CH 06 Microbial Growth

9/18/2022
Microbiology an Introduction
Thirteenth Edition, Global Edition
Chapter 6
Microbial Growth
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Serratia Marcescens Bacteria
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The Requirements for Growth (2 of 3)

• Physical requirements
– Temperature
– pH
– Osmotic pressure
• Chemical requirements
– Carbon
– Nitrogen, sulfur, and phosphorous
– Trace elements
– Oxygen
– Organic growth factors
Physical Requirements (1 of 2)
• Temperature
– Minimum growth temperature
– Optimum growth temperature
– Maximum growth temperature
• Temperature
– Psychrophiles-cold-loving
– Mesophiles-moderate-temperature-loving
– Thermophiles-heat-loving
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Figure 6-1 Typical Growth Rates of Different Types

of Microorganisms in Response to Temperature
For Long description, see slide 92: Appendix 1.

Temperature (1 of 2)
• Psychrotrophs
– Grow between 0C and 20 to 30C
– Cause food spoilage
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Figure 6-2 Food Preservation Temperatures
Figure 6-3
The Effect of the Amount of Food on Its Cooling Rate in a Refrigerator and Its Chance of
Spoilage
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Temperature (2 of 2)
• Thermophiles
– Optimum growth temperature of 50 to 60C
– Found in hot springs and organic compost
• Hyperthermophiles
– Optimum growth temperature >80C
Ph
• Most bacteria grow between pH 6.5 and 7.5
• Molds and yeasts grow between pH 5 and 6
• Acidophiles grow in acidic environments
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Osmotic Pressure
• Hypertonic environments (higher in solutes than inside
the cell) cause plasmolysis due to high osmotic
pressure
• Extreme or obligate halophiles require high osmotic
pressure (high salt)
• Facultative halophiles tolerate high osmotic pressure
Figure 6-4 Plasmolysis
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The Requirements for Growth (3 of 3)
Chemical Requirements (1 of 3)
• Carbon
– Structural backbone of organic molecules
– Chemoheterotrophs use organic molecules as energy
• Autotrophs use CO 2
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• Nitrogen
– Component of proteins, DNA, and ATP
– Most bacteria decompose protein material for the
nitrogen source
– Some bacteria use NH4 or NO3  from organic
material
– A few bacteria use N2 in nitrogen fixation
• Sulfur
– Used in amino acids, thiamine, and biotin
– Most bacteria decompose protein for the sulfur source
– Some bacteria use SO4 2 or H2S
• Phosphorus
– Used in DNA, RNA, and ATP
– Found in membranes
– PO4 is a source of phosphorus

3
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Trace Elements
• Inorganic elements required in small amounts
• Usually as enzyme cofactors
• Include iron, copper, molybdenum, and zinc
Oxygen (1 of 2)
• Obligate aerobes+-require oxygen
• Facultative anaerobes-grow via fermentation or
anaerobic respiration when oxygen is not available
• Anaerobes-unable to use oxygen and most are harmed
by it
• Aerotolerant anaerobes-tolerate but cannot use oxygen
• Microaerophiles-require oxygen concentration lower
than air
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Table 6-1 the Effect of Oxygen on the

Growth of Various Types of Bacteria
Oxygen (2 of 2)
• Singlet oxygen: ( O2 ) boosted to a higher-energy
1
state and is reactive
• Superoxide radicals: O 2
O 2  O 2  2H   H 2 O 2  O 2
• Peroxide anion: O22–

2H 2 O2  2H 2 O  O2
H 2 O 2  2H   2H 2 O
• Hydroxyl radical (OH•)
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Organic Growth Factors

