Chapter 7

Microbial Growth

When we talk about microbial growth, we are really referring to the number of cells, not the size
of the cells. Microbes that are “growing” are increasing in number, accumulating into colonies (groups of
cells large enough to be seen without a microscope) of hundreds of thousands of cells or populations of
billions of cells. Although individual cells approximately double in size during their lifetime, this change is
not very significant compared with the size increases observed during the lifetime of plants and animals.
Microbial populations can become incredibly large in a very short time. By understanding the
conditions necessary for microbial growth, we can determine how to control the growth of microbes that
cause diseases and food spoilage. We can also learn how to encourage the growth of helpful microbes
and those we wish to study.
In this chapter we will examine the physical and chemical requirements for microbial growth, the
various kinds of culture media, bacterial cell division, the phases of microbial growth, and the methods of
measuring microbial growth.

REQUIREMENTS FOR GROWTH

Learning Objectives:
a. Classify microbes on the basis of pH requirements, preferred temperature range, and oxygen
requirements
b. Explain the importance of osmotic pressure to microbial growth
c. Provide a use for each of the four elements (carbon, nitrogen, sulfur, and phosphorus) needed in
large amounts for microbial growth

Growth Factors - Microbes can exist in a great many environments because they are small, easily
dispersed, need only small quantities of nutrients, are diverse in their nutritional requirements.

A. Physical Factors

1. pH – bacteria can classified as:

a. acidophiles (acid-loving) – grow best at a pH of 1 to 5.4; Ex. Lactobacilllus (ferments milk)

b. neutrophiles – exist from pH to 5.4 to 8.5; most bacteria that cause human disease are in
this category.
c. alkaliphiles (base loving) – exist from pH to 7.0 to 11.5; ex. Vibrio cholerae (causes cholera)

2. Temperature – bacteria can be classified as:

a. psychrophiles (cold-loving) 15oC to 20oC; some can grow at 0oC.

b. mesophiles - grow best between 25oC and 40 C; human body temp is 37oC.
c. thermophiles (heat-loving) – 50oC to 60oC; found in compost heaps and in boiling hot springs.
3. Moisture – only the spores of sport-forming bacteria can exist in a dormant state in a dry environment.

4. Hydrostatic pressure – pressure exerted by standing water (ex. lakes, oceans, etc.); some bacteria can
only survive in high hydrostatic pressure environments (ex. ocean valleys in excess of 7000 meters); the
high pressure is necessary to keep their enzymes in the proper 3-D shape; without it, the enzymes lose
their shape and denature and the cell dies.

5. Tonicity (hypotonic, hypertonic, isotonic) – The use of salt as a preservative in curing meats and the
use of sugar in making jellies is based on the fact that a hypertonic environment kills or inhibits microbial
growth. Halophiles (salt lovers) inhabit the oceans.

6. Radiation – UV rays and gamma rays can cause mutations in DNA and even kill microorganisms. Some
bacteria have enzyme systems that can repair some mutations.

B. Oxygen Requirements

1. strict or obligate anaerobes – oxygen kills the bacteria; ex. Clostridium tetani
2. strict or obligate aerobes – lack of oxygen kills the bacteria; ex. Pserdomonas
3. facultative anaerobes – can shift their metabolism (anaerobic if oxygen is absent or aerobic if
oxygen is present); ex. E. coli, Staphylococcus
4. aerotolerant – the bacteria don’t use oxygen, but oxygen doesn’t harm them; ex. Lactobacillus
 5. microaerophiles – like low oxygen concentrations and higher carbon dioxide concentrations;
ex. Campylobacter

C. Nutritional (Biochemical) Factors – Nutrients needed by microorganisms include:

Carbon – carbon containing compounds are needed as an energy source (ex. glucose) and for
building blocks.

Nitrogen - needed for amino acids and nucleotides; some can synthesize all 20 amino acids;
others have to have some provided in their medium.

Sulfur – needed for amino acids, coenzymes,

Phosphorus – needed for ATP, phospholipids, and nucleotides

Vitamins – a vitamin is an organic substance that an organism requires in small amounts and that
is typically used as a coenzyme; many bacteria make their own, but some are required in the
medium; microbes living in the human intestine manufacture vitamin K, needed for blood clotting,
and some of the B vitamins, thus benefiting their host.

