Undergraduate lab series using the K562 human leukemia cell line: Model for cell growth, death, and differentiation in an advanced cell biology course.

This sequence of labs was developed for an upper level undergraduate cell biology course at Fairfield University. The labs are based on the use of the K562 human erythroleukemia cell line, a model system that is exceptionally amenable to an undergraduate cell biology lab course due to its ease of maintenance and propagation and usefulness for studies of growth, death, and differentiation. The sequence of labs is conducted over a 6-week period, following a series of weekly cell biology labs covering basic cell and molecular biology techniques. Together, the lab series has four primary objectives 1) to teach students how to culture and maintain mammalian cells; 2) to build student competency in standard cell biology techniques; 3) to demonstrate the role of growth factors on cell proliferation and viability; and 4) to provide students with an opportunity to use these cells in an independent investigation on cell differentiation. We provide examples of student data and offer a range of experimental measurements depending on institutional capacity and facilities. Our assessment data suggest that students find great value in this lab series, enhancing their comprehension of key concepts, acquisition of important lab skills, and depth of understanding of the research process. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):263-271, 2019.

An Overview of Seafood Supply, Food Safety and Regulation in New South Wales, Australia.

Seafood consumption is increasing in Australia, especially in New South Wales (NSW). Average per capita seafood consumption in NSW is higher than the national average. Seafood supply in NSW comes from domestic (wild catch and aquaculture) and overseas (seafood imports) sources. The contribution of wild catch and aquaculture in domestic seafood production (2012-2013) was 73.42% and 26.52%, respectively. Seafood-associated foodborne illness outbreaks are not common and on an average four outbreaks occur each year in NSW. Most of the outbreaks in 2015 and 2016 were related to ciguatera poisoning. The regulation of the seafood industry and the management of food safety is an example of the coordinated work of multiple government agencies and organizations in which NSW Food Authority is responsible for managing the overall risks through the Seafood Safety Scheme. Overall, seafood supply in NSW is of high quality and poses low food safety risk to consumers.

Toxoplasma gondii in the Food Supply.

Toxoplasmosis is caused by infection with the protozoan parasite Toxoplasma gondii. Infections are usually either asymptomatic or develop mild symptoms that are self-limited, but infections in immunosuppressed persons can be severe. Infections in pregnant women can cause serious health problems in the child such as mental retardation and blindness. Infection with T. gondii in immunocompetent adults can lead to impaired eyesight. Toxoplasmosis has ranked very highly in two studies of death and disability attributable to foodborne pathogens. The consumption of raw or undercooked meat containing T. gondii tissue cysts and the consumption of raw vegetables or water contaminated with T. gondii oocysts from cat feces is most frequently associated with human illness. The risk of acquiring a Toxoplasma infection via food varies with cultural and eating habits in different human populations.

A survey of total hydrocyanic acid content in ready-to-eat cassava-based chips obtained in the Australian market in 2008.

Cassava (Manihot esculenta Crantz) is a widely consumed food in the tropics that naturally contains cyanogenic glycosides (cyanogens, mainly composed of linamarin, acetone cyanohydrin, and hydrocyanic acid). If cassava is not adequately processed to reduce the level of cyanogens prior to consumption, these compounds can lead to the formation of hydrocyanic acid in the gut. Exposure to hydrocyanic acid can cause symptoms ranging from vomiting and abdominal pain to coma and death. In 2008, a survey of ready-to-eat (RTE) cassava-based snack foods was undertaken to determine levels of cyanogens measured as total hydrocyanic acid. This survey was undertaken in response to the New South Wales Food Authority being alerted to the detection of elevated levels of cyanogens in an RTE cassava-based snack food. This survey took 374 samples of RTE cassava chips available in the Australian marketplace. Significant variation in the levels of total hydrocyanic acid were observed in the 317 samples testing positive for cyanogens, with levels ranging from 13 to 165 mg of HCN equivalents per kg (mean value, 64.2 mg of HCN eq/kg for positive samples). The results from this survey serve as a timely warning for manufacturers of RTE cassava chips and other cassava-based snack foods to ensure there is tight control over the levels of cyanogens in the cassava ingredient. Evidence from this survey contributed to an amendment to the Australia New Zealand Food Standards Code, which now prescribes a maximum level for hydrocyanic acid in RTE cassava chips of 10 mg of HCN eq/kg, which aligns with the Codex Alimentarius Commission international standard for edible cassava flour.

Toward validation of process criteria for high-pressure processing of orange juice with predictive models.

