Measuring Microbial Growth

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Measuring Microbial Growth

• Lab 7

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Measuring Cell Growth

• Number of Cells per milliliter of liquid
• cells/ml
• Bacterial cultures contain millions of cells
• Direct Vs. indirect counts
• Direct Counts mean actually counting cells
• More accurate, but takes a lot longer
• Indirect Counts mean estimating the number of
  cells based on turbidity in liquid or dry weight.
• Less accurate, but quick
• Total Count vs. Viable count
• Total count?all cells in a culture
• Viable count?only live cells

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Turbidity Method

• Spectrophotometer
• Measure transmittance
• Amount of light going through culture
• Transmittance is inversely proportional to
  turbidity (cell concentration)
• As turbidity increases cell number increases and
  transmittance decreases
• Indirect and Total Count

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Turbidity and Transmittance
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LEARN YOUR SCIENTIFIC NOTATION BY THURSDAY

• http//a-s.clayton.edu/furlong/BIOL2250LAB/mediagu
  ide/Scienctific20Notation20Help.doc
• Or there is a link on the course web.

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Plate Counts

• Direct Viable counts
• The idea is to evenly spread the culture across a
  plate and count the colonies
• Each colony represents a cell in the original
  culture.
• Problem If we have a culture that has 1x106
  cells/ml and we put 1 ml on the plate will we
  have isolated colonies?

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Plate Count

• We must dilute first
• If we put 1ml of culture into 9 ml of sterile
  media we have performed a 1/10 serial dilution
  (SD)
• Formula SDAT/TV
• ATamount of culture transferred
• TVTotal volume after transfer (1ml 9 ml)
• What if we did 5 serial dilutions on that culture
  that had 1 x 106 cells/ml

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1
2
3
5
4
We can multiply the SD to get total dilution TD
at each tube. Tube 11/10 Tube 21/10 x 1/10
1/100 0.01 1 x 10-2 Tube 31/10 x 1/10 x 1/10
1/1000 0.001 1 x 10-3 You do the next one
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• Now if we have 1 x 106 cells/ml in the original
  culture and we diluted it by 1/100,000 or 1 x
  10-5 (as in Tube 5) then how many cells per ml do
  we have in tube 5?
• (1 x 106 cells/ml) X (1 x 10-5 ) 1 x 101 10
  cells/ml
• Now we must plate it out!

• If I plate 1 ml of this solution I am plating 10
  cells.
• Or 1ml x 10 cells/ml!
• What if I plated 0.1 ml?

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Practice

• I have a pond water with 1 x 108 cells/ml
• I do the following dilution.
• Transfer 1ml from pond water to A which has 9 ml
  of sterile media.
• Transfer 0.1 ml from A to B which has 9.9 ml of
  sterile media.
• Transfer 0.1 ml from B to plate.
• How many colonies should I expect to see on the
  plate?

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The Reverse

• Rarely do we actually know the concentration of
  the starting culture. Our point is to find this
  out!
• Lets look at the dilution scheme used to get the
  colony count that you counted Tuesday.

1ml
1ml
1ml
Determine the SDs for the 3 and the TD of the
third tube.
0.1ml
1
2
3
99 ml
original
99 ml
99 ml
A
B
C
TD A1x10-2 B1x10-4 C1x10-6
TD A1/100 B1/10,000 C1/1,000,000
SD A1/100 B1/100 C1/100
Easier!!!
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Step 1 Take the number of colonies on the plate
and divide by the volume applied to the
plate. EX)200 colonies/0.1ml2,000 c/ml?this is
the number of cells/ml in tube C! Step 2 Take
the cells/ml in tube C and DIVIDE by the total
dilution of C. EX)2,000 c/ml ? 1 x 10-6 1 x 109
cells/ml in the original Now you do it with your
numbers!
HINT you never want to count a plate that has
under 30 colonies or you will get an inaccurate
count! Dont bother trying to count a plate that
has over 300 colonies because it will be
difficult, which will result in inaccuracies!
1ml
1ml
1ml
0.1ml
1
2
3
99 ml
original
99 ml
99 ml
A
B
C
TD A1/100 B1/10,000 C1/1,000,000
SD A1/100 B1/100 C1/100
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PRACTICE

• You have practice problems on the web!
• Practice problems on the objectives!
• Practice problems on the data sheet!