# Comparative Study of Fresh and Frozen Broiler Neck Skin Sampled for Process Hygiene Purposes
## 4 Discussion  
Overall, our results showed that freezing and storing for eight weeks at \(-\)30 \({}^{\circ}\)C had no significant effect on the microbial load (TVC or EC) of broiler neck skin samples. The detected differences in bacteria levels on fresh and frozen neck skin samples were within the maximum accepted measurement of uncertainty in microbiology laboratory analyses. The observed differences can be considered to be methodologically and biologically irrelevant [39] cited in [40,41]. Similar results regarding the effect of freezing on TVC and EC were shown in other investigations on different food products, vegetables, milk, and meat products [21,32,33,42,43,44,42,44]. Freezing places bacteria in a dormant state, but is also a common method of storing bacterial cultures. [PERSON] et al. (2007) [29] showed recoveries of up to 98% for cultures of Gram-positive and Gram-negative bacteria after storage in \(-\)80 \({}^{\circ}\)C in glycerol for 564 days. Similar to glycerol or other media used for storage of highly concentrated bacterial suspensions [31], our results and those of other authors for frozen vegetables, meat products, and other food products showed that the food matrix seems to have a certain protective effect during freezing [21,33,44]. The bacterial load, TVC or EC, did not change when the foods were stored frozen. In our study, we suspect that the fat present in the neck skin had a protective effect, and, in addition, we observed that the samples were entirely frozen very quickly due to their low volume, so we presume that the bacteria present were not destroyed and could start proliferating again after thawing. This assumption is in concordance with [PERSON] and [PERSON] (1977) [14], who reported that rapid freezing resulted in small cellular ice crystals which did not damage the bacteria as much as slow freezing, which results in large ice crystals. Additionally, rapid thawing results in less lethal damage to bacteria [14]. These two factors can be assumed to be the case for the examined frozen neck skin samples in our study, as they had a low volume and mass and, thus, were frozen and thawed quickly. Therefore, the temperature of \(-\)30 \({}^{\circ}\)C is suitable for freezing neck skin samples and for limited storage of such samples before further analysis, even if this temperature is not common in food storage or for laboratory purposes. However, freezing can result in the death of individual bacteria fractions while other fractions of bacteria can proliferate after thawing [19]. Consequently, the detectable numbers of cfu/g in quantitative TVC and EC studies can remain almost the same. Anyway, individual neck skin samples in our study with EC below the minimum limit of detection showed that Enterobacteriaceaeae (or Gram-negative bacteria in general) enumerated with the EC technique are more sensitive to freezing than Gram-positive bacteria. This is in concordance with other studies looking at the effects of freezing at temperatures around \(-\)20 to \(-\)25 \({}^{\circ}\)C on bacteria numbers in different kinds of foods [19,21,28,33,44]. [PERSON] et al. (2012) [45] showed that freezing at different temperatures for at least 16 weeks did not influence the survival of common mastitistits pathogens in mastitistits milk samples in South Africa. Lower detection rates for _E. coli_ when compared to Gram-positive bacteria were observed, but the authors suggest that storing milk samples at \(-\)20 or \(-\)196 \({}^{\circ}\)C can be used in South Africa for improving under health management [45]. Altogether, we conclude that, in our study, the effect of freezing the broiler neck skin samples on the mean microbial loads was small, as the tests for equivalence, comparingmean loads on fresh and frozen samples, showed equality. Similar results were reported by [PERSON] et al. (2008) [34] for the investigation of cream and bakery products with non-heated fillings.