Certain trace elements – ex. copper, iron, zinc, sodium, chloride, potassium, calcium, etc.; often
serve as cofactors in enzymatic reactions.
CULTURE MEDIA
Learning Objectives:
a. Distinguishing between chemically defined and complex media
b. Justify the use of each of the following: anaerobic techniques, candle jars

A nutrient material prepared for the growth of microorganisms in a laboratory is called a

culture medium. Some bacteria can grow well on just about any culture medium; others require
special media, and still others cannot grow on any nonliving medium yet developed. Microbes
that are introduced into a culture medium to initiate growth are called an inoculum. The microbes
that grow and multiply in or on a culture medium are referred to as a culture.
Suppose we want to grow a culture of a certain microorganism, perhaps the microbes
from a particular clinical specimen. What criteria must the culture medium meet? First, it must
contain the right nutrients for the specific microorganism we want to grow. It should also contain
sufficient moisture, a properly adjusted pH, and a suitable level of oxygen, perhaps none at all.
The medium must initially be sterile—that is, it must initially contain no living microorganisms—
so that the culture will contain only the microbes (and their offspring) we add to the medium.
Finally, the growing culture should be incubated at the proper temperature.
A wide variety of media are available for the growth of microorganisms in the laboratory.
Most of these media, which are available from commercial sources, have premixed components
and require only the addition of water and then sterilization. Media are constantly being
developed or revised for use in the isolation and identification of bacteria that are of interest to
researchers in such fields as food, water, and clinical microbiology.
When it is desirable to grow bacteria on a solid medium, a solidifying agent such as agar
is added to the medium. A complex polysaccharide derived from a marine alga, agar has long been
used as a thickener in foods such as jellies and ice cream.
Agar has some very important properties that make it valuable to microbiology, and no
satisfactory substitute has ever been found. Few microbes can degrade agar, so it remains solid.
Also, agar liquefies at about 100°C (the boiling point of water) and at sea level remains liquid until
the temperature drops to about 40°C. For laboratory use, agar is held in water baths at about
50°C. At this temperature, it does not injure most bacteria when it is poured over. Once the agar
has solidified, it can be incubated at temperatures
approaching 100°C before it again liquefies; this property is particularly useful when thermophilic
bacteria are being grown.
Agar media are usually contained in test tubes or Petri dishes. The test tubes are called
slants when their contents are allowed to solidify with the tube held at an angle so that a large
surface area for growth is available. When the agar solidifies in a vertical tube, it is called a deep.
Petri dishes, named for their inventor, are shallow dishes with a lid that nests over the bottom
to prevent contamination; when filled, they are called Petri (or culture) plates.
 Slants Deep

A. Types of Media

1. Synthetic medium – prepared in the lab from materials of precise or reasonably well-defined
composition.

2. Complex medium – contains certain reasonably familiar materials but varies slightly in
chemical composition from batch to batch (contains extracts from beef, yeasts, blood); ex.
nutrient agar, nutrient broth

B. Selective & Differential Media (we will learn about these in detail in lab!)

1. Selective – one that encourages the growth of some bacteria but suppresses the growth of
others.

2. Differential – has an ingredient that causes an observable change in the medium when a
particular biochemical reaction occurs (ex. a color or pH change).

C. Controlling Oxygen Content of Media

1. Candle jars – the inoculated tube or plate is placed in a jar; a candle is lit before the jar is
sealed; the burning candle uses the oxygen in the jar and adds carbon dioxide to it; when the
carbon dioxide extinguishes the flame, condition are optimum for the growth of
microorganisms that require small amounts of carbon dioxide (ex. Neisseria gonorrhoeae)
2. Thioglycollate medium – oxygen-binding agent added to the medium to prevent oxygen from
exerting toxic effects on anaerobes; media is usually dispensed in sealed screw-cap tubes.

3. Anaerobic Chamber (Brewer Jar) – A catalyst is added to a reservoir in the lid of the jar. Water
is added to the gas-pak. Water is converted into hydrogen gas and carbon dioxide. The hydrogen
gas can then bind with any oxygen in the jar to form water. A methylene blue test strip is included
in the jar to ensure that anaerobic conditions are reached. When oxidized (oxygen is present) the
strip is blue; when reduced (no oxygen), the strip is clear.

H2O -----------> CO2 + H2

H2 + O2 -----------> H2O
CULTURING BACTERIA
Learning Objectives:
a. Define colony
b. Describe how pure cultures can be isolated by using t streak plate method

Most infectious materials, such as pus, sputum, and urine, contain several different kinds of bacteria;
so do samples of soil, water, or food. If these materials are plated out onto the surface of a solid medium,
colonies will form that are exact copies of the original organism. A visible colony theoretically arises from
a single spore or vegetative cell or from a group of the same microorganisms attached to one another in
clumps or chains. Estimates are that only about 1% of bacteria in ecosystems produce colonies by
conventional culture methods. Microbial colonies often have a distinctive appearance that distinguishes
one microbe from another (see Figure 6.10). The bacteria must be distributed widely enough so that the
colonies are visibly separated from each other.
 Colony

A. Methods of Obtaining Pure Cultures (a culture that contains only 1 species of organism)

1. The Streak Plate Method – Bacteria are picked up on a sterile wire loop, and the wire is moved lightly
along the agar surface, depositing streaks of bacteria on the surface. The loop is flamed and a few bacteria
are picked up from the region already deposited and streaked onto a new region. Fewer and fewer
bacteria are deposited as the streaking continues, and the loop is flamed after each streaking. Individual
organisms (individual cells) are deposited in the region streaked last. After the plate is incubated at a
suitable growth temperature for the organism, small colonies (each derived from a single bacterial cell)
appear. The loop is used to pick up a portion of an isolated colony and transfer it to another medium for
study. The use of aseptic technique assures that the new medium will contain organisms of a single
species. We’ll do this in lab.
 Streak Plate Method

