Conventional and rapid analytical microbiology 189 sterile diluent(the sample to diluent ratio is usually 1: 10); this mixture is then homogenised using a homogeniser (e.g. stomacher or pulsifier)that breaks the ample apart, releasing any organisms into the diluent. The correct choice of diluent is important. If the organisms in the sample are stressed by incorrect pH or low osmotic strength, then they could be injured or killed, thus affecting the final result obtained from the microbiological test the diluent must be well buffered at a pH suitable for the food being tested and be osmotically balanced When testing some foods (e.g. dried products) which may contain highly stressed microorganisms, then a suitable recovery period may be required before the test commences, in order to ensure cells are not killed during the initial phase of the test procedure(Davis and Jones 1997) 8.3.1 Conventional quantitative procedures The enumeration of organisms in samples is generally done by using plate count, or most probable number(MPN)methods. The former are the most widely used whilst the latter tend to be used only for certain organisms(e.g. Escherichia coll) or groups(e.g. coliforms) Plate count method The plate count method is based on the deposition of the sample, in or on an agar ayer in a Petri dish. Individual organisms or small groups of organisms will occupy a discrete site in the agar, and on incubation will grow to form discrete colonies that are counted visually. Various types of agar media can be used in this form to enumerate different types of microorganisms. The use of a non- selective nutrient medium that is incubated at 30oC aerobically will result in a total viable count or mesophilic aerobic count. By changing the conditions of incubation to anaerobic. a total anaerobe count will be obtained. altering the incubation temperature will result in changes in the type of organism capable of growth, thus showing some of the flexibility in the conventional agar approach If there is a requirement to enumerate a specific type of organism from the ample, then in most cases the composition of the medium will need to be djusted to allow only that particular organism to grow. There are three approaches used in media design that allow a specific medium to be produced he elective, selective and differential procedures Elective procedures refer to the inclusion in the medium of reagents, or the e of growth conditions, that encourage the development of the target organisms, but do not inhibit the growth of other microorganisms. Such reagents may be sugars, amino acids or other growth factors. Selective procedures refer to he inclusion of reagents or the use of growth conditions that inhibit the development of non-target microorganisms. It should be noted that, in many cases, selective agents will also have a negative effect on the growth of the target microorganism, but this will be less great than the effect on non-target cells. Examples of selective procedures would be the inclusion of antibiotics in a medium or the use of anaerobic growth conditions. Finally, differentialsterile diluent (the sample to diluent ratio is usually 1:10); this mixture is then homogenised using a homogeniser (e.g. stomacher or pulsifier) that breaks the sample apart, releasing any organisms into the diluent. The correct choice of diluent is important. If the organisms in the sample are stressed by incorrect pH or low osmotic strength, then they could be injured or killed, thus affecting the final result obtained from the microbiological test. The diluent must be well buffered at a pH suitable for the food being tested and be osmotically balanced. When testing some foods (e.g. dried products) which may contain highly stressed microorganisms, then a suitable recovery period may be required before the test commences, in order to ensure cells are not killed during the initial phase of the test procedure (Davis and Jones 1997). 8.3.1 Conventional quantitative procedures The enumeration of organisms in samples is generally done by using plate count, or most probable number (MPN) methods. The former are the most widely used, whilst the latter tend to be used only for certain organisms (e.g. Escherichia coli) or groups (e.g. coliforms). Plate count method The plate count method is based on the deposition of the sample, in or on an agar layer in a Petri dish. Individual organisms or small groups of organisms will occupy a discrete site in the agar, and on incubation will grow to form discrete colonies that are counted visually. Various types of agar media can be used in this form to enumerate different types of microorganisms. The use of a non￾selective nutrient medium that is incubated at 30ºC aerobically will result in a total viable count or mesophilic aerobic count. By changing the conditions of incubation to anaerobic, a total anaerobe count will be obtained. Altering the incubation temperature will result in changes in the type of organism capable of growth, thus showing some of the flexibility in the conventional agar approach. If there is a requirement to enumerate a specific type of organism from the sample, then in most cases the composition of the medium will need to be adjusted to allow only that particular organism to grow. There are three approaches used in media design that allow a specific medium to be produced: the elective, selective and differential procedures. Elective procedures refer to the inclusion in the medium of reagents, or the use of growth conditions, that encourage the development of the target organisms, but do not inhibit the growth of other microorganisms. Such reagents may be sugars, amino acids or other growth factors. Selective procedures refer to the inclusion of reagents or the use of growth conditions that inhibit the development of non-target microorganisms. It should be noted that, in many cases, selective agents will also have a negative effect on the growth of the target microorganism, but this will be less great than the effect on non-target cells. Examples of selective procedures would be the inclusion of antibiotics in a medium or the use of anaerobic growth conditions. Finally, differential Conventional and rapid analytical microbiology 189