MicrobialGrowth:Analysis of a BacterialGrowthCurve

acterial growth is usually defined as the increase in numbersof viable vegetative cells in a population. For optimal growth, sev-eral physical factors must be met. These include an optimal pH

for the growth medium, an optimal temperature, and adequate oxygen gas(for aerobic microbes). Growth of heterotrophic microorganisms alsorequires specific nutritional factors, including carbon, nitrogen, sulfur,phosphorus, cofactors, vitamins, and, of course, water. These nutritionalrequirements are supplied in the environment or by the culture medium inwhich the bacteria are inoculated.

Under these conditions, bacterial cells will undergo rapid binary fis-sion. The dynamics of bacterial growth follow a predictable pattern visu-alized as a population growth curve. A growth curve is generated byplotting the increase in cell number versus time of exposure or length ofincubation in the growth medium. The curve then can be used to determinethe generation time, the time required for a microbial population to dou-ble in cell number. In the time allotted for this lab, you will not have timeto follow a complete growth curve. However, you can study that portion ofthe curve (log phase) needed to estimate the generation time.

You can appreciate how hard it would be to count individual bacterialcells! Therefore, a simpler but indirect method to estimate bacterial cellnumber is to use a spectrophotometer. This instrument uses a beam of lightthat is projected at a tube of liquid (growth medium in this case) placed in theinstrument. The medium and any bacterial present will absorb some of thelight. The light not absorbed will pass through the tube and be detected bya photoelectric device that converts the light intensity into a measurable

B

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number. For the light absorbed, this is called the absorbance. As growth pro-gresses and the number of cells increase, the absorbance will increase also.

Bacterial Growth Dynamics

Under a set of permissive growth conditions as described in the earlierlab exercises in this section, bacterial cells in a broth culture will reproduceat a steady rate. Such growth follows a specific set of phases or stages thatare described and drawn as a growth curve (Figure 15.1).

pecial Materials

• 5-hour brain-heart infusion (BHI) broths of Escherichia coli

• 1 tube with 5.3 ml sterile BHI broth• 1 tube with 5 ml sterile BHI broth• 2 sterile spec tubes• 20° and 37° C incubators• Spectrophotometer• 1 ml sterile pipettes

rocedure

1. This experiment is best done in lab groups of 2–3 individuals. The exercisedescribed here examines the effect of temperature on growth. Your instruc-tor may have different groups examine other physical or chemical fac-tors governing bacterial growth.

2. For this exercise, odd-numbered groups will incubate their broth culturetubes at 20°C while even-numbered groups will incubate their tubes at 37°C.

3. Pour one tube containing 5.3 ml sterile BHI broth into a sterile spec-trophotometer (spec) tube. Standardize the spectrophotometer as directedby your instructor.

4. Label the tube as Stnd. Keep this tube because you will need to restandard-ize the spectrophotometer before every new reading.

P

S

A.

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PURPOSE: to examine thepopulation growth dynamicsof bacterial cultures growingunder specified growth conditions.

1098765

4

3

2

0 5 10 15 20 25 30 35 400

(a) Lag phase

Time (hours)

(b) Log (exponential

growth) phase

(c) Stationary phase (d) Decline phase

Some cells

remainviable

Few cells

Live cells

Dead cell

Total cells inpopulation:

Log

arith

m (

10n )

of v

iab

le c

ells

F I G U R E 1 5 . 1Four phases of a bacterial growth curve.

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5. Label another sterile spec tube with your group’s identification. Add 0.3 mlof 5-hour E. coli into a tube containing 5 ml of BHI broth. Carefully, butquickly pour the suspension into the labeled Spec tube.

6. Immediately determine the absorbance of this suspension at 590 nm asdemonstrated by the instructor. This will represent the zero time readingfor the growth curve. The absorbance reading should be between 0.05and 0.15. Record your absorbance in the 0 min time box in Table 15.1 in theResults section.

7. After recording the absorbance, incubate the tube at the designated tem-perature for 20 minutes.

8. Now, every 20 minutes, remove your Spec tube from the incubator anddetermine the absorbance. Remember to first standardize the spec-trophotometer with your Stnd tube! Record the absorbance in Table15.1 and then return the tube to the incubator for another 20 minutes.

9. You will need absorbance readings every 20 minutes through 140 minutes.

Plotting a Growth Curve and Determiningthe Generation Time

The dynamics and phases of microbial growth can be followed and plottedas a population growth curve by graphing the absorbance readings versustime. In other words, the growth curve represents the change in cell num-bers with time of incubation.

The curve also can be used to determine the populations generationtime. This indirect determination of doubling is made by extrapolatingfrom the log phase of the growth curve. You select two points on theabsorbance scale, such as 0.2 and 0.4 that represent a doubling in turbidity(Figure 15.2). As shown with the arrows in the graph, extrapolate to theirrespective time intervals. The difference is the generation time; in thisexample it would be approximately 30 minutes.

B.

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0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

Abs

orba

nce

at 5

90 n

m

0 40 80 120 160Time (min)

F I G U R E 1 5 . 2The estimation of the generationtime from the log phase of a bacter-ial growth curve.

PURPOSE: to plot a growthcurve for such a bacterialculture and estimate thegeneration time.

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rocedure

1. As you get your absorbance readings, take the data from your table and plotthe data on the graph paper provided in the Results section. You need toplot absorbance (590 nm) versus time (min) of incubation.

2. After you have determined that the bacterial growth curve is in log phase,calculate the generation time for your culture conditions. Extrapolate fromthe absorbance scale on the plotted growth curves as explained above.Record your results in Table 15.2 in the Results section.

uestions

1. Why do variations in generation time exist in the plotted curves?

2. Does the phrase microbial growth, such as in bacteria, mean the samething as when describing human growth? Explain.

3. Propose an explanation for the growth differences at 20°C and 37°C.

4. Answer each of the following questions based on the growth curves (A, B,and C) illustrated in the graph.

Q

P

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9

8

7

6

5

4

3

2

1

0

Log

Num

ber

of C

ells

/ml

0 10 20 30 40Time (min)

50

B

A

C

a. Which growth curve (A, B, or C) most closely matches that of a normalbacterium like Escherichia coli when growing in the colon? From thegrowth curve, figure out this bacterium’s generation time.

b. Which growth curve (A, B, or C) would most likely match that of a psy-chrotrophic bacterium growing at 20°C? From the growth curve, figureout this bacterium’s generation time.

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Name

Date Section

Exercise Results

Microbial Growth: Analysis of a Bacterial Growth Curve

A. Bacterial Growth Dynamics

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Time(min)

0

Group 1 Group 3 Group 5 Group 7 Group 2 Group 4 Group 6 Group 8

20

40

60

80

100

120

140

BHI (20C) BHI (37C)

Absorbance (590 nm)

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B. Plotting a Growth Curve and Determining the Generation Time

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

Sample Calculated Generation Time (min)

BHI (20°C)

BHI (37°C)

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