Skim Milk Broth Research Articles

In Vitro Anticoagulant Activity of Crude Protease of Bacillus tequilensis HSFI-5

obtained from the fermented intestine of Holothuria scabra (sand sea cucumber). Strain HSFI-5 had been reported to be able to produce proteases, which had shown several characteristics of an antithrombotic agent, i.e., fibrinolytic and clot-lysis activities. However, its anticoagulation activity test is yest to be done. This study aimed to determine the anticoagulant activity of the crude protease HSFI-5 in vitro. The study design was a completely randomized design with a sample size of 90 calculated using the Federer formula. The material used was crude protease from B. tequilensis in skim milk broth. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma recalcification time (PRT) were carried out to test the anticoagulant activity. Citrated platelet poor plasma samples were divided into positive control, normal control, direct examination with crude enzyme in volumes of 50 and 100 µL and pre-incubation at 37ºC for 5, 10, and 15 min with crude enzyme volumes of 50 and 100 µL. The data normality was tested with the Kolmogorov-Smirnov test and the different tests were analyzed by one-way ANOVA with the Post hoc LSD test. The results of one-way ANOVA both on PT, aPTT, and PRT examinations showed that there was a significant difference between the treatment groups (p<0.05). The longest results of PT, aPTT, and PRT are positive controls, and the shortest results are normal controls for PT, and 15’ 50 group for aPTT and PRT. It is clear that crude protease B. tequilensis HSFI-5 exhibits anticoagulant as well as thrombolytic action, raising the possibility that it could function as an antithrombotic drug.

Characteristics and Substrate Specificity of Semi-Purified Bacterial Protease ofBacillusthuringiensisHSFI-12 with Potential as Antithrombotic Agent

Commercial proteases, such as Nattokinase (NK), Staphylokinase (SAK), and Streptokinase (SK) play an important role in the destruction of thrombus, the main cause of death in cardiovascular disease. The latest technology combining enzymes with certain drugs is the target of new research in the thrombolytic area. The first step is to develop protease from Bacillus thuringiensis HSFI-12 bacteria as an antithrombotic agent, characterization of the bacterial enzyme is necessary. This study aims to determine the specificity of protease from Bacillus thuringiensis HSFI-12 to explore its potential as an antithrombotic agent in terms of anticoagulant and fibrinolytic activities. The molecular weight and specificity of bacterial protease were determined with a zymographic method with casein as substrate. Bacillus thuringiensis HSFI-12 was first cultured on Nutrient Agar (NA) media and then on Skim Milk Agar (SMA) media. The obtained crude protease from Skim Milk Broth (SMB) was then concentrated as dialysate. Both crude and dialysate proteases were tested for their specific activity, as well as anticoagulant and fibrinolytic activities. Next, the dialysate’s molecular weight and specificity on the casein substrate were investigated using the zymographic method. As result, protease activity in crude form is lower than that in dialysate, which was 0.5570 ± 0,004 U/mL and 2.1767 ± 0,005 U/mL, respectively. The molecular weight of the obtained bacterial protease was between 117 – 133 kDa and the enzyme is capable of degrading casein as shown on the zymogram. Overall, both crude and dialysate proteases of Bacillus thuringiensis HSFI-12 show potential as an antithrombotic agent for exhibiting anticoagulant and antiplatelet activities. Yet, it could not exhibit direct fibrinolytic activity implying the possibility that the enzyme plays a role as a plasminogen activator, which can dissolve fibrin by activating plasmin.

Development of a Monte Carlo simulation model to predict pasteurized fluid milk spoilage due to post-pasteurization contamination with gram-negative bacteria

