Post on 25-Mar-2020
BIOL 3702L - Microbial Numbers Spring 2020
Dr. Cooper 1
BIOL 3702L:Determining
Microbial Numbers
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Purpose of This Week’s Labs
1) Determine the number of cells in a yeast culture using three different methods:a) Serial dilution platingb) Direct counts using a
hemocytometerc) Indirect counts using a
spectrophotometer
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Purpose of This Week’s Labs
1) Understand dilutions, viable vs. total cell counts and direct vs. indirect counts
2) Analyze ”real-time” data3) Learn to use different
instruments
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Today’s Exercise1) To be performed in two different groups
with each group divided into three smaller subgroupsa) Group A – first two benchesb) Group B – second two benches
2) Groups A and B shall break into three smaller groups with assigned functionsa) Serial dilution platingb) Direct counts with a hemocytometerc) Indirect counts with a spectrophotometer
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Today’s Exercise (cont.)
Serial Dilution Plating1) Prepare dilution bottles with 90 ml of
Butterfield buffer2) Place 10 ml of yeast culture into first tube
and serial dilute3) Transfer 0.1 ml of designated dilutions to
each of two YPD plates4) Spread cells and incubate plates at 37°C
for 3-5 days
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Today’s Exercise (cont.)Serial Dilution Scheme
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BIOL 3702L - Microbial Numbers Spring 2020
Dr. Cooper 2
Today’s Exercise (cont.)Spread plate technique
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Today’s Exercises (cont.)
Direct Count with a Hemocytometer1) Mix 100 µl of the 10-1
dilution with 100 µl 1% methylene blue, then add 10 µl of the mixture to Chamber A of a hemocytometer
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Today’s Exercises (cont.)
2) Mix 100 µl of the 10-2 dilution with 100 µl 1% methylene blue, then add 10 µl of the mixture to Chamber B of a hemocytometer
3) Count cells in four large corner squaresa) Count all cellsb) Count blue cells
4) Calculate total cell concentration per ml and percent viable cells
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Today’s Exercises (cont.)
Copyright © C. R. Cooper, Jr.
http://www.rci.rutgers.edu/~microlab/applied%20micro/schedulelinks/budding%20yeast%20cells.htm and http://braukaiser.com/blog/blog/category/science/yeast/
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Today’s Exercise (cont.)
Measuring Turbidity1) Prepare serial dilutions in 13 mm x 100
mm test tubes using DYN medium – 3 ml per tube
2) Place 3 ml of yeast culture in tube 1 and mix
3) Transfer 3 ml from tube 1 to tube 2, mix, then 3 ml from tube 2 to tube 3, mix, etc.
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Today’s Exercises (cont.)
4) Measure absorbance of each tube dilution using the Spectronic200E spectro-photometer.
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BIOL 3702L - Microbial Numbers Spring 2020
Dr. Cooper 3
Lab Report Expectations
• SHARE DATA WITHIN YOUR GROUP!!!!•Work together to analyze data.• Answer essay questions, but DO NOT
PLAGIARIZE!!!! Remember, where appropriate, YOUR ANSWERS MUST BE CLEAR, CONCISE, AND GRAMMATICALLY SOUND SENTENCES!• Retain this lab report. Your laboratory
instructor may not call for it at this time, but may do so in the near future.
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Questions?
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