Microbial Nutrition and Growth

Microbial Population Growth

Microbiology of the Health Sciences

Objectives:
Classify microbes into five groups on the basis of preferred temperature range.

 Explain the importance of osmotic pressure to microbial growth.

 Provide a use for each of the four elements (C, N, S, P) needed in large amounts for microbial growth.

Explain how microbes are classified on the basis of O 2 needs.

Identify ways in which aerobes avoid damage by toxic forms of O2.

Describe the formation of biofilms and their potential for causing infection.

Distinguish between chemically defined and complex media.

Justify the use of each of the following: anaerobic techniques, living host cells, candle jars, selective, differential, and
enrichment media.

Define colony and CFUs and describe how pure cultures can be isolated with streak plates.

Explain how microbes are preserved by deep-freezing and lyophilization.

Distinguish between binary fission and budding.

Define generation time and explain the bacterial growth curve.

Review some direct and indirect methods of measuring bacterial cell growth.
 Factors Affecting Microbial Growth
• Physical Requirements of Microbes
• Temperature (optimal enzyme operation)
• Psychrophiles, mesophiles, thermophiles
• pH (optimal enzyme operation)
• Using buffers in media
• Molds & yeasts versus bacteria
• Chemical Requirements
• Carbon source in medium
• Nitrogen, sulfur, phosphorous, trace elements
• Oxygen requirements
• Obligate aerobes, anaerobes, facultative anaerobes
• Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes
• Culture Media for Microbes
• Chemically defined vs. complex media
• Anaerobes: reducing media/Brewer jar
• Other: animals, eggs, tissue culture, CO2
• Media types
• Selective, Differential, Enrichment
• Bacterial Population Growth
• Growth Curve: Lag, Log, Stationary, Death
• Quantifying Growth
Microbial Nutrition Growth Requirements

[INSERT FIGURE 6.1]

Temperature Growth Ranges and Food Safety

“2-40-140”
If > 2 hrs at 40-140oF, don’t eat it!

Figure 6.2
 Microbes & Oxygen

Aerobic and anaerobic bacteria can be identified by growing them in liquid culture:
1: Obligate aerobic bacteria gather at top of test tube to absorb maximal amount of oxygen.
2: Obligate anaerobic bacteria gather at bottom to avoid oxygen.
3: Facultative anaerobes gather mostly at the top, since aerobic respiration is most beneficial; but as
lack of oxygen does not hurt them, they can be found all along the test tube.
4: Microaerophiles gather at upper part of test tube, not at top. Require O2, but at low concentration.
5: Aerotolerant bacteria are not affected by oxygen, and they are evenly spread along the test tube.

From the Virtual Microbiology ClassroomFrom the Virtual Microbiology Image: Microbial oxygen requirements, Pixie
Figure 7.13
 Generation Time Under Optimal Conditions
(at 37oC)

Organism Generation
Time
Bacillus cereus 28 min

Escherichia coli 12.5 min

Staphylococcus aureus (causes many types of infections) 27-30 min

Mycobacterium tuberculosis (agent of Tuberculosis) 18 – 24 hrs

Treponema pallidum (agent of Syphilis) 30 hrs

Images: B. cereus, E. coli & S. aureus by T. Port;

TB culture, Dr. George Kubica PHIL #4428,
Treponema pallidum, Dr. Edwin P. Ewing, Jr., PHIL #836 From the Virtual Microbiology ClassroomFrom the Virtual Microbiology
 Stages in the Normal Growth Curve
Data from an entire growth
period typically
produce a curve with a series
of phases

🞂 Lag Phase
🞂 Exponential Growth Phase
🞂 Stationary Growth Phase
🞂 Rapidly Declining Phase
🞂 Death Phase
 Lag Phase
• Relatively “flat” period
• Newly inoculated cells
require a period of
adjustment, enlargement, and
synthesis
• The cells are not yet
multiplying at their maximum
rate
• The population of cells is so
sparse that the sampling
misses them
• Length of lag period varies
from one population to
another
Exponential Growth (Logarithmic or log) Phase