Psychrotolerant gram-negative bacteria introduced as post-pasteurization contamination (PPC) are a major cause of spoilage and reduced shelf life of high-temperature, short-time pasteurized fluid milk. To provide improved tools to (1) predict pasteurized fluid milk shelf life as influenced by PPC and (2) assess the effectiveness of different potential interventions that could reduce spoilage due to PPC, we developed a Monte Carlo simulation model that predicts fluid milk spoilage due to psychrotolerant gram-negative bacteria introduced as PPC. As a first step, 17 gram-negative bacterial isolates frequently associated with fluid milk spoilage were selected and used to generate growth data in skim milk broth at 6°C. The resulting growth parameters, frequency of isolation for the 17 different isolates, and initial concentration of bacteria in milk with PPC, were used to develop a Monte Carlo model to predict bacterial number at different days of shelf life based on storage temperature of milk. This model was then validated with data from d 7 and 10 of shelf life, collected from commercial operations. The validated model predicted that the parameters (1) maximum growth rate and (2) storage temperature had the greatest influence on the percentage of containers exceeding 20,000 cfu/mL standard plate count on d 7 and 10 (i.e., spoiling due to PPC), which indicates that accurate data on maximum growth rate and storage temperature are important for accurate predictions. In addition to allowing for prediction of fluid milk shelf life, the model allows for simulation of "what-if" scenarios, which allowed us to predict the effectiveness of different interventions to reduce overall fluid milk spoilage due to PPC through a set of proof-of-concept scenario (e.g., frequency of PPC in containers reduced from 100% to 10%; limiting distribution temperature to a maximum of 6°C). Combined with other models, such as previous models on fluid milk spoilage due to psychrotolerant spore-forming bacteria, the data and tools developed here will allow for rational, digitally enabled, fluid milk shelf life prediction and quality enhancement.

Protease Production from Bacillus sp. Isolated from Gastrointestinal Tract of Catfish (Clarias sp.) with Different Medium

The need for protease in the industrial field has been increasing. Candidates for producing these enzymes can be isolated from the digestive tract of catfish (Clarias sp.). The purpose of this study was to obtain bacterial isolates that produce proteolytic from the gastrointestinal tract of catfish and determine the effect of different production media on the activity of proteolytic. The first step of this study was isolation, screening, and identification of bacteria. The second step was to test the effect of the media Luria Bertani, trypticase soy broth, and skim milk broth on proteolytic activity. Nineteen isolates were obtained from the screening process of proteolytic bacteria. Isolate no 1, was known as the best isolate in producing enzymes and was known as Bacillus sp. Tests with different growth media gave results that semi-quantitative, nutrient growth media produced the highest activity with a proteolytic index value of 2.09 ± 0.41. In addition, based on quantitative tests, the media Luria Bertani Broth produced the highest specific activity with a value of 36.479 U/mg. The conclusion of this study, Bacillus sp. from the gastrointestinal tract of catfish that cultured on the Luria Bertani Broth medium produced the best activity.

Evaluation of Proteolytic and Chitinolytic Activities of Indigenous Bacillus Speciesfrom Crab Shell Waste

The increase of crab meat export activities in Indonesia leads to the accumulation of crab shell waste in a massive amount that can naturally degrade and causing bad odor. Naturally, microorganisms will degrade this waste through fermentation and enzymatic reaction, including protease and chitinase due to its high content of chitin and protein. Bacillus is the most potential bacteria to degrade crab shell waste, and indigenous Bacillus from this waste can increase the degradation rates. The aims of this study were to evaluate the proteolytic and chitinolytic activities of indigenous Bacillus species from crab shell waste. Bacillus cereus BP14 and Bacillus licheniformis CK20 as the chitinolytic bacteria, and Bacillus subtilis AP9 and Bacillus licheniformis AP5 as the proteolytic bacteria were isolated from crab shell waste and identified based on the 16S rDNA sequences. The bacterial isolates were grown in skim milk broth for proteolytic characterization and colloidal chitin broth for chitinolytic characterization. The growth rates of each bacteria were determined through the growth curves. The enzymatic activities were determined based on the international standard for protease and chitinase enzyme activities together with growth curve sampling to determine the best incubation time for obtaining the highest enzymatic activities. From the shortest to the longest generation times of the Bacillus species obtained were B. subtilis AP9, B. licheniformis CK20, B. cereus BP14, and B. licheniformis AP5, respectively. The best incubation time for producing the highest enzymatic activity varied among species. However, the end of the logarithmic phase was similar. All of the Bacillus species obtained from this study exhibited chitinolytic and proteolytic activity. Therefore, it can be used as promising candidates of biodegradation agents inenhancing the degradation rates of crab shell wastes.

Isolation and characterization of Streptococcus thermophilus possessing prtS gene from raw milk in Japan.

