There are Four Methods that conducted on the laboratories in order to detect the presence of microorganisms. There are Rodac Method, Swab Method, Rinse Method, and lastly Open Dish Method and it will be discussed in detail below. 2. 1. 1 Rodac Method The purpose of this Standard Operating Procedure is to describe a program that will adequately measure the efficacy of disinfection of Rooms and equipment in each laboratory, RODAC plates can detect the presence or absence of live microorganisms (Longree and Armbruster 1996).
This Method is used to monitor the contamination level of personnel gowns and Personal Protective Equipment (PPE) before or during manufacturing production. The advantages of the RODAC method are that it may be prepared and stored for weeks prior to use (Harrigan 1986). Additional advantages of the RODAC method include relatively low cost, consistent and precise recovery, effective use by personnel without extensive training, and the elimination of laboratory manipulation after sampling (Marriott and Gravani 2006).
On the other hand, the disadvantages of this method are the spreading of the colonies and applicable to only limited to low levels of surface contaminants. 2. 1. 2 Swab Method The Swab method is among the most Reproducible Methods used to determine the population of microorganisms present on equipment or food products (Marriott and Gravani 2006). It may be used to assess the amount of contamination from the air, water, surfaces, facilities and food products. By using this technique the equipment surfaces, facilities and food products which to be analyzed are swabbed.
The swab are diluted in a dilutant such as peptone water or phosphate buffer, according to the anticipated amount of contamination and subsequently applied to a growth medium containing agar in a sterile, covered plate (David, Richard and R. 2004). There are many advantages to the cotton swab method. These include the ease with which any health care provider can procure the necessary items: a CTA or culturette transport medium (Longree and Armbruster 1996). In addition, the method requires little expertise, with minimal training time required, and very little time required to actually perform the procedure. On the other hand,
Disadvantages of the swab method are that sampling and technique can affect the results and that the method requires manipulation to culture the sample. Swabs are designed for hard-to-reach places, and can fit easily into equipment recesses, nooks, and crevices (Tamime 2008). After collection of the sample, it is recommended that a standard membrane filtration of the rinse solution be conducted. 2. 1. 3 Rinse Method The Rinse Method use elution of contamination by rinsing to permit a microbial assay of the resultant suspension (Forsythe 2008). A sterile fluid is manually or mechanically agitated over an entire surface.
The rinse fluid then diluted and subsequently plated, this method are more precise compared to the swab method, because a larger surface area can be tested (David, Richard and R. 2004). While the disadvantages is that it requires time and labor to prepare solutions and media, dilute samples, pour plate samples, and count colony-forming units on the plates. 2. 1. 4 Open Dish Method The principle behind this method is that the bacteria carrying particles are allowed to settle onto the medium for a given period of time and incubated at the required temperature.
A count of colonies formed shows the number of settled bacteria containing particles (David, Richard and R. 2004). In this method petri dishes containing an agar medium of known surface area are selected so that the agar surface is dry without any moisture. Choice of the medium depends upon the kind of microorganisms to be enumerated. For an overall count of pathogenic, commensal and saprophytic bacteria in air blood agar can be used (Longree and Armbruster 1996). For detecting a particular pathogen which may be present in only small numbers, an appropriate selective medium may be used. Malt extract agar can be used for molds.
The plates are labeled appropriately about the place and time of sampling, duration of exposure etc. Then the plates are uncovered in the selected position for the required period of time. The optimal duration of exposure should give a significant and readily countable number of well isolated colonies, for example about 30-100 colonies (McLandsborough 2003). Usually it depends on the dustiness of air being sampled. In occupied rooms and hospital wards the time would generally be between 10 to 60 ‘minutes (McLandsborough 2003). During sampling it is better to keep the plates about I meter above the ground.