GROWTH OF MICROBIAL CULTURE

Learning Objectives:
a. Define bacterial growth including binary fission
b. Compare the phases of microbial growth, and describe their relation to generation time

Two Types of Asexual Reproduction in Microbes:

1.) Binary Fission - Bacterial reproduction occurs through fission, a primitive form of cell division
that does not employ a spindle fiber apparatus. [A spindle fiber apparatus made of protein
filaments is responsible for moving the chromosomes around during cell division (mitosis &
meiosis) in most eukaryotic cells. Bacteria do not have these structures.] The bacterial cell
doubles in size and replicates its chromosome. Following DNA replication, the two
chromosomes attach to separate sites on the plasma membrane, and the cell wall is laid
down between them, producing two daughter cells.
 Binary Fission

2.) Budding - A few bacteria and some eukaryotes (including yeasts) may also replicate by
budding, forming a bubble-like growth that enlarges and separates from the parent cell.

A. Phases of Growth - A microbial lab culture typically passes through 4 distinct, sequential phases of
growth that form the standard bacterial growth curve: (Not all growth phases occur in all cultures). See
graph; be able to draw & label.

1. Lag Phase - In the lag phase, the number of cells doesn't increase. However, considerable
metabolic activity is occurring as the cells prepare to grow. (This phase may not occur, if the
cells used to inoculate a new culture are in the log phase & provided conditions are the same).

2. Log Phase (logarithmic or exponential phase) - cell numbers increase exponentially; during
each generation time, the number of cells in the population increases by a factor of two). The
number of microbes in an exponentially increasing population increases slowly at first, then
extremely rapidly. Organisms in a tube of culture medium can maintain log growth for only a
limited time, as nutrients are used up, metabolic wastes accumulate, microbes suffer from oxygen
depletion.

3. Stationary Phase - The number of cells doesn't increase, but changes in cells occur: cell become
smaller and synthesize components to help them survive longer periods without growing (some
may even produce endospores); the signal to enter this phase may have to do with overcrowding
(accumulation of metabolic byproducts, depletion of nutrients, etc.).
 4. Death Phase - In this phase, cells begin to die out. Death occurs exponentially, but at a low
rate. Death occurs because cell have depleted intracellular ATP reserves. Not all cells necessarily
die during this phase!

Bacterial Growth Curve

B. Continuous Culture of Microbes

In the lab, cultures usually pass through all growth phases - not in nature. In nature, nutrients
continuously enter the cell's environment at low concentrations, and populations grow continually at a
low but steady rate. The growth rate is set by the concentration of the scarcest or limiting nutrient, not
by the accumulation of metabolic byproducts - in nature there is always some other microbe that can use
these metabolic byproducts for their own metabolism. In the lab, we have to continually replace the
media.

MEASURING NUMBERS OF MICROBES

Learning Objectives:
a. Explain four direct methods of measuring cell growth

A. Indirect Measurements (measure a property of the mass of cells and then ESTIMATE the number of
microbes)

1. Turbidity – Can hold tube up to the light and look for cloudiness as evidence of growth (difficult
to detect slight growth). A spectrophotometer can measure how much light a solution of
 microbial cell transmits; the greater the mass of cells in the culture, the greater its turbidity
(cloudiness) and the less light that will be transmitted. Disadvantages: Not sensitive in terms of
numbers of bacterial cells & not useful for detecting minor contamination.

2. Metabolic Activity - 3 ways:

a. The rate of formation of metabolic products, such as gases or acids, that a culture
produces.

b. The rate of utilization of a substrate, such as oxygen or glucose.

c. The rate of reduction of certain dyes. Ex. methylene blue becomes colorless when
reduced.

B. Direct Measurements - Give more accurate measurements of numbers of microbes.

1. Direct Counts - Coulter Counter - electronic counter; rapid & accurate only if bacterial cells
are the only particles present in the solution. [gives a total count - live & dead cells].

3. Plate Count – most common used methods.

 It measures the number of viable cells

 It takes time for visible colonies to form
 When a plate count is performed, it is important that only limited number of
colonies develop in a plate (25-250)
 To ensure that some colony counts will be within range, the original inoculum is
diluted several times in a process called “serial dilution”

Plate Counts: Perform serial dilutions of a sample

4. Filtration - A known volume of liquid or air is drawn through a membrane filter by vacuum.
The pores in the filter are too small for microbial cells to pass through. Then the filter is placed
on an appropriate solid medium and incubated. The number of colonies that develop is the
number of viable microbial cell in the volume of liquid that was filtered. This technique is great
for concentrating a sample, ex. a swimming pool, where small populations may go undetected
using some other methods. [gives a viable count]