Liu Yuanwei (SET130704) Lab Report Growth Curves

Microbial Growth in Homogeneous Batch Culture: Growth Curves

Information about Group Members

Name Matric Number
Liu Yuanwei SET130704
Maxwell SET130008
Razil Bin Tahir SET130019

1. Introduction

Growing E.coli at different temperatures and measuring growth by turbidity with a

spectrophotometer will provide the data for drawing growth curves, which can be further

analyzed mathematically to reflect a typical entire growth cycle of a new microbial

population in a batch culture.

2. Objectives

To measure microbial growth rate by spectrophotometers

To draw microbial growth curves

To calculate microbial growth rates

3. Background

3.1 The Growth Cycle

Microbial growth is defined as an increase in the number of cells in a population. A

growth curve represents growth as a function of time. The growth curve describes an

entire growth cycle, including the lag phase, the exponential or log phase, the stationary

phase, and the death or decline phase. The terms lag phase, exponential phase, stationary

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Liu Yuanwei (SET130704) Lab Report Growth Curves

phase, and death phase have no meaning with respect to individual cells but only to cell

populations.

3.2 Lag Phase

When a microbial population is inoculated into a fresh medium, growth usually does

not begin immediately but only after a period of time called the lag phase. This interval

may be brief or extended, depending on the history of the culture and the growth

conditions.

3.3 Exponential Phase

The lag phase is followed by the exponential phase. During the exponential phase of

growth each cell divides to form two cells, each of which also divides to form two more

cells, and so on, for a brief or extended period, depending on the available resources and

other factors. Cells in exponential growth are usually in their highest state.

Most unicellular microorganisms grow exponentially, but rates of exponential growth

vary greatly. The rate of exponential growth is influenced by environmental conditions

(temperature, composition of the culture medium), as well as by genetic characteristics of

the organism itself.

3.4 Stationary Phase

In a batch culture, such as in a tube or a flask, exponential growth is limited. Typical-

ly, one or both of two things occurs to limit growth: an essential nutrient of the culture

medium is used up, or some waste product(s) of the organism accumulates in the medium

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Liu Yuanwei (SET130704) Lab Report Growth Curves

to inhibit growth. Either way, exponential growth ceases, and the population reaches the

stationary phase.

3.5 Death Phase

If incubation continues after a population reaches the stationary phase, the cells may

remain alive and continue to metabolize, but they will eventually die. When this occurs,

the population enters the death phase of the growth cycle. In some cases death is

accompanied by actual cell lysis. Occasionally, the death phase of the growth cycle is

also exponential. Typically, however, the rate of cell death is much slower than that of

exponential growth.

3.6 Measuring Microbial Growth

Microbial growth can be measured in different ways. One way is to measure the

optical density (OD) of a broth culture by spectrophotometer in time. In that way, the

change in apparent absorbency (actually the light loss due to scattering of light by the

particles) is a measure of the generation time of the population.

4. Materials and Methods

4.1 Materials

E. coli cultures pregrown at r.t. plastic cuvettes, 1 cm pathlength

and 37C photospectrometer

LB medium, preheated to r.t. and 2 shakers

37C respectively bunsen burner

pipettors (5ml, 1ml and 200l) 2x 250ml Erlenmeyer flasks

with tips dH2O

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Liu Yuanwei (SET130704) Lab Report Growth Curves

waste beaker (for autoclaving) aluminium foil

technical alcohol matches

household paper marker pens

kleenex timer

gloves ev. water quentch ev.

parafilm tripod 2 clamps

4.2 Methods

The experiment took place on October 1, 2014 in the University of Malaya biology

lab. Our professor cultured the E.coli in several plastic petri dishes a few days in advance.

To prepare for this experiment, each group of students was given one petri dish with

several E.coli colonies and four flasks with the same LB medium. Each group was

required to grow two cultures in the flasks at room temperature (20C) and 37C,

respectively. Students first gathered a single colony from the given petri dish, and

inoculated it into a flask. The other flasks were then inoculated and labeled with the

temperature, number (A, or B), and group members name. Once the inoculation process

was completed, 2-3 ml of samples from all flasks were transferred into cuvettes to

measure their initial OD at 600 nm by a spectrophotometer. At the same time, two flasks

containing E.coli were put on a shaker kept in the incubator at 37C and the rest two

flasks were left on the shaker placed in the lab (room temperature). All shakes had been

adjusted to the same revolutions per minute (RPM) before the flasks were put on them.

We measured the absorbency of the E. coli cultures at 600 nm every 15 min, as the same

procedure mentioned before. We also recorded the time and the OD at 600 nm against

uninoculated LB medium as a blank. If the OD exceeds 0.7, dilute the sample in the

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Liu Yuanwei (SET130704) Lab Report Growth Curves

cuvette with medium. After the measurement, we poured the sample into the waste

beaker and washed the cuvette, then shook out any moisture and put the cuvette upside

down on a household paper until the next measurement.

5. Results

Absorbency: OD at 600 nm by a spectrophotometer

Time (min) Room Temperature 37C
A B Mean A B Mean
0
15 0.303 0.294 0.299 0.159 0.157 0.158
30 0.147 0.149 0.148 0.132 0.141 0.137
45 0.137 0.139 0.138 0.127 0.165 0.146
60 0.121 0.109 0.115 0.537 0.342 0.440
75 0.127 0.133 0.130 0.146 0.325 0.236
90 0.139 0.124 0.132 0.125 0.126 0.126

Chart Title
0.5
0.45
0.4
0.35
0.3
Absorbency

0.25
0.2
0.15
0.1
0.05
0
0 10 20 30 40 50 60 70 80 90 100
Time / min

Room Temperature 37C

6. Discussion

In this experiment, we sought to discover the growth rate of a new inoculated microbial

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Liu Yuanwei (SET130704) Lab Report Growth Curves

population. We predicted that there were four phases during the growth cycle. However,

the results are distinct from our prediction. The number of E.coli that grew at room

temperature showed a decrease trend during the bath culture, whereas those growing at

37C had a similar tendency as the standard curve.

Population growth is measured by following changes in the number of cells or the weight

of some component of cell mass.

References
Madigan, M. T., & Martinko, J. M. (2006). Brock biology of microorganisms. Upper Saddle River,
N.J. : Pearson Prentice Hall / Pearson Education.