Assessment of Microbial Contamination and Spoilage

Introduction

Microbial contamination and spoilage can be forensically determined by the following methods:

1. Monitoring the physical and chemical changes of the product 
2. Assessment of sterility (sterility tests)
3. Identification and quantification of microorganisms in non-sterile products
4. Estimation of pyrogens and microbial toxins

1. Monitoring the physical and chemical changes of the product

Monitoring the physical and chemical changes of the product by:

1. Olfactory detection: Several common metabolites with extreme intense taste or small can be detected as an indication of active microbial attack in medicines.
2. HPLC (High performance liquid chromatography) or Gas chromatography can be used to distinguish microbial spoilage from other non-biological deteriorative processes. 
3. Conventional instrumentation: When microbial spoilage promotes physicochemical changes such that the use of specific instrumentation process can be employed for the detection of the physiochemical changes. Examples of these case whereby conventional instrumentation can be used include the following;

• Surfactant biodeterioration detected by measurement of reduction in surface tension. 
• Emulsion spoilage can be monitored by changes in viscosity, surface tension, pH and rates of creaming or particle sedimentation.
• Metabolism of particular ingredients (alkaloids or steroids) might be assessed using analytical techniques such as thin layer chromatography.

2. Assessment of Sterility (Sterility Tests

When a medicinal product is “sterile” that means that it is devoid of all viable microorganisms, although this is hardly obtainable.
A reliable performance of sterility test is to cultivate a sample of the product from selected items of a batch in a culture media and incubate the media to detect visible growth of microorganism. The achievement of an adequate level of sterility assurance is only obtained with a fully operational quality appearance programme which includes proper design, validation and in process control of manufacture.
Sterility testy detect total failure of a sterlisation process. It does not detect borderline cases, because:

1. To detech borderline failture would require sampling so many items of a batch that only little would remain for sale.
2. Several ingredients of standard sterility test media such as thioglycollate or polysorbete 80 can be toxic to damaged microorganism.
3. Nutritionally rich media is normally used in sterility takes. However, damaged but still potentially viable microbes generally require mild, non-growth-inducing conditions favouring cellular repair processes to operate before subjection to the stresses of attempted replication in the nutritionally rich medium.

3. Identification and quantification of microorganisms in non-sterile products

Non-sterile products are expected to carry a limited number of different types of microorganism and a limited overall numbers of microbial contamination to reduce spoilage risks. 
However, it is known that methods for counting (or even detecting viable microorganism have poor accuracy and precision to estimate the true microbial levels in a complex formulation that is meant not to be sterile. It is also not known which is more important vis-à-vis the number or type of organism to determine spoilage.  A product can have a high load of a non-pathogenic organism and not spoil the product. An uneven distributed growth of microorganism in viscous formulations or around particulate ingredients can provide serious sampling problems. Spoilage contaminant can be present in a small amount or low numbers at the time of testing which can grow to high levels during storage. The composition of culture media for viable counting and the incubation temperature can greatly influence the number of colony forming units (CFU) obtained leading to counts of varying numbers with different media and even from different batches of the same medium.
As a result of these problems, the UK and E.E. C food regulatory authorities have abandoned the use of microbial counts as the legally enforceable standards of food quality. E.E.C will soon introduce conventional and quantitative microbial limit tests for medicines. There standards will then become the statutory requirements in all the E.E.C  member states.
Convert test method in UK are as follows 

1. Some medicine are tested for presence of specific pathogens (such as salmonella spp in Pancreatin), Ecoli, staph aureus etc. by incubation in a medium favouring the particular the pathogen, and their cultivation on several “selective” and diagnostic media. 
2. Known spoilage organism such as pseudomonads are sought for by using selective media developed for food and diary industries. 
3. Many manufacturers carry out “in home” estimation of microbial population viable counts. The CFU are determined using non-specific solid media inoculated with serial dilutions of the test material. 

Other methods for estimating microbial content in a non-sterile formulation are 

1. estimation of total microbial content at low levels (around 103  bacterial colonies) by assay of a metabolite present in relatively fixed quantities per viable cell. For example Adenosine triphosphate (ATP) can be measured by the quantum of light emission with luciferase enzymes. 
2. Release of CO2 from added radiolabelled substrates indicating the level of microbial metabolism.
3. Direct ultraviolet (UV) epifluorescence microscopic counting of microbes within a product, when “dead” cells fluoresce orange with ethidium bromide or acidine orange and “viable” cells fluoresce green with fluorescein diacetate. Note: ethidium bromide and acridine orange do not readily penetrate intact “viable” membranes.

Recent developments for the detection of extremely low number of microbes have made use of monoclonal antibodies chemically linked to enzymes which yield detectable products such as fluorescent chemicals when reacted with contaminants on a filter membrane.

4. Estimation of pyrogens and microbial toxins 

Pyrogens are estimated in pharmaceuticals by measuring febrile response following their injection into rabbits. A less expensive, rapid and very sensitive in vitro assay relies upon the highly specific interaction of amoebocyte lysate from the horsehoe crab (Limulus polyphemus) with microbial lipopolysaccharide (but not other pyrogens) yielding a gelling and opacification of the lysate, or a colour with a chromogenic substrate.
However, some therapeutic agents such as of polymyxins are known to interfere with the chromogenic test. 
Microbial toxins are now usually detected in foodstuffs using animal moculations, mass spectroscopic, chromatographic or immunological techniques. These are not commonly applied to pharmaceutical except for the examination of vegetable ingredients for aflatoxins by extraction, concentration with ion-exchange chromatography and detection under UV light by their strong fluorescence