🞂 When the growth curve

increases geometrically
🞂 Cells reach the
maximum rate of cell
division
🞂 Will continue as long as
cells have adequate
nutrients and the
environment is
favorable
🞂 The number of cells
growing greatly out
number the number of
cells dying.
 Stationary Growth Phase
🞂 The population enters a survival
mode in which cells stop
growing or grow slowly
◦ The rate of cell inhibition or
death balances out the rate of
multiplication
◦ Depleted nutrients and
oxygen
◦ Excretion of organic acids
and other biochemical
pollutants into the growth
medium
◦ The number of cells growing
will equal the amount of cells
dying.
◦ Endospores begin to form in
this phase.
 Rapidly Declining Phase

🞂 The curve dips downward

🞂 Cells begin to die at an
exponential rate
🞂 The amount of cells dying
out numbers the amount of
cells growing.
🞂 The dead cells become
nutrients for the growing
cells.
Death Phase

🞂 The curve continues

to dips downward
🞂 Most cellular activity
stops
🞂 Endospores are
formed and released
from the parent cells.
Potential Importance of the Growth Curve

• Implications in microbial control, infection, food microbiology, and culture

technology

• Growth patterns in microorganisms can account for the stages of infection

• Understanding the stages of cell growth is crucial for working with

cultures

• In some applications, closed batch culturing is inefficient, and instead,

must use a chemostat or continuous culture system
Culture Media
🞂 Culture medium: Nutrients prepared for
microbial growth
🞂 Have to be sterile (not contain living
microbes)
🞂 Inoculum: Microbes introduced into medium
🞂 Culture: Microbes growing in/on culture
medium
🞂 Chemically defined media: Exact chemical
composition is known (for research purposes
only)
🞂 Complex media: Extracts and digests of
yeasts,
meat, or plants, e.g.:
◦ Nutrient broth
◦ Nutrient agar
◦ Blood agar
Agar
🞂 Complex polysaccharide
🞂 Used as solidifying agent for
culture media in Petri plates,
slants, and deeps
🞂 Generally not metabolized by
microbes
🞂 Liquefies at 100°C
🞂 Solidifies ~40°C
Anaerobic Culture Methods
🞂Use reducing media, containing chemicals
(e.g.: thioglycollate) that combine with O2

🞂Are heated shortly before use to drive off O2

🞂Use anaerobic jar

🞂Novel method in clinical labs:
Add oxyrase to growth media
⇒ OxyPlate (no need for anaerobic jar)

Fig 6.5
Capnophiles: Aerobic Bacteria Requiring High CO2
🞂Low oxygen, high CO2 Candle
conditions resemble those jar Fig 6.7
found in
◦ intestinal tract
◦ respiratory tract and
◦ other body tissues where
pathogens grow

🞂E.g: Campylobacter jejuni

🞂Use candle jar, CO2-
generator packets, or CO2
incubators
Selective Media and Differential Media
Selective medium: Additives
suppress unwanted and
encourage desired microbes –
e.g. EMB, mannitol salt agar
etc.
Differential medium:
changed in recognizable
manner by some bacteria ⇒
Make it easy to distinguish Mannitol

colonies of different microbes salt

– e.g. α and β hemolysis on

MacConkey agar,(LF and
NLF) mannitol salt agar.
Enrichment Media/Culture
🞂 Contain rich supply of special nutrients that promotes the growth of desired
microbe.
◦ Blood agar (NA + 5% sheep blood).
◦ Chocolate agar (NA + powdered hemoglobin)
🞂 Example: Assume soil sample contains a few phenol-degrading bacteria and
thousands of other bacteria
◦ Inoculate phenol-containing culture medium with the soil and incubate
◦ Transfer 1 ml to another flask of the phenol medium and incubate
◦ Transfer 1 ml to another flask of the phenol medium and incubate
◦ Only phenol-metabolizing bacteria will be growing
 Pure Cultures
Contain only one species or strain.
Most patient specimens and
environmental samples contain
several different kinds of bacteria
Streak-plate method is commonly used
Colony formation: A population of cells arising from a
single cell or spore or from a group of attached cells
(also referred to as CFU).
Only ~1% of all bacteria can be successfully cultured
 Streak Plate Method

3 or 4
quadrant
methods
Culturing Microorganisms

[INSERT TABLE 6.3]

Culturing Microorganisms