Introduction
Pathogens are present all over the place, reaching on every aspect of life. Majority of human infection and death are attributed to food borne pathogens. There has been great improvement on methods for rapid and reliable detection of these pathogens. Microbiology food analysis for particular species has become a challenging task in food technology (Mandal et al, 2011p.87). This paper discusses microbiological examination for specific food borne pathogens using Australian standards (and modern rapid methods).
Aims
- To understand enrichment processes
- To learn microbiological examination processes
- To learn different types of media
- To learn species numeration methods
Methods
Total plate count was done by preparing pour plate according to AS5013 and AS1766 method 1.3with serial dilutions of food sample up to 10 -7. The dilutions were made using 0.1% peptone solution. Detection of S. aureus using spread plate method (AS5013.12.1-2004) and triplicate tube method (AS1766.2.4) method as described from the practical manual. Detection and enumeration of Salmonellae species was done through use of selective media to culture the bacteria. Biochemist test were performed as described by the practical manual (Deakin University, 2011).
Results
First, the meat sample had varying plate counts. For the total microbial load, the spread plate and pour plate methods yielded the count of 8.9×105 and 7.7×106 CFG/g. The count for S. aureus using the spread plate method was 1.21×104 CFG/g. The biochemical test was done to detect contamination from salmonella. The Lysine decarboxylase (LD) test was positive with a purple color, ONPG tested negative and was colorless. The serology test for both Anti sera H and Anti sera (O) were both positive with coagulation. Coagulase test to detect contamination from S. aureus was negative in the sterile control. The test was positive in the three typical colonies, apart from the positive control. The Staphytect test was done to check whether S. aureus was present in S. epidermidis. It was positive in the S. aureus and negative in S. epidermidis. The results obtained were recorded as follows:
Plates count for meat sample
Biochemical tests to detect salmonella contamination
Coagulase test to detect S. aureus contamination
Antibody test to detect Staphytect test S.aureus from S.epidermidis.
Discussion
Often infinitesimal numbers of these microorganisms are sufficient to cause a food poisoning, therefore only are small numbers of these bacteria may be present in the food product. The amount of food sample used was 10g. The amount differs with different micro-organism because factors such as Ph, moisture concentration and other metabolic activity do vary, and will influence the microorganism concentration. Thus for E.coli, food sample could be more or lower than 10g.
To identify this microorganism, an enrichment step is usually required Enrichment technique principles lies on the provision of favorable growth condition for species of interest and unfavorable conditions for competing organisms. In this case, culture pre-enrichment broth is inoculated into Rappaport vassiliadis (RVS) broth and Muler Kauffmann tetrathionate/ novobiocin (MKTTn broth). The purpose of enrichment process is done to recover fully functional bacterial cells as well as viable cells that have been damaged by food conditions.
Peptone water is a minimal growth medium. It is the bacterium source of carbon, nitrogen, vitamins and minerals. Sodium chloride maintains the high Ph. It is used for non-selective pre-enrichment of salmonella species. This allows injured cells due to processes of food preservation to resuscitate (Edel and Hampelmacher.1973, p. 174).
The optimum temperature for most of bacteria is 37°C. Therefore, broth incubation at 37°C is for 16 to 20 hours is mainly to optimize bacterial growth. Temperature below or above this range may denature or inhibit of the enzymes activity and other metabolic processes. For example, selenite inhibitory effect lowers within 6-12 hours of incubation.
Rappaport vassiliadis is the best a selective media for salmonella detection and enumeration of samples with small levels of Salmonella. RVS broths have a low pH and facilitate bacteria growth with minimal nutritional requirements. Manitol selenite cystine broths are also enrichment broths that contain modified SF. The carbohydrate source is from Mannitol. These two broths differ in that the RVS peptone is soya peptone and Mannitol broth peptone is bacteriological peptone (Andrews, 1996).
Bismuth Sulphite agar is useful for the isolation of lactose fermenting Salmonella. Freshly precipitated Bismuth sulphite and brilliant green serve as a selective agent for Salmonella species. They suppress coliforms. The metallic compounds present induce colonies to produce medium black to brown sheen due to presence of hydrogen sulphide (Cook, 1952, p.59).
XLD agar stands for xylose lysine deoxycholate agar. It is a selective media for Salmonella species. It’s a phenol red indicator. Hydrogen sulfide production gives colonies with black centers (Satler and Gragas, 1977, p.200).
The typical biochemist tests used to identify Salmonella species using Australian methods include; Serology tests, its principle involve specific antibody antigen reaction. When the suspended organism is mixed with antiserum containing Salmonella specific antibodies, homologous antiserum present causes agglutination. The bacterium LD test principle lies on the ability of decarboxylation of lysine by the species to produce a designated color, usually purple (Phirke, 1977, p.453). ONPG (ortho-nitrophenyl-b-D-galacto-pyranose) principle tests for beta galactosidase production.
Rapid methods are best designed for preliminary screening. Multiple pathogen analysis using these methods is tedious and more costly. PCR is a powerful technology, but its procedures are complicated and require very high standard of hygiene. More so, it cannot differentiate the live and dead cells and is more likely to give false negatives. Therefore the Australian method becomes the best alternative due to its ability to detect the food pathogens and is more sensitive to indicate presence of infinitesimal pathogen and as fast as possible with the utmost accuracy species identification.
Conclusion
The food analysis challenges is attributed to uneven distribution of bacteria’s in the food, heterogeneity of food matrices and in their varying concentration of proteins, carbohydrates, fats and oils, chemical and their physical state, whether the food is in liquid, powder semisolid or solid which then interferes with its viscosity. Quality assurance in food management must be emphasized. This needs a clean working environment to enhance clean and hygienic environments. The food industry needs more rapid methods which are more sensitive to indicate presence of infinitesimal pathogen and as fast as possible with the utmost accurate species identification (Mandal et al, 2011p.87).
Reference list
Andrews, W. 1996. Evaluation of methods for the detection of Salmonella in foods. J.AOAC Int 79, 4-12. Web.
Cook, G. 1952. J.Path. Bact.64.559. Web.
Deakin University, 2011. Faculty of Health sciences: Food microbiology and HACCP. Burwood: Deakin University.
Edel, W., and Kampelmacher, E, H. 1973. Bull world Health Org.48:164-167. Web.
Mandal et al, 2011. Methods for rapid detection of food borne pathogens: an overview American journal of food technology6:87-102. Web.
Phirke, P. 1977. Application of the rapid lysine decarboxylase test for early isolation and detection of salmonellae in sewage and other waste waters. Nehru.
Salter, J., Gragas, A. 1977. Xylose lysine deoxycholate agar for the isolation of Salmonella from clinical specimens. Zentralbl Bakteriol (Origin) 237(2-3): 196 -200.