• Organic compounds obtained from the environment
• Vitamins, amino acids, purines, and pyrimidines
Biofilms (2 of 3)
• Microbial communities
• Form slime or hydrogels that adhere to surfaces
– Bacteria communicate cell-to-cell via quorum
sensing
– Bacteria secrete an inducer (signaling chemical) to
attract other bacterial cells
• Share nutrients
• Shelter bacteria from harmful environmental factors
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Figure 6-5 Biofilms
Biofilms (3 of 3)
• Found in digestive system and sewage treatment
systems; can clog pipes
• 1000x resistant to microbicides
• Involved in 70% of infections
– Catheters, heart valves, contact lenses, dental caries
For Long description, see slide 98:

Appendix 7.
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Culture Media (2 of 4)
• Culture medium: nutrients prepared for microbial growth
• Sterile: no living microbes
• Inoculum: introduction of microbes into a medium
• Culture: microbes growing in or on a culture medium
• Agar
– Complex polysaccharide
– Used as a solidifying agent for culture media in Petri
plates, slants, and deeps
– Generally not metabolized by microbes
– Liquefies at 100C
– Solidifies at ~ 40C
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• Chemically defined media: exact chemical composition
is known
– Fastidious organisms are those that require many
growth factors provided in chemically defined media
• Complex media: extracts and digests of yeasts, meat, or
plants; chemical composition varies batch to batch
– Nutrient broth
– Nutrient agar
Table 6-2 A Chemically Defined Medium for

Growing a Typical Chemoheterotroph, Such as
Escherichia Coli
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Table 6-3 Defined Culture Medium for

Leuconostoc Mesenteroides (1 of 2)
Table 6-3 Defined Culture Medium for

Leuconostoc Mesenteroides (2 of 2)
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Table 6-4 Composition of Nutrient Agar, a Complex

Medium for the Growth of Heterotrophic Bacteria
Constituent Amount
Peptone (partially digested protein) 5.0 g
Beef extract 3.0 g
Sodium chloride 8.0 g
Agar 15.0 g
Water 1 liter
Anaerobic Growth Media and Methods
• Reducing media
– Used for the cultivation
of anaerobic bacteria
– Contain chemicals
(sodium thioglycolate)
that combine O2 to
deplete it
– Heated to drive off O2

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Figure 6-7 An Anaerobic Chamber
Special Culture Techniques (1 of 2)

• Capnophiles
– Microbes that require high CO2 conditions
– CO2 packet
– Candle jar
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Special Culture Techniques (2 of 2)

• Biosafety levels
– BSL-1: no 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
Figure 6-8 Technicians in a Biosafety Level

4 (BSL-4) Laboratory
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Selective and Differential Media (1 of 2)

• Selective media
– Suppress unwanted microbes and encourage desired
microbes
– Contain inhibitors to suppress growth
• Differential media
– Allow distinguishing of colonies of different microbes
on the same plate
• Some media have both selective and differential
characteristics
Figure 6-9 Blood Agar, a Differential Medium

Containing Red Blood Cells
For Long description, see slide 104: For Long description, see slide
Appendix 13. 105: Appendix 14.
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Enrichment Culture
• Encourages the growth of a desired microbe by
increasing very small numbers of a desired organism to
detectable levels
• Usually a liquid
Table 6-5 Culture Media
Type Purpose
Chemically Defined Growth of chemoautotrophs and photoautotrophs; microbiological assays
Complex Growth of most chemoheterotrophic organisms
Reducing Growth of obligate anaerobes
Selective Suppression of unwanted microbes; encouraging desired microbes
Differential Differentiation of colonies of desired microbes from others
Enrichment Similar to selective media but designed to increase numbers of desired

microbes to detectable levels
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Obtaining Pure Cultures (2 of 2)

• A pure culture contains only one species or strain
• A colony is a population 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
Figure 6-11 The Streak Plate Method for

Isolating Pure Bacterial Cultures
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Preserving Bacterial Cultures (2 of 2)