Immediately after exposure for the given period of time, the plates are closed with the lids. Then the plates are incubated for 24 hours at 37°C for aerobic bacteria and for 3 days at 22°C for saprophytic bacteria (McLandsborough 2003). 2. 2 Group of microbes that often exist in the room and equipment The normal tendency of a microbial cell when it comes in contact with a solid surface is to attach itself to the surface in an effort to compete ef? ciently with other microbial cells for space and nutrient supply and to resist any unfavorable environ-mental conditions (Adams and Moss 2000).
Under suitable conditions, almost all microbial cells can attach to solid surfaces, which are achieved through their ability to produce extracellular polysaccharides. As the cells multiply, they form micro colonies, giving rise to a bio? lm on the surface containing microbial cells, extracellular polysaccharide glycocalyx, and entrapped debris. In some situations, instead of forming a bio? lm, the cells may attach to contact surfaces and other cells by thin, thread like exopolysaccharide materials, also called ? mbriae (Lappin-Scott and J. 1995).
Attachment of microorganisms on solid surfaces has several implications on the overall microbiological quality of food. Microbial attachment to and bio? lm formation on solid surfaces provide some protection of the cells against physical removal of the cells by washing and cleaning. These cells seem to have greater resistance to sanitizers and heat. Thus, spoilage and pathogenic microorganisms attached to food surfaces, such as carcasses, ? sh, meat, and cut fruits and vegetables, cannot be easily removed by washing, and later they can multiply and reduce the safety and stability of the foods (Hui 2003).
Similarly, microbial cells attached to a culture broth. These places, in turn, can be a constant source of undesirable microorganisms to foods handled in the environment. The concept and importance of microbial attachment and bio? lm formation in solid food, equipment, and food environments are now being recognized (Loken 1995). Limited studies have shown that under suitable conditions, many of the microorganisms important in food can form a bio? lm. Several species and strains of Pseudomonas were found to attach to stainless steel surfaces, some within 30 min at 25oC to 2 hour at 4oC (Stanga 2009).
Listeria monocytogenes was found to attach to stainless steel, glass, and rubber surfaces within 20 min of contact. Attachment of several pathogenic and spoilage bacteria has also been demonstrated on meat and carcasses of poultry, beef, pork, and lamb (Stanga 2009). The microorganisms found to attach to meat surfaces include Lis. monocytogenes, Micrococcus spp. , Staphylococcus spp. , Clostridium spp. , Bacillusspp. , Lactobacillus spp. , Brochothrix thermosphacta, Salmonella spp. , Escherichiacoli, Serratia spp. , and Pseudomonas spp (Tamime 2008).
It is apparent from the limited data that microbial attachment to solid food and food contact surfaces is quite wide and needs to be considered in controlling the microbiological quality of food. Several possible mechanisms by which microbial cells attach and form a bio? lm on solid surfaces have been suggested. One suggestion is that the attachment occurs in two stages. In the ? rst stage, which is reversible, a cell is held to the surface by weak forces (Cramer 2006). In the second stage, a cell produces complex polysaccharide molecules to attach its outer surface to the surface of a food or equipment, and the process is irreversible.
A three-step process that includes adsorption, consolidation, and colonization has been suggested by others (Cramer 2006). In the reversible adsorption stage, which can occur in 20 min, the cells attach loosely to the surface. During the consolidation stage, the microorganisms produce threadlike exopolysaccharides ? mbriae and ? rmly attach the cells to the surface. At this stage, the cells cannot be removed by rinsing (Marriott and Gravani 2006). In the colonization stage, which is also irreversible, the complex polysaccharides may bind to metal ions on equipment surfaces and the cells may metabolize products that can damage the surfaces.
The level of attachment of microorganisms to food-processing equipment surfaces is found to be directly related to contact time. As the contact time is prolonged, more cells attach to the surface, the size of the microcolony increases, and attachment between cells increases (Loken 1995). Fimbriae formation by the cells occurs faster at optimum temperature and pH of growth. Limited studies also showed that when microorganisms such as Pseudomonas fragi and Lis. monocytogenes are grown together, they form a more complex bio? lm than when either is grown separately (Stanga 2009).
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