agar and poured into a sterile plate. The plate is then incubated until the colonies appear, and the number of colonies is counted. It is important that the number of colonies developing on the plates should be neither too large nor too small. To obtain the... 

A viable count via a pour plate or spread plate should be obtained for the final dilution of each micro-organism to verify the challenge level. 

If 30 is accepted as the lowest reliable number to count and a pour plate method uses a 1.0-ml sample, it follows that the procedures described above are unsuitable for any sample that is expected to contain <30 CFU ml-1, e.g. water samples where the count may be 1 CFU ml-1 or less. Here, membrane filter methods are used in which a large, known volume of sample is passed through the membrane which is placed, without inversion, on the agar surface. Nutrients then diffuse up through the membrane and allow the retained cells to grow into colonies on it just as they would on the agar itself. 

Pour plate Requires no pre-drying of the agar surface Will detect lower concentrations than surface spread/surface drop methods Very small colonies of strict aerobes at the base of the agar may be missed Colonies of different species within the agar appear similar —so it is difficult to detect contaminants... 

Surface spread and surface drop methods Surface spread often gives larger colonies than pour plates—thus they are easier to count Easier to identify contaminants by appearance of the colonies Agar surface requires pre- drying to absorb sample Possibility of confluent growth, particularly with moulds, masking individual colonies... 

The TAC can be conducted using a number of microbiological methods. These are the pour-plate, membrane filtration, and multiple tube methods. The TYMC is conducted by using either the pour-plate or membrane filtration method. The TAC for bioburden is performed by adding 10 g, 10 mL, or 10 units in SCD broth or lactose broth to make 100 mL. Aliquots of this sample preparation are transferred into four standard size (15 x 100 mm) Petri dishes. Into two of the plates 15-20 mL of molten SCD agar is poured, and into the other two the same volume of Sabouraud dextrose agar (SAB) agar is poured. 

Pour plate method. An aliquot of the neat or diluted sample is added to a sterile Petri dish and mixed with the selected molten agar medium. When the agar has solidified, the plates are incubated for a predetermined time at the specified temperature. Surface or subsurface colonies will develop in some of the agar plates, which can be counted to provide a quantitative value for the bacterial density of the original sample. They can also be picked for further qualitative study. 

Liquid samples can be analysed directly by the spread plate (A) or the pour plate method (B) but they can also be filtered to retain the micro-organism on a filter which is than placed on the culture medium (C). Suspensions need to be filtrated or centrifuged afterwards the filtrate or the filter can be analysed. Solids (e.g. food) must be minced before filtration. Incubation at a given temperature can be aerobic or anaerobic. The result of the counting will depend on the ability to isolate the target organisms with the best recovery rate. 

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. 

Table VII. Enumeration of Groundwater Bacteria by Pour Plate Counts on Groundwater-Yeast Extract Agar...

LB-Amp plates Dissolve 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar in 1L deionized water and sterilize by autoclaving. Add lmL of l,000x (100mg/mL) ampicillin stock to cool (<50°C), mix and pour plates. 

Testkits Muta-ChromoPlate (a microplate version of the traditional pour plate Ames test, Environmental Bio-detection Products Incorporated (EBPI), Brampton, Canada). 

Pour plate serial dilutions on MSAI (100 ppm) agar for bacteria SDAI agar for fungi... 

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