Sample Preparation And Treatments

One of the most important steps for successful microbiological analysis of any material is sample preparation. With the advancement of microbiological techniques and miniaturization of kits and test systems to ever smaller sizes, proper sample preparation becomes critical. Chapter 24 discusses in detail various sample preparation, detection, and enumeration methods for fruits and vegetables. Some novel methods are discussed in this chapter.

Fruits and vegetables are considered solid food. The most efficient method to prepare the samples for enumeration and detection is to use the Stomacher instrument where a known weight of solid sample is placed in the stomacher bag, and a volume of sterile diluent is added to make a 1:10 dilution of the sample. Then the sample is "stomached" for one to two minutes before an aliquot is taken out for viable cell count, differential count, or pathogen count and detection. Dr. Anthony Sharpe invented the Stomacher about 25 years ago, and now more than 40,000 units are in use worldwide. Recently he introduced a new instrument called the Pulsifier (Microgen BioProducts Ltd, Surrey, U.K.) for dislodging microorganisms from foods without excessively breaking the food structure. The Pulsifier has an oval ring that can house a plastic bag with sample and diluent. When the instrument is activated the ring will vibrate vigorously for a predetermined time (30 to 60 seconds). During this time microorganisms on the food surface or in the food will be dislodged into the diluent with minimum destruction of the food. Fung et al. [2] evaluated the Pulsifier against the Stomacher with 96 food items (including beef, pork, veal, fish, shrimp, cheese, peas, a variety of vegetables, cereal, and fruits) and found that both systems gave essentially the same viable cell count in the food, but the "pulsified" samples were much clearer than the "stomached" samples. Kang et al. [3] found that the Pulsifier and Stomacher had a correlation coefficient of 0.971 and 0.959 for total aerobic count and coliform count, respectively, with 50 samples of lean meat tissues. More recently, Wu et al. [4] made a comprehensive study of the Pulsifier versus the Stomacher on 30 vegetables and reported no difference in total count and coliform between the two methods (Table 25.1 for total count). However, there were distinct differences in the liquids between the methods with pulsified samples having less turbidity, less total solids, and higher pH than the stomached samples (Table 25.2). The superior quality of microbial suspensions with minimum food particles and inhibitors from the Pulsifier has positive implications for general microbial analysis such as ease of pipetting samples and ease of filtration through bacteriological membrane filters, as well as for techniques such as adenosine triphosphate (ATP) bioluminescence tests, DNA/RNA hybridization, polymerase chain reaction (PCR) amplifications, enzymatic assays, etc.

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