Spread-plate

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. 

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

Place 0.1 mL from each dilution blank onto a sterile Petri dish containing the appropriate sohdihed medium. 

Holding the handle, dip a glass hockey stick into 70% v/v ethanol, flame, and allow flame to bum itself out and cool. Spread the 0.1 mL around on the plate using the hockey stick. 

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

In this method, molten agar is poured into the petri dish, allowed to set and then dried thoroughly. The liquid sample is pipetted onto the plate and immediately 

Spread plates have certain advantages over pour plates for aerobic counts. The agar is not hot and therefore cells are not killed or heat damaged. Secondly, because all colonies are on the surface of the plate the availability of air to each colony is maximised. 

Both spread and pour plates should be counted using a plate counter equipped with magnification, illumination, and a centimetre grid. A large number of electronic counters are available. The colonies on the plate should be counted promptly after the appropriate incubation period, or if this is not possible they should be stored in a cold room. If necessary, the edges of the plates may be sealed with cellotape to stop them drying out. 

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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... 

The chemicals come from cracks on the ocean floor called hydrothermal vents. Hydrothermal vents are most often found where the sea floor is spreading due to the movement of sections of Earths crust. Seawater enters the cracks produced by the spreading plates and is heated by hot magma lying under the surface. The water can reach temperatures as high as 750°F (400°C). 

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. 

While most studies of seafloor hydrothermal systems have focused on the currently active plate boundary ( 0-1 Ma crust), pooled heat-flow data from throughout the world s ocean basins (Figure 1) indicate that convective heat loss from the oceanic lithosphere actually continues in crust from 0-65 Ma in age (Stein et al, 1995). Indeed, most recent estimates would indicate that hydrothermal circulation through this older (1-65 Ma) section, termed flank fluxes, may be responsible for some 70% or more of the total hydrothermal heat loss associated with spreading-plate boundaries—either in the form of warm (20-65 °C) altered seawater, or as cooler water, which is only much more subtly chemically altered (Mottl, 2003). 

Spread plate method. A pre-selected small volume of the neat or diluted sample is inoculated onto a solid selective or non-selective medium. It is spread uniformly over the surface by mechanical or manual methods e.g. by holding a sterile stick at a set... 

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. 

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). 

At regular time intervals, a 2.5-mL of culture was taken out by a catheter for the measurements of the cell density. The cell density was determined by the conventional spread plate method. 

Exposure of the spread plates to fluorescent light in the absence of RB did not cause a substantial change in CFU. However, in the presence of RB, there was a dramatic decrease in CFU (Fig. 1). RB prevented completely the growth of E.coli (reaching 0 % of survival), when illuminated at 7500 lux for 40 min. 

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. 

In this method, 25 ml of a molten sterile medium is poured into presterilised Petri dishes and allowed to solidify at room temperature (RT). The agar plates are seeded with 0.1 ml of (1 X 10 spores/ml) of a fungal/bacterial culture using the spread plate method. Subsequently, 10 mm wide wells are bored into these agar plates using a sterile cork borer. 250 pi of a stock solution of an antimicrobial compound is filled into the wells and the plates are incubated at 25 2 °C. The antimicrobial activity is interpreted from the size of the zone of inhibition(s) (ZOI) measured to the nearest millimetre, i.e., the clear zones surrounding the wells, as shown in Figure 11.1 [20-23]. 

Fig. 6.12 Survival curve of E. coli JM 109 over exposure time to cast and electrospun P(3HB)-50 wt% TiOi films under different illumination sources. The initial cell concentration used was 3.6 X 10 CPU mL . Bacterial suspension was sampled at 3 h intervals for 12 h. The samples were spread plated on LB agar and incubated at 37 °C for 24 h...

Inocula from frozen stock cultures was cultivated in Wilkins-Chalgren (W-C) broth (Oxoid Ltd. Basingstoke, Hampshire, England) at 37°C in ambient atmosphere, after being screened by Gram-staining to confirm purity. Loopful inoculations of Streptococcus mutans and Enterococcus faecalis were transferred to 10 ml of appropriate broth and incubated at 37 °C under anaerobic conditions. Bacterial suspensions were prepared to 0.5 MacFarland standard and diluted to a 1 10 concentration with W-C broth. Two hundred ml of the 1 10 dilution were then taken and spread-plated using a hockey stick on a turntable to ensure confluent bacterial distribution on the plates. 

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. 

Both previous methods suffer from the requirement of initially small sample volumes. In the case of pour plates, the volume of inoculum must be limited, lest the agar not become sufficiently solid, whereas in the spread-plate method, excess surface moisture permits spreading and coalescence of colonies. Because of this, it is necessary to plate a range of dilutions in order to have countable plates. 

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. 

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