**3.4.** *Enterobacteriaceae* **and coliform bacteria**

330 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

72 hours, depending on the medium used.

**3.2. Mesophilic aerobic bacteria** 

**3.3. Yeasts and molds** 

cycloheximide (100 mg/L) as inhibitors of eukaryotic organisms (to prevent yeasts and molds growth) or with sodium azide (200 mg/L) as Gram-negative bacteria inhibitor. A number of differential and selective media were created for the isolation and characterization of certain groups of LAB. HHD medium is the most used for the differential enumeration of homofermentative and heterofermentative LAB. This medium contains fructose which is reduced to mannitol by heterofermentative but not by homofermentative LAB. Differences in the colour of the colonies are based on differences in the amounts of acids produced by these bacterial groups (Schillinger & Holzapfel, 2003). So in this medium, homofermentative LAB are blue to green, while heterofermentative LAB colonies are white (Fleming et al., 2001). M17 medium should be used for lactococci isolation, but isolation of these LAB is more frequent when analysing dairy products rather than fermented vegetables. All LAB isolation requires anaerobic conditions and incubation at 30-32°C for 48-

The mesophilic count, or standard plate count, is generally obtained on Plate Count agar (PCA) which is a generic medium for aerobic microorganisms that grow at mesophilic temperatures. Aerobic plate counts are poor indicators of safety in some products such as those fermented which commonly show a high aerobic count. However, this count gives information about the hygienic and sensorial quality, about the adherence to good

Alternatively to PCA, similar generic media are available as Nutrient agar. The growth

Yeasts and molds are widely diffused eukaryotic microorganisms because of their adaptation to different environmental conditions. Yeasts and molds can cause various degrees of food decomposition. Invasion and growth may occur on virtually any type of food (Beuchat & Cousin, 2001). Their contamination of food can lead to product losses but, in particular, the highest risk is due to the mycotoxin production by the molds. Several yeasts genera may be important for the sensorial properties of fermented products and for their interrelation with *Lactobacillus* bacteria (see section 2.1) in fermented olives. However, film-forming yeasts as *Debaryomyces, Candida, Pichia* and *Endomycopsis*, are often associated with pickled products and vegetable brines (Fleming et al., 2001), representing the cause of

Available media for yeasts and molds count are several: Rose Bengal Chloramphenicol agar, YM agar, Oxytetracycline Glucose Yeast Extract agar, Sabouraud agar and others. When not included in the medium, the addition of 100 mg/L chloramphenicol is recommended to inhibit the growth of bacteria. For simple enumeration of yeasts and molds, the plates

manufacturing practice and shelf life of the product (Morton, 2001).

conditions are 25-30°C for 24-48 hours, aerobically.

olive defects and consequent product losses.

should be incubated aerobically at 25°C for 48 hours.

The *Enterobacteriaceae* family consists of Gram-negative, facultative anaerobic rods widely distributed in the environment, but which are usually associated with intestinal infections.

**Figure 2.** *E. coli* strain detected by fluorescence microscopy.

Numerous studies have determined that *Escherichia coli*, coliforms, faecal coliforms and *Enterobacteriaceae* are unreliable when used as index of pathogen contamination of food. So National and International advisory committees invalidated the prediction of food safety based on levels of *Enterobacteriaceae*, coliforms, faecal coliforms and *E. coli* (Kornacki & Johnson, 2001)*.* The most important application of *Enterobacteriaceae* and coliforms is their enumeration to assess if pasteurization has been adequately performed for example in pasteurized milk, because a proper pasteurization under appropriate conditions inactivates *E. coli* cells present in raw material.

Total coliform bacteria belong to the *Enterobacteriaceae* family, and include *E. coli* as well as various members of the genera *Enterobacter, Klebsiella* and *Citrobacter*. All ferment lactose with gas and acid production in 48 hours at 35-37°C. While faecal coliforms are coliforms which can ferment lactose to acid and gas within 48 hours at 45°C and they are so called because are more closely associated with faecal pollution (Manafi, 2003). The term coliform is based only on lactose hydrolysis and has no taxonomy validity. In order to perform the best examination of olive samples, it is preferable enumerate both *Enterobacteriaceae* and coliforms, because some lactose-negative bacteria, such as *Salmonella,* are pathogens.

The most probable number (MPN) is a method which indicates the most likely number of microorganisms present in the analysed sample. This method, used for several years, has now been replaced with techniques on agar media because the MPN is based on a statistical approximation. So, in addition to Violet Red Bile agar or Violet Red Bile Glucose agar, other differential media based on a chromatic response were developed.

Mac Conkey's MUG agar and Eosin Methylene Blue agar (EMB) are differential and selective media, suitable to obtain at the same time the isolation of *Salmonella, Shigella* and coliform bacteria, in particular *E. coli*. In the first medium, lactose-negative colonies are colourless, lactose-positive colonies are red and often surrounded by a turbid zone due to the precipitation of bile acids. *E. coli* can be identified by fluorescence in UV due to its β-Dglucoronidase production. In EMB agar, lactose-fermenters form colonies with dark-blue centres (*E. coli* may also have a green metallic sheen) while the non-lactose fermenters form completely colourless colonies.

Microbiological Aspects of Table Olives 333

which contains mannitol. Coagulase-positive staphylococci grow and produce acid from mannitol showing a yellow colony and halo in a red medium. For all these media, the chemical inhibitors usually used are not completely selective, so additional diagnostic tests may be necessary to identify *S. aureus* colonies. Microscopic examination, catalase test and coagulase test may be rapidly executed to identify *S. aureus* from isolates. Common MPN procedures may be used also for enumeration of *S. aureus*. In most cases, the methodologies need a liquid enrichment procedure to detect low numbers of staphylococci (<100 UFC/g).

Clostridia are a widespread heterogeneous group of bacteria showing metabolic and nutritional differences. They easily contaminate foods because they produce resistant spores which can survive under mild processing conditions. The group of sulphite reducing clostridia may be used as marker of raw material quality and hygiene of manufacturing practices. Of particular concern for public health are *C. botulinum*, which forms a deadly toxin in foods, and *C. perfringens* which causes enteritis when present in high numbers. Other species or strains are also known to be toxinogenic or neurotoxinogenic (Bredius & Ree, 2003). *C. perfringens* is the agent of a food poisoning usually associated with consumption of cooked meats or poultry products. Sporulating cells of *C. perfringens* produce a heat-labile enterotoxin which appears to be released *in vivo* in the intestine

Most isolation media include sulphite and an appropriate iron salt. Sulphite is reduced to sulphide by the clostridial enzyme sulphite reductase; the sulphide will then precipitate as a black deposit in the presence of iron salt. This causes a blackening of the liquid media and clostridia will appear as black colonies. To obtain a higher selectivity for *C. perfringens*  isolation, the Oleandomycin Polymixin Sulphadiazine Perfringens (OPSP) agar medium was performed. It contains selective antibiotics and utilises sodium metabisulphite and liver

Culture conditions are usually 37°C for 48 hours aerobically.

**Figure 4.** *S. aureus* strain detected by fluorescence microscopy.

**3.6. Sulphite reducing clostridia** 

(Labbe, 2001).
