CH 6 & 7 - MICROBIAL GROWTH
AND CONTROL
Stephanie Lanoue
REQUIREMENTS FOR GROWTH
Learning Objectives
6-1 Classify microbes into five groups on the
basis of preferred temperature range.
6-2 Identify how and why the pH of culture
media is controlled.
6-3 Explain the importance of osmotic pressure to
microbial growth.
ENVIRONMENTAL FACTORS FOR GROWTH
(OVERVIEW)

Physical requirements
1.
2.
3.

__________________
_____
Osmotic pressure
Chemical Requirements
1.
2.
3.
CHONPKS
Trace elements-Fe, Cu, Zn, Mb
Organic growth factors
PHYSICAL REQUIREMENTS, TEMPERATURE

Temperature

Minimum growth temperature


Optimum growth temperature


The _________ temperature at which the species will grow
Species grow _______
Maximum growth temperature

The __________ temperature at which growth is possible
PHYSICAL REQUIREMENTS, TEMPERATURE




Temperature (optimal enzyme operation)
Psychrophiles, _____-loving microbes (-10oC to 20o C)
Mesophiles, __________ temperature-loving microbes (10o C to 50o
C)
 Pathogens
Thermophiles, _____-loving microbes, (40o C to 70o C)
 Extreme thermophiles (65o C to 110o C)
PHYSICAL REQUIREMENTS, PH

Most bacteria grow between pH ______ and 7.5
(best pH for most bacteria)

Molds and yeasts grow between pH ____ and 6

Acidophiles grow in ______ environments
pH 1.0 - 5.5
 Ex. Lactobacillus acidophilus, H. pylori


Alkalophiles
pH 8.5 - 11.5
 Ex. Bacillus alcalophilus

PHYSICAL REQUIREMENTS, OSMOTIC
PRESSURE (OP)

Bacteria are better adapted to _____ OP

High OP remove water from a cell


Environment is hypertonic
 High ______ or sugar concentration in environment (solute)
Plasmolysis occurs at high OP

Shrinkage of cells
Figure 6.4 Plasmolysis.
Plasma
membrane
Cell wall
Plasma
membrane
H2O
Cytoplasm
NaCl 0.85%
Cell in isotonic solution. Under these
conditions, the solute concentration in the
cell is equivalent to a solute concentration
of 0.85% sodium chloride (NaCl).
Cytoplasm
NaCl 10%
Plasmolyzed cell in hypertonic solution.
If the concentration of solutes such as NaCl
is higher in the surrounding medium than in
the cell (the environment is hypertonic), water
tends to leave the cell. Growth of the cell
is inhibited.
REQUIREMENTS FOR GROWTH
Learning Objectives
6-4 Name a use for each of the four elements
(carbon, nitrogen, sulfur, and phosphorus)
needed in large amounts for microbial
growth.
6-5 Explain how microbes are classified on the
basis of oxygen requirements.
6-6 Identify ways in which aerobes avoid damage
by toxic forms of oxygen.
CHEMICAL REQUIREMENTS

Carbon
Structure of ________ molecules
 Most important requirements for microbial growth


Nitrogen, sulfur, phosphorous


To synthesize cellular material such as _____ and
protein
trace elements, small amount of other
chemical

such as, zinc
 Function of enzymes as cofactors
We have talked about cofactors before in a previous chapter. What
is a cofactor?
CHEMICAL REQUIREMENTS, OXYGEN
 Obligate
aerobes—_______ oxygen
 Facultative anaerobes—grow via
fermentation or anaerobic respiration
when oxygen is available
 Obligate anaerobes—grow occurs where
there is ___ oxygen
Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria
CHEMICAL REQUIREMENTS, ORGANIC
GROWTH FACTORS
Organic compounds obtained from the
____________
 Vitamins, amino acids, purines, and pyrimidines

CULTURE MEDIA
Learning Objectives
6-8
Distinguish chemically defined and complex
media.
6-9
Justify the use of each of the following:
anaerobic techniques, living host cells,
candle jars, selective and differential media,
enrichment medium.
6-10 Differentiate biosafety levels 1, 2, 3, and 4.
CULTURE MEDIA

Agar





__________ polysaccharide
Used as a solidifying agent for culture media in Petri
plates, slants, and deeps
Generally ____ metabolized by microbes
Liquefies at 100C
Solidifies at ~40C
https://learning.uonbi.ac.ke/courses/SBT202/scormPackages/path_2/culture_media.jpg
CULTURE MEDIA
https://learning.uonbi.ac.ke/courses/SBT202/scormPackages/path_2/culture_media.jpg
CULTURE MEDIA
Culture medium: nutrients prepared for microbial
__________
 Sterile: ___ living microbes
 Inoculum: ___________ of microbes into a medium
 Culture: microbes growing in or on a culture
medium

CULTURE MEDIA


Chemically defined media: exact chemical
composition is ________
Complex media: extracts and digests of yeasts,
meat, or plants; chemical composition varies
batch to batch
 Nutrient _______
 Nutrient agar
Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli
SPECIAL CULTURE TECHNIQUES