Streptococcus thermophilus is widely used for producing fermented dairy products such as yogurt and cheese. Some S. thermophilus strains possessing the cell-wall protease PrtS show high proteolytic activity and fast acidification properties, which are very useful in industrial starters. However, few S. thermophilus strains possessing the prtS gene have been isolated from the environment. To clarify whether or not S. thermophilus strains possessing the prtS gene are present in Japan, we isolated S. thermophilus from raw milk collected in Japan from 2011 to 2017 and investigated the strains for the presence of prtS by PCR. A total of 172 S. thermophilus strains were isolated, and 59 strains were confirmed to possess prtS. We measured fermentation times of 59 prtS-positive strains in skim milk broth and found that 53 strains showed fast acidification properties, finishing fermentation within 10 hr. However, the remaining 6 prtS-positive strains showed slow acidification properties, and they had several amino acid mutations in PrtS compared with fast acidifying S. thermophilus LMD-9 and 4F44. These results demonstrate that S. thermophilus strains possessing prtS are prevalent in Japan and that some prtS-positive strains could lose their fast acidifying properties through mutations in PrtS.

Efficient inhibition of Cronobacter biofilms by chitooligosaccharides of specific molecular weight.

Biofilms enable Cronobacter spp. to contaminate food, infect infants and resist different environmental stresses, especially desiccation, which is the main reason why Cronobacter can survive in powdered infant formula (PIF) for a long time. Considering the high lethality of Cronobacter infection in infants, it is important to find efficient and safe inhibitors of Cronobacter biofilms. In this study, we found that chitooligosaccharides (COS) with a molecular weight of 2000Da efficiently inhibited Cronobacter biofilms, especially in skim milk broth. The minimum biofilm inhibitory concentration (MBIC77) of COS was as low as 20μg/mL, which is lower than that reported in most previous studies. Besides, the elimination rate of COS for Cronobacter mature biofilms was 50% when the concentration was 10mg/mL. COS could significantly inhibit soluble polysaccharide secretion and biofilm cell growth, as well as change the cell membrane permeability of Cronobacter. These might be the possible reasons for COS's efficient inhibition of Cronobacter biofilms. However, during the inhibition, five important genes-related to biofilm formation-flhD, flgJ, luxR, ompA, and wcaJ-were all up-regulated after COS treatment, except the gene bcsA. In summary, our findings showed that COS could be used as an efficient and safe inhibitor against Cronobacter biofilms for better control of Cronobacter contamination and infection.

Genes Associated With Psychrotolerant Bacillus cereus Group Isolates.

The Bacillus cereus group comprises 18 different species, including human pathogens as well as psychrotolerant strains that are an important cause of fluid milk spoilage. To enhance our understanding of the genetic markers associated with psychrotolerance (defined here as > 1 log10 increase in cfu/mL after 21 days incubation at 6°C) among dairy-associated B. cereus group isolates, we used genetic (whole genome sequencing) and phenotypic methods [growth in Skim Milk Broth (SMB) and Brain Heart Infusion (BHI) broth] to characterize 23 genetically-distinct representative isolates from a collection of 503 dairy-associated isolates. Quality threshold clustering identified three categories of psychrotolerance: (i) 14 isolates that were not psychrotolerant in BHI or SMB, (ii) 6 isolates that were psychrotolerant in BHI but not in SMB, and (iii) 2 isolates that were psychrotolerant in BHI and SMB. One isolate, which was psychrotolerant in BHI broth but was just below the cut-off of >1 log10 cfu/mL increase in SMB was not assigned to a cluster. A maximum likelihood phylogeny constructed with core genome single nucleotide polymorphisms classified all psychrotolerant isolates (i.e., psychrotolerant in BHI) into clade VI (representing B. mycoides/weihenstephanensis). Analysis of correlations between gene ortholog presence or absence patterns and psychrotolerance identified 206 orthologous gene clusters that were significantly overrepresented among psychrotolerant strains, including two clusters of cold shock proteins, which were identified in 8/9 and 7/9 psychrotolerant isolates. Gene ontology analyses revealed 36 gene ontology terms that were overrepresented in psychrotolerant isolates, including putrescine catabolic processes and putrescine transmembrane transporter activity. Lastly, Hidden Markov Model searches identified three protein family motifs, including cold shock domain proteins and fatty acid hydroxylases that were significantly associated with psychrotolerance in BHI broth. Analyses of CspA sequences revealed a positive association between psychrotolerant strains and a previously identified “psychrotolerant” CspA sequence. Overall, our data highlight genetic and phenotypic differences in psychrotolerance among B. cereus group dairy-associated isolates and show that psychrotolerance is dependent on the growth medium. We also identified a number of gene targets that could be used for specific detection or control of psychrotolerant B. cereus group isolates.

