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Flow cytometry analysis of cell viability of Lactobacillus reuteri during growth under different conditions

Sen, Anirudh LU (2020) KMBM01 20192
Applied Microbiology
Biotechnology
Abstract
Lactobacillus reuteri is a probiotic bacterium which when consumed or administered in certain recommended dosages can provide a number of health benefits to the consumer. To have an effect on the consumer, these bacteria must survive until they reach the gut. Process conditions during production of the cells determine their ability to adapt to subsequent environmental shifts during storage and administration. In this project, different conditions during shake flask cultivation were studied to determine the best conditions with respect to growth rate and cell viability as determined by flow cytometry.
Specifically, the effect of growth temperature, agitation and supplementation of citrate and acetate to MRS medium was studied. 44°C gave... (More)
Lactobacillus reuteri is a probiotic bacterium which when consumed or administered in certain recommended dosages can provide a number of health benefits to the consumer. To have an effect on the consumer, these bacteria must survive until they reach the gut. Process conditions during production of the cells determine their ability to adapt to subsequent environmental shifts during storage and administration. In this project, different conditions during shake flask cultivation were studied to determine the best conditions with respect to growth rate and cell viability as determined by flow cytometry.
Specifically, the effect of growth temperature, agitation and supplementation of citrate and acetate to MRS medium was studied. 44°C gave the highest growth rate and cell titers, as well as a survivability of 96% of the cells during the first 8 hours of fermentation. Agitation of the cells in a shake flask incubator was found to be inhibitory for growth as well as detrimental to the survivability of the cells.
In addition, a mixture of CFDA (carboxy fluorescein diacetate) and PI (propidium iodide) as fluorescent dyes to determine cell viability were benchmarked to SYBR green and PI. It was found that CFDA can be used instead of SYBR green to reach comparable results. The best assay conditions were to use an OD620 of 0.1, 10 mM CFDA, and 1.5 mM PI in a PBS buffer. (Less)
Popular Abstract
Lactobacillus reuteri is a probiotic bacterium which when consumed in certain recommended dosages can provide a number of health benefits to the consumer. To have an effect on the consumer, these bacteria must survive until they reach the gut. Process conditions during production of the cells determine their ability to adapt to subsequent environmental shifts during storage and administration. In this project, different conditions were studied to determine the best conditions with respect to rate of growth and viability of the cells, determined using an analytical technique called flow cytometry.
Specifically, the effect of growth temperature, agitation and supplementation of the metabolites - citrate and acetate to the growth medium... (More)
Lactobacillus reuteri is a probiotic bacterium which when consumed in certain recommended dosages can provide a number of health benefits to the consumer. To have an effect on the consumer, these bacteria must survive until they reach the gut. Process conditions during production of the cells determine their ability to adapt to subsequent environmental shifts during storage and administration. In this project, different conditions were studied to determine the best conditions with respect to rate of growth and viability of the cells, determined using an analytical technique called flow cytometry.
Specifically, the effect of growth temperature, agitation and supplementation of the metabolites - citrate and acetate to the growth medium (MRS) used was studied. 44°C gave the highest growth rate and, as well as a survivability of 96% of the cells during the first 8 hours of cell growth. Agitation of the cells in a shake flask incubator was found to be inhibitory for growth as well as detrimental to the survivability of the cells.
In addition, a mixture of the two fluorescent dyes, CFDA (carboxy fluorescein diacetate) and PI (propidium iodide) to determine viability of the cells using Flow cytometry were benchmarked to another mixture of fluorescent dyes, SYBR green and PI. It was found that CFDA can be used instead of SYBR green to give comparable results. The best assay conditions were to use an Optical Density (measured for the cells) of 0.1, 10 mM concentration CFDA, and 1.5 mM concentration PI in a PBS buffer. (Less)
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author
Sen, Anirudh LU
supervisor
organization
course
KMBM01 20192
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Lactobacillus reuteri, Flow cytometry, cFDA, Applied microbiology, Teknisk mikrobiologi
language
English
id
9003820
date added to LUP
2020-02-17 13:25:10
date last changed
2020-02-17 13:25:10
@misc{9003820,
  abstract     = {{Lactobacillus reuteri is a probiotic bacterium which when consumed or administered in certain recommended dosages can provide a number of health benefits to the consumer. To have an effect on the consumer, these bacteria must survive until they reach the gut. Process conditions during production of the cells determine their ability to adapt to subsequent environmental shifts during storage and administration. In this project, different conditions during shake flask cultivation were studied to determine the best conditions with respect to growth rate and cell viability as determined by flow cytometry. 
Specifically, the effect of growth temperature, agitation and supplementation of citrate and acetate to MRS medium was studied. 44°C gave the highest growth rate and cell titers, as well as a survivability of 96% of the cells during the first 8 hours of fermentation. Agitation of the cells in a shake flask incubator was found to be inhibitory for growth as well as detrimental to the survivability of the cells. 
In addition, a mixture of CFDA (carboxy fluorescein diacetate) and PI (propidium iodide) as fluorescent dyes to determine cell viability were benchmarked to SYBR green and PI. It was found that CFDA can be used instead of SYBR green to reach comparable results. The best assay conditions were to use an OD620 of 0.1, 10 mM CFDA, and 1.5 mM PI in a PBS buffer.}},
  author       = {{Sen, Anirudh}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Flow cytometry analysis of cell viability of Lactobacillus reuteri during growth under different conditions}},
  year         = {{2020}},
}