Learning Module - Periods 2A and 2B

BACTERIAL GROWTH

Bacterial growth is logarithmic:

Optical Density is a measure of transmitted light:

Absorbance (O.D.) is proportional to cell number over a limited range.

There are four phases in bacterial growth:

• LAG phase (cells recover from stationary phase)
• LOG phase (cells are dividing by binary fission)
• STATIONARY phase (nutrients have been consumed)
• DEATH phase (cells die from lack of food and accumulation of toxic waste products)

Doubling time is the time it takes for the O.D. to double.

DILUTIONS

Serial dilution:

Overall dilution is the product of the individual dilutions.

A 1:10 or ten-fold dilution:

If there are 100 cells / mL in the first tube, then there are 10 cells / mL in the second tube.

PLATING OF BACTERIA

1) Apply 0.1 mL of solution containing bacteria.

2) Spread evenly over plate.

DATA TABULATION

Use a spreadsheet to organize and plot the data

Calculate the number of viable cells per mL of culture:

For example: At 30 min, 40 colonies were observed.

Hence, 40 colonies / 0.1 ml x 10^4 dilution = 4 x 10^6 CFU / mL.

           DILUTION      # OF COLONIES

TIME        FACTOR         ON PLATE      CFU / ML

----       --------       ----------     ----------

  0          10^4              20        2.0 x 10^6 

 15          10^4              25        2.5 x 10^6 

 30          10^4              40        4.0 x 10^6 

 45          10^4              75        7.5 x 10^6 

 60          10^5              14        1.4 x 10^7 

 80          10^5              30        3.0 x 10^7 

100          10^5              60        6.0 x 10^7 

120          10^5             120        1.2 x 10^8 

140          10^6              22        2.2 x 10^8 

160          10^6              40        4.0 x 10^8 

180          10^6              80        8.0 x 10^8 

etc.         etc.           etc.             etc.

Question: Procede to LON-CAPA to answer the question for this Learning Module.