Purification and characterization of a broad spectrum bacteriocin produced by a selected Lactococcus lactis strain 63 isolated from Indian dairy products.

Lactococcus lactis subsp. lactis strain 63 was isolated from Indian dairy products, produced a bacteriocin with broad spectrum inhibition against several of food pathogens like Listeria monocytogenes and Bacillus cereus as well as Gram negative bacteria viz., E. coli, Yersinia, Citrobacter, Proteus, Enterobacter, Klebsiella and Serratia strains. Bacteriocin production was higher in GM-17 and MRS as compared to TYGE broth and enriched skim milk broth and reached the maximum level during the early stationary phase. The bacteriocin was purified by performing ammonium sulfate precipitation. The bacteriocin was able to survive 90°C/10min but not 100°C/10min. Complete inactivation of bacteriocin was observed after autoclaving. The bacteriocin maintained its activity over a wide range of pH (3-9). The antimicrobial compound produced by the isolate 63, was sensitive to papain, pepsin, trypsin and amylase but was resistant to detergents like SDS and urea. Tween 20, Tween-80 as well as Triton X-100 enhanced its activity. Since the treatment with proteolytic enzymes resulted in loss of activity, this shows that the proteinaceous nature of the antimicrobial substance. Tentative molecular weight of the bacteriocin was found to be between 3.5 and 5kDa by Tricine SDS-PAGE. Finally, we confirmed the presence of gene for nisin, and the sequence thus obtained, was identical to the sequences previously described for nisin Z. Lactococcus lactis subsp. lactis 63 or its bacteriocin, which has a wide inhibitory spectrum, has the potential for use as a starter or protective culture in the manufacture of fermented products.

Psychrotolerant spore-former growth characterization for the development of a dairy spoilage predictive model

Psychrotolerant spore-forming bacteria represent a major challenge regarding microbial spoilage of fluid milk. These organisms can survive most conventional pasteurization regimens and subsequently germinate and grow to spoilage levels during refrigerated storage. To improve predictions of fluid milk shelf life and assess different approaches to control psychrotolerant spore-forming bacteria in the fluid milk production and processing continuum, we developed a predictive model of spoilage of fluid milk due to germination and growth of psychrotolerant spore-forming bacteria. We characterized 14 psychrotolerant spore-formers, representing the most common Bacillales subtypes isolated from raw and pasteurized milk, for ability to germinate from spores and grow in skim milk broth at 6°C. Complete growth curves were obtained by determining total bacterial count and spore count every 24 h for 30 d. Based on growth curves at 6°C, probability distributions of initial spore counts in bulk tank raw milk, and subtype frequency in bulk tank raw milk, a Monte Carlo simulation model was created to predict spoilage patterns in high temperature, short time-pasteurized fluid milk. Monte Carlo simulations predicted that 66% of half-gallons (1,900 mL) of high temperature, short time fluid milk would reach a cell density greater than 20,000 cfu/mL after 21 d of storage at 6°C, consistent with current spoilage patterns observed in commercial products. Our model also predicted that an intervention that reduces initial spore loads by 2.2 Log10 most probable number/mL (e.g., microfiltration) can extend fluid milk shelf life by 4 d (end of shelf life was defined here as the first day when the mean total bacterial count exceeded 20,000 cfu/mL). This study not only provides a baseline understanding of the growth rates of psychrotolerant spore-formers in fluid milk, it also provides a stochastic model of spoilage by these organisms over the shelf life of fluid milk, which will ultimately allow for the assessment of different approaches to reduce fluid milk spoilage.

English

Protease from Bacillus subtilis has wide applications in industrial sectors.A total of 28 bacteria were isolated from marine mud sample, of which 3 isolates exhibited constructive results for protease production in skim milk, gelatin and casein agar. Among three isolates, MMK 2/8 had superlative enzymatic activity in skim milk broth (170 U/mL) and was subjected for taxonomical identification, which was found to be Bacillus subtilis. The finest conditions were found to be at pH 6.0, temperature 40 C and 1% NaCl concentration and their maximal enzyme activitywas 354 U/mL, 437 U/mL and 263U/mL respectively, at 36 hrs of incubation. The molecular weight of the protease enzyme was considered to be approximately 50 kDa by SDS-PAGE. The enzyme activity was enriched by exposing it to ultra violet radiation [UV] for 60 secand it was observed to be 610 U/mL. Hence, we charted that the production of acidic protease treated with ultra violet radiation was maximum when compared with the parental strain.

