The objective of this work was to assess and compare the impact of three different freezing methods on the physiology of probiotic bacteria based on evaluation of vitality, membrane integrity and special metabolic properties. Lactobacillus rhamnosus GG (LGG) was used as a model strain and was analysed with the plate enumeration method and flowcytometric analyses (FCM) before and after treatment in phosphate buffer saline (PBS) and 20% (w/v) skim milk (RSM), respectively. To get insight into the induced changes in esterase activity and membrane integrity combined staining with carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) was applied. Furthermore the ability of the cells to extract intracellular accumulated cF in presence of glucose was investigated as an indicator for ATP driven membrane transport processes. The freezing methods applied were static air freezing (AF) at -30 degrees C for 3 h, high pressure shift freezing (PSF) at -21 degrees C/210 MPa and spray freezing (SF) at -30 degrees C. Spray freezing was accomplished by spraying the bacteria suspension in a cold air stream using a two fluid atomizer. The atomizing gas was air. After each treatment all samples were stored at -20 degrees C for one day before they were analysed. Minimum losses in viability occurred when spray freezing LGG in presence of 20% (w/v) RSM whereas air freezing in PBS resulted in the lowest survival rates. For all treatments higher survival rates were achieved when 20% (w/v) RSM was used as protecting media. Comparing the results of live cell count with the FCM data gave evidence for a fraction of viable but not culturable cells. LGG cells frozen by PSF showed viability comparable to AF samples, but physiological characteristics of pressure treated bacteria. (c) 2008 Elsevier Ltd. All rights reserved.