Biosafety levels
BSL-1: __ special precautions; basic teaching labs
 BSL-2: lab coat, gloves, eye protection
 BSL-3: biosafety cabinets to prevent airborne
transmission
 BSL-4: sealed, negative pressure; "hot zone"


Exhaust air is filtered twice through HEPA filters
MEDIA TYPES

Selective, suppress growth of _________
bacteria

Contain inhibitors to suppress growth
1.
Mannitol _______ agar : selective for halophiles
with 7% salt (osmotic challenge) and differential
for mannitol fermenters. Selects for Gram(+) and
inhibits Gram(-) bacteria.
2.
Eosin Methylene Blue Agar (EMB) Agar:
kills Gram(+) with eosin and methylene blue,
selective for Gram(-). Differential for lactose
fermenters.
3.
McConkey Agar: suppresses Gram(+) with
crystal violet and bile salts and selects Gram(-).
Differential for lactose fermenters.
MEDIA TYPES (CONT’D)

Differential - distinguishes between ________
species (different microbes on the same plate)
Blood agar
(sheep’s blood) reveals if hemolytic
MEDIA TYPES (CONT’D)

Enrichment - __________ nutrients to favor
growth of special bacteria
Lysed red blood cells provide unique
nutrients in blood/chocolate agar
OBTAINING PURE CULTURES
Learning Objectives
6-11 Define colony.
6-12 Describe how pure cultures can be isolated
by using the streak plate method.
PURE CULTURES
• A pure culture contains only ____ species or strain
• A colony is a ___________ of cells arising from a
single cell or spore or from a group of attached cells
• A colony is often called a colony-forming unit
(CFU)
The streak plate method is used to isolate pure
cultures
THE GROWTH OF BACTERIAL CULTURES
Learning Objectives
6-14 Define bacterial growth, including binary
fission.
6-15 Compare the phases of microbial growth,
and describe their relation to generation
time.
BACTERIAL GROWTH
 Refers
to bacterial __________, not the cell
size!
 Generation time, time required for cell
________ (1 hr to 3hr, for some 24hr)
 Growth
Curve: Lag, Log, Stationary,
Death
 Quantifying Growth
BACTERIAL DIVISION, _______ FISSION
GENERATION TIME
 Time

required for a cell to ______
20 minutes to 24 hours
 Binary
fission _______ the number of cells
each generation
 Total number of cells = 2number of generations
 Growth curves are represented
logarithmically
Figure 6.13a Cell division.
Figure 6.13b Cell division.
PHASES OF GROWTH (IN ORDER)
_____ phase
 Log phase
 Stationary phase
 Death phase

Figure 6.15 Understanding the Bacterial Growth Curve.
THE GROWTH OF BACTERIAL CULTURES
Learning Objectives
6-16 Explain four direct methods of measuring
cell growth.
6-17 Differentiate direct and indirect methods of
measuring cell growth.
MEASURING BACTERIAL GROWTH

Direct measurement
1.
2.
Plate _______ of viable bacterial forming colonies
Direct microscopic count
Counts are performed on bacteria mixed into a dish with agar (pour
plate method) or spread on the surface of a plate (spread plate
method)
After incubation, count colonies on plates that have 30-300 colonies
(CFUs)
Figure 6.17 Methods of preparing plates for plate counts.
Thepour
pour
plate
method
The
plate
method
1.0 or 0.1 ml
The
Thespread
spreadplate
plate method
method
0.1 ml
Inoculate plate
containing
solid medium.
Inoculate
empty plate.
Bacterial
dilution
Spread inoculum
over surface
evenly.
Add melted
nutrient agar.
Swirl to mix.
Colonies grow
only on surface
of medium.
Colonies
grow on and
in solidified
medium.
Plate counts
DIRECT MICROSCOPIC COUNT
Volume of a bacterial suspension placed on a
________
 Average _______ of bacteria per viewing field is
calculated
 Uses a special Petroff-Hausser cell counter

Number of cells counted
Number of bacteria/ml =
Volume of area counted
Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter.
Grid with 25 large squares
Cover glass
Slide
Bacterial suspension is added here
and fills the shallow volume over the
squares by capillary action.
Bacterial
suspension
Microscopic count: All cells in
several large squares are
counted, and the numbers are
averaged. The large square
shown here has 14 bacterial cells.
Cover glass
Slide
Location of squares
Cross section of a cell counter.
The depth under the cover glass and the area
of the squares are known, so the volume of the
bacterial suspension over the squares can be
calculated (depth × area).
The volume of fluid over the
large square is 1/1,250,000
of a milliliter. If it contains 14
cells, as shown here, then
there are 14 × 1,250,000 =
17,500,000 cells in a milliliter.
Measuring by Indirect Methods
Example: Turbidity—measurement of ___________ with a spectrophotometer
More turbid = More bacteria
Light source
Spectrophotometer
Light
Blank
Scattered light
that does not
reach detector
Light-sensitive
detector
Bacterial suspension