Identification and characterization of psychrotolerant coliform bacteria isolated from pasteurized fluid milk

The presence of coliform bacteria in pasteurized fluid milk typically indicates that product contamination occurred downstream of the pasteurizer, but it may also indicate pasteurization failure. Although coliform detection is frequently used as a hygiene indicator for dairy products, our understanding of the taxonomic and phenotypic coliform diversity associated with dairy products is surprisingly limited. Therefore, using Petrifilm Coliform Count plates (3M, St. Paul, MN), we isolated coliforms from high-temperature, short-time (HTST)-pasteurized fluid milk samples from 21 fluid milk processing plants in the northeast United States. Based on source information and initial characterization using partial 16S rDNA sequencing, 240 nonredundant isolates were obtained. The majority of these isolates were identified as belonging to the genera Enterobacter (42% of isolates), Hafnia (13%), Citrobacter (12%), Serratia (10%), and Raoultella (9%); additional isolates were classified into the genera Buttiauxella, Cedecea, Kluyvera, Leclercia, Pantoea, and Rahnella. A subset of 104 representative isolates was subsequently characterized phenotypically. Cold growth analysis in skim milk broth showed that all isolates displayed at least a 2-log increase over 10 d at 6°C; the majority of isolates (n=74) displayed more than a 5-log increase. In total, 43% of the representative isolates displayed lipolysis when incubated on spirit blue agar at 6°C for 14 d, whereas 71% of isolates displayed proteolysis when incubated on skim milk agar at 6°C for 14 d. Our data indicate that a considerable diversity of coliforms is found in HTST-pasteurized fluid milk and that a considerable proportion of these coliforms have phenotypic characteristics that will allow them to cause fluid milk spoilage.

A standard bacterial isolate set for research on contemporary dairy spoilage

Food spoilage is an ongoing issue that could be dealt with more efficiently if some standardization and unification was introduced in this field of research. For example, research and development efforts to understand and reduce food spoilage can greatly be enhanced through availability and use of standardized isolate sets. To address this critical issue, we have assembled a standard isolate set of dairy spoilers and other selected nonpathogenic organisms frequently associated with dairy products. This publicly available bacterial set consists of (1) 35 gram-positive isolates including 9 Bacillus and 15 Paenibacillus isolates and (2) 16 gram-negative isolates including 4 Pseudomonas and 8 coliform isolates. The set includes isolates obtained from samples of pasteurized milk (n=43), pasteurized chocolate milk (n=1), raw milk (n=1), cheese (n=2), as well as isolates obtained from samples obtained from dairy-powder production (n=4). Analysis of growth characteristics in skim milk broth identified 16 gram-positive and 13 gram-negative isolates as psychrotolerant. Additional phenotypic characterization of isolates included testing for activity of β-galactosidase and lipolytic and proteolytic enzymes. All groups of isolates included in the isolate set exhibited diversity in growth and enzyme activity. Source data for all isolates in this isolate set are publicly available in the FoodMicrobeTracker database (http://www.foodmicrobetracker.com), which allows for continuous updating of information and advancement of knowledge on dairy-spoilage representatives included in this isolate set. This isolate set along with publicly available isolate data provide a unique resource that will help advance knowledge of dairy-spoilage organisms as well as aid industry in development and validation of new control strategies.

Identification and Characterization of Psychrotolerant Sporeformers Associated with Fluid Milk Production and Processing

Psychrotolerant spore-forming bacteria represent a major challenge to the goal of extending the shelf life of pasteurized dairy products. The objective of this study was to identify prominent phylogenetic groups of dairy-associated aerobic sporeformers and to characterize representative isolates for phenotypes relevant to growth in milk. Analysis of sequence data for a 632-nucleotide fragment of rpoB showed that 1,288 dairy-associated isolates (obtained from raw and pasteurized milk and from dairy farm environments) clustered into two major divisions representing (i) the genus Paenibacillus (737 isolates, including the species Paenibacillus odorifer, Paenibacillus graminis, and Paenibacillus amylolyticus sensu lato) and (ii) Bacillus (n = 467) (e.g., Bacillus licheniformis sensu lato, Bacillus pumilus, Bacillus weihenstephanensis) and genera formerly classified as Bacillus (n = 84) (e.g., Viridibacillus spp.). When isolates representing the most common rpoB allelic types (ATs) were tested for growth in skim milk broth at 6°C, 6/9 Paenibacillus isolates, but only 2/8 isolates representing Bacillus subtypes, grew >5 log CFU/ml over 21 days. In addition, 38/40 Paenibacillus isolates but only 3/47 Bacillus isolates tested were positive for β-galactosidase activity (including some isolates representing Bacillus licheniformis sensu lato, a common dairy-associated clade). Our study confirms that Paenibacillus spp. are the predominant psychrotolerant sporeformers in fluid milk and provides 16S rRNA gene and rpoB subtype data and phenotypic characteristics facilitating the identification of aerobic spore-forming spoilage organisms of concern. These data will be critical for the development of detection methods and control strategies that will reduce the introduction of psychrotolerant sporeformers and extend the shelf life of dairy products.

Hydrolytic potential of a psychrotrophic Pseudomonas isolated from refrigerated raw milk

The production of extracellular hydrolases by a psychrotrophic bacterium isolated from refrigerated raw milk, and identified as a Pseudomonas sp. belonging to the Pseudomonas jenssenii group, was studied. This bacterium produced proteolytic and lipolytic enzymes in all media investigated (skim milk, cheese whey, casein broth, and tryptone soy broth). High levels of α-glucosidase were produced in skim milk broth. Hydrolytic enzymes detected in skim milk broth are of particular concern, indicating that these enzymes could be produced by Pseudomonas sp. during the cold storage of raw milk, contributing to the spoilage problem in milk and dairy products.

Hydrolytic potential of a psychrotrophic Pseudomonas isolated from refrigerated raw milk.

The production of extracellular hydrolases by a psychrotrophic bacterium isolated from refrigerated raw milk, and identified as a Pseudomonas sp. belonging to the Pseudomonas jenssenii group, was studied. This bacterium produced proteolytic and lipolytic enzymes in all media investigated (skim milk, cheese whey, casein broth, and tryptone soy broth). High levels of α-glucosidase were produced in skim milk broth. Hydrolytic enzymes detected in skim milk broth are of particular concern, indicating that these enzymes could be produced by Pseudomonas sp. during the cold storage of raw milk, contributing to the spoilage problem in milk and dairy products.

NaCl‐Activated Extracellular Proteinase from Virgibacillus sp. SK37 Isolated from Fish Sauce Fermentation

Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 degrees C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca(2+) activated the enzyme activity but reduced enzyme stability at 65 degrees C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation.

Relationship between thickness, chapping and Staphylococcus aureus colonization of bovine teat tissue.

Post-milking measurements of teat skin chapping score, teat thickness and colonization by Staphylococcus aureus were determined for 11.5 d. Three teats on each of twelve Holstein cows, free from Staph. aureus intramammary infections, were immersed in 1 M-NaOH solution to induce teat chapping; the fourth teat served as a control. To achieve different degrees of chapping, one teat per cow received one immersion after each milking for three consecutive milking periods, a second teat received two immersions, and the third teat received one immersion which coincided with the last immersion of the other two teats. All teats were challenged twice with a skim milk broth culture of Staph. aureus (5 x 10(6) cfu) after the first and second milking following NaOH treatment. Measures were initiated with the milking following the last Staph. aureus challenges. Tissue thickness of the lateral side of the teat (barrel) and colonization by Staph. aureus declined with time. Thickness of the teat end varied more erratically. Teat skin chapping score was positively correlated (P < 0.001) with tissue thickness of the barrel and Staph. aureus colonization. Thickness of the barrel was not significantly correlated with Staph. aureus colonization. Barrel thickness as a covariate had a significant effect (P < 0.05) on teat skin colonization of Staph. aureus, whereas teat skin score and teat end thickness had no significant effect. Thus, thickness of the lateral side of the teat explained the greatest variation in Staph. aureus teat skin colonization in the model tested.