Mathematical models were developed to predict time to inactivation (TTI) by high-pressure processing of Salmonella in Australian Valencia orange juice (pH 4.3) and navel orange juice (pH 3.7) as a function of pressure magnitude (300 to 600 MPa) and inoculum level (3 to 7 log CFU/ml). For each model, the TTI was found to increase with increasing inoculum level and decrease with increasing pressure magnitude. The U.S. Food and Drug Administration Juice Hazard Analysis and Critical Control Point Regulation requires fruit juice processors to include control measures that produce a 5-log reduction of the pertinent microorganism of public health significance in the juice. To achieve a 5-log reduction of Salmonella in navel orange juice at 20 degrees C, the models predicted hold times of 198, 19, and 5 s at 300, 450, and 600 MPa, respectively. In Valencia orange juice at 20 degrees C, a 5-log reduction of Salmonella was achieved in 369, 25, and 5 s at 300, 450, and 600 MPa, respectively. At pressures below 400 MPa, Salmonella was more sensitive to pressure in the more acidic conditions of the navel orange juice and TTIs were shorter. At higher pressures, little difference in the predicted TTI was observed. Refrigerated storage (4 degrees C) of inoculated navel orange juice treated at selected pressure/time/inoculum combinations showed that under conditions in which viable Salmonella was recovered immediately after high-pressure processing, pressure-treated Salmonella was susceptible to the acidic environment of orange juice or to chill storage temperature. These TTI models can assist fruit juice processors in selecting processing criteria to achieve an appropriate performance criterion with regard to the reduction of Salmonella in orange juice, while allowing for processing flexibility and optimization of high-pressure juice processing.

Effect of prior growth temperature, type of enrichment medium, and temperature and time of storage on recovery of Listeria monocytogenes following high pressure processing of milk.

A five-isolate cocktail of Listeria monocytogenes (10(3) cfu/ml in skim or whole raw milk) was subjected to 450 MPa for 900 s or 600 MPa for 90 s. The effects of prior growth temperature, type of milk (skim vs. whole), type of recovery-enrichment media (optimized Penn State University [oPSU] broth, Listeria Enrichment Broth [LEB], Buffered LEB [BLEB], Modified BLEB [MBLEB], and milk), storage temperature and storage time on the recovery of L. monocytogenes were examined. Optimized PSU broth significantly increased the recovery of L. monocytogenes following high pressure processing (HPP), and was 63 times more likely to recover L. monocytogenes following HPP, compared to LEB, BLEB and MBLEB broths (p<0.05; Odds Ratio=63.09, C.I. 23.70-167.96). There was a significant main effect for prior growth temperature (p<0.05). However, this relationship could not be interpreted given the significant interaction effects between temperature and both pressure and milk type. HPP-injured L. monocytogenes could be recovered using both LEB and oPSU broths after storage of milk at 4, 15 and 30 degrees C, with recovery being maximal after 24 to 72 h of storage; however, recovery yield dropped to 0% after prolonged storage of milk at 4 and 30 degrees C. In contrast, storage of milk at 15 degrees C yielded the most rapid rate of recovery and the highest recovery yield (100%), which remained high throughout the 14 days of storage at 15 degrees C. The above factors need to be taken into consideration when designing challenge studies to insure complete inactivation of L. monocytogenes and possibly other foodborne pathogens during high pressure processing of foods.

The effect of growth atmosphere on the ability of Listeria monocytogenes to survive exposure to acid, proteolytic enzymes and bile salts.

Four isolates of Listeria monocytogenes from food, human and environmental sources were grown separately in broth (pH 6.0 at 8 degrees C) under atmospheres of air, 100% N(2), 40% CO(2):60% N(2) or 100% CO(2). Exponential and stationary phase cells were harvested to determine if growth atmosphere and growth phase influenced this pathogen's ability to survive exposure to an acid environment coupled with proteolytic enzymes, and the activity of bile salts. In general, isolates were more resistant to the acid environment than the bile salts environment and stationary phase cells were significantly more resistant to both environments than exponential phase cells. Irrespective of prior growth atmosphere, none of the isolates when in exponential phase remained detectable following full exposure to the acid environment (110 min at 37 degrees C) or the bile environment (3 h at 37 degrees C). With the exception of one isolate grown under the atmosphere of 40% CO(2):60% N(2), all isolates when in stationary phase were detectable following full exposure to the acid environment but death rates varied significantly. Stationary phase cells of all isolates grown under 40% CO(2):60% N(2) and 100% CO(2) were highly susceptible to the bile salts environment: cells were not detectable after a 2-min exposure whereas stationary phase cells grown under air or 100% N(2) were recovered following full exposure to the bile environment. Survival curves were characterised by a population decline of at least 3 log(10)/ml (from an initial level of 7 log(10) CFU/ml) in the first 15 min; thereafter a constant population number of approximately 4 log(10)/ml was maintained over the remaining exposure period. No survival was observed when stationary phase cells of L. monocytogenes FRRB 2538 grown in air and 100% N(2) were subjected to the acid environment followed by immediate exposure to the bile salts environment. The results showed that growth atmosphere and growth phase could influence survival of this pathogen against conditions that imitate the extremes of the most important nonspecific defence mechanisms against microbial infection: the acid environment of the stomach coupled with the activity of proteolytic enzymes, and the activity of bile salts in the small intestine.