• Deep-freezing: −50 to − 95C
• Lyophilization (freeze-drying): frozen (− 54 to − 72C)
and dehydrated in a vacuum
The Growth of Bacterial Cultures
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Bacterial Division
• Increase in number of cells, not cell size
• Binary fission
• Budding
• Conidiospores (actinomycetes)
• Fragmentation of filaments
Figure 6-12a Binary Fission in Bacteria
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Figure 6-12b Binary Fission in Bacteria
Generation Time
• Time required for a cell to divide
– 20 minutes to 24 hours
• Binary fission doubles the number of cells each
generation
• Total number of cells = 2number of generations
• Growth curves are represented logarithmically
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Figure 6-13a Cell Division
Figure 6-13b Cell Division
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Figure 6-14
A Growth Curve for an Exponentially Increasing Population, Plotted

Logarithmically (Dashed Line) and Arithmetically (Solid Line)
Phases of Growth
• Lag phase
• Log phase
• Stationary phase
– Bacteria approach the carrying capacity
• Death phase
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Figure 6-15 Understanding the Bacterial

Growth Curve
For Long description, see slide 112: Appendix 21
Direct Measurement of Microbial Growth
• Direct measurements-count microbial cells

– Plate count
– Filtration
– Most probable number (m PN) method eter
– Direct microscopic count
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Plate Counts
• Count colonies on plates that have 30 to 300 colonies (C
FUs)
• To ensure the right number of colonies, the original
inoculum must be diluted via serial dilution
• 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)
Figure 6-16 Serial Dilutions and Plate

Counts
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Figure 6-17 Methods of Preparing Plates for

Plate Counts
Filtration
• Solution passed through a filter that collects bacteria
• Filter is transferred to a Petri dish and grows as colonies
on the surface

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The Most Probable Number (MPN)

Method
• Multiple tube test

• Count positive tubes
• Compare with a statistical table
Figure 6-19b The Most Probable Number (M

PN) Method
For Long description, see slide 116:

Appendix 25 For Long description, see slide 117:
Appendix 26
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Direct Microscopic Count

• Volume of a bacterial suspension placed on a slide
• Average number 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
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Estimating Bacterial Numbers by Indirect

Methods
• Turbidity-measurement of cloudiness with a

spectrophotometer
• Metabolic activity-amount of metabolic product is
proportional to the number of bacteria
• Dry weight-bacteria are filtered, dried, and weighed; used
for filamentous organisms
Figure 6-21 Turbidity Estimation of Bacterial

Numbers
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Appendix 1
Temperature is on the x-axis from -10 degrees Celsius to
110 degrees Celsius. Rate of growth is on the y-axis. The
table below summarizes the data: Values approximate.
Microorganism, Temperature range in degrees Celsius,
Optimal Growth Temperature. Psychrophiles, minus 10 to
20, 10. Psychrotrophs, 0 to 30, 25. Mesophiles, 10 to 50,
35. Thermophiles, 40 to 72, 64. Hyperthermophiles, 65 to
110, 95.
Return to Presentation
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Appendix 2
A scale of temperatures and its effect on bacteria is as follows. Values
approximate. 260 to 145 degrees Fahrenheit. 130 to 60 degrees
Celsius. Effect on bacterial growth: Temperatures in this range destroy
most microbes, although lower temperatures take more time. 145 to
130 degrees Fahrenheit. 62 to 50 degrees Celsius. Effect on bacterial
growth: Very slow bacterial growth. 130 to 60 degrees Fahrenheit. 50 to
15 degrees Celsius. Effect on bacterial growth: Danger zone! Rapid
growth of bacteria; some may produce toxins. 60 to 40 degrees
Fahrenheit. 15 to 5 degrees Celsius. Effect on bacterial growth: Many
bacteria survive; some may grow. 40 to 30 degrees Fahrenheit. 5 to 0
degrees Celsius. Effect on bacterial growth: Refrigerator temperatures;
may allow slow growth of spoilage bacteria; very few pathogens. 30 to
minus 20 degrees Fahrenheit. 0 to minus 30 degrees Celsius. Effect on
bacterial growth: No significant growth below freezing.
Appendix 3
The x-axis is hours from 0 to 8; the y-axis is temperature in degrees
Celsius. from minus 10 to 80. Two curves are drawn. The first curve
corresponds to a 5 centimeter (2 inch) deep pan of rice. The
temperature of this pan exponentially decreases over time until it
reaches 0 degrees Celsius, which is equal to the temperature of the
refrigerator air (shown with a line that fluctuates around 0 degrees
Celsius). The second curve corresponds to a 15 centimeter (6 inch)
deep pan of rice. The temperature of this pan slowly decreases over
time, only reaching 15 degrees Celsius after eight hours. A darker band
within the graph shows approximate temperature range (15 to 43
degrees Celsius) at which Bacillus cereus multiples in rice.
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Appendix 4
a. Cell in isotonic solution. Under these conditions, the solute
concentration in the cell is equivalent to a solute concentration of
0.85% sodium chloride (N a C l). The bacterial cell is drawn with the
cell wall, plasma membrane, and cytoplasm labeled. There is no net
movement of water into or out of the cell. b. Plasmolyzed cell in
hypertonic solution. If the concentration of solutes such as N a C l 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.
In the drawing, the cell’s internal solute concentration remains 0.85%,
but N a C l concentration in the surrounding environment is now 10%.
As water flows out of the cell, the plasma membrane shrinks away from
the cell wall.
Appendix 5
A table has 4 rows and 6 columns. The columns have the following headings from left to right.
Category. A, Obligate Aerobes. B, Facultative Anaerobes. C, Obligate Anaerobes. D, Aerotolerant
Anaerobes, e. Microaerophiles. The row entries are as follows. Row 1. Category. Effect of Oxygen on
Growth. A, only aerobic growth; oxygen required. B, Both aerobic and anaerobic growth; greater
growth in presence of oxygen. C, Only anaerobic growth; growth ceases in presence of oxygen. D,
only anaerobic growth; but growth continues in presence of oxygen. E, only aerobic growth; oxygen
required in low concentration. Row 2. Category. Bacterial Growth in Tube of Solid Growth Medium. A,
bacteria form a cluster at the top of the medium. B, bacteria cluster at the top of the medium and
spread throughout the medium in smaller quantities. C, bacteria form a cluster at the bottom of the
medium. D, bacteria are evenly distributed throughout the medium. E, bacteria form a small cluster at
the center of the medium. Row 3. Category. Explanation of Growth Patterns. A, Growth occurs only
where high concentrations of oxygen have diffused into the medium. B, Growth is best where most
oxygen is present, but occurs throughout tube. C, Growth occurs only where there is no oxygen. D,
Growth occurs evenly; oxygen has no effect. E, Growth occurs only where a low concentration of
oxygen has diffused into medium. Row 4. Category. Explanation of Oxygen’s Effects. A, Presence of
enzymes catalase and superoxide dismutase (S O D) allows toxic forms of oxygen to be neutralized;
can use oxygen. B, Presence of enzymes catalase and S O D allows toxic forms of oxygen to be
neutralized; can use oxygen. C, lacks enzymes to neutralize harmful forms of oxygen; cannot tolerate
oxygen. D, Presence of one enzyme, S O D, allows harmful forms of oxygen to be partially
neutralized; tolerates oxygen. E, produce lethal amounts of toxic forms of oxygen if exposed to normal
atmospheric oxygen.
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Appendix 6
Water currents move, as shown by the blue arrow, among pillars of
slime formed by the growth of bacteria attached to solid surfaces. This
allows efficient access to nutrients and removal of bacterial waste
products. Individual slime-forming bacteria or bacteria in clumps of
slime detach and move to new locations. In the drawing, biofilms
(colored white) take the form of a forest-like group of pillars with puffed-
out tops; the pillars attach to a surface at their bases. Clumps of
bacteria adhere to the surface of the pillars. Water currents flow
through and around the base of the pillars in one direction. Migrating
clumps of bacteria surrounded by biofilm move through the pillars. The
scale shows 10 micrometers, or about half the size of the pillars.
Appendix 7
Serratia liquefaciens forms cluster of rod shaped cells,
exuding numerous filaments labeled capsular material. The
filaments anchor the bacteria to plastic. The scale bar
indicates 3 micrometers, which is about half the length of
one of the cells (S E M).
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Appendix 8
A table has 6 rows and 2 columns. The columns have the
following headings from left to right. Constituent, Amount.
The row entries are as follows. Row 1. Constituent,
Glucose. Amount, 5.0 grams Row 2. Constituent,
Ammonium phosphate, monobasic (N H 4 H 2 P O 4).
Amount, 1.0 gram Row 3. Constituent, Sodium chloride (N
a C l). Amount, 5.0 grams Row 4. Constituent, Magnesium
sulfate (M g S O 4, dot, 7 H 2 O). Amount, 0.2 grams Row
5. Constituent, Potassium phosphate, dibasic (K 2 H P O
4). Amount, 1.0 gram Row 6. Constituent, Water. Amount,
1 liter.
Appendix 9
The following list provides defined culture mediums. Category 1. Carbon and
energy. Glucose, 25 grams. Category 2. Salts. N H 4 C l, 3.0 grams. K 2 H P O
4, which also serves as a buffer, 0.6 grams. K H 2 P O 4, 0.6 grams. M g S O
4, 0.1 grams. Category 3. Amino Acids, 100 to 200 milligrams each. Alanine,
arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, valine. Category 4. Purines and Pyrimidines, 10
milligrams of each. Adenine, guanine, uracil, xanthine. Category 5. Vitamins,
0.01 to 1 milligram each. Biotin, folate, nicotinic acid, pyridoxal, pyridoxamine,
pyridoxine, riboflavin, thiamine, pantothenate, p-aminobenzoic acid. Category
6. Trace Elements, 2 to 10 milligrams each. F e, C o, M n, Z n, C u, N i, M o.
Category 7. Buffer, p H 7. Sodium acetate, 25 grams. Category 8. Distilled
Water, 1000 milliliters.
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Appendix 10
A table has 5 rows and 2 columns. The columns have the
following headings from left to right. Constituent, Amount.
The row entries are as follows. Row 1. Constituent,
Peptone (partially digested protein). Amount, 5.0 grams.
Row 2. Constituent, Beef extract. Amount, 3.0 grams. Row
3. Constituent, Sodium chloride. Amount, 8.0 grams. Row
4. Constituent, Agar. Amount, 15.0 grams. Row 5.
Constituent, Water. Amount, 1 liter.
Appendix 11
Inside the jar is a stack of Petri plates, the anaerobic
indicator (with methylene blue), and an envelope containing
inorganic carbonate, activated carbon, ascorbic acid, and
water. C O 2 and H 2 exit the envelope
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Appendix 12
An inset photograph shows hands working with Petri dishes
in the chamber through the arm ports.
Appendix 13
The bacterial colonies present as dark dots, surrounded by
large clear area labeled hemolysis surrounds all bacterial
colonies.
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Appendix 14
1. uninoculated (red media); 2. red media with a pink streak
labeled Staphylococcus epidermidis; and 3. a yellow streak
labeled Staphylococcus aureus with a thick margin of
yellow media.
Appendix 15
A. The diagram shows 4 sets of zigzagging streaks, each in a different
direction and taking up roughly 1 fourth of the plate. Series 1 is a line
that zigzags up and down while moving from left to right; series 2
zigzags left and right while moving downward; series 3 zigzags left to
right but moving generally downward; and series 4 zigzags up and
down while moving generally from right to left. Each set of streaks
overlaps slightly, increasing in overlap to the point where streak series
4s overlaps with half of streak series 3. B. A photograph of a Petri dish
shows the four-streak series described in part A. Streak series 1 and 2
are thick, bright red lines, and streak 3 is a thick line that varies from
red to. Streak 4 consists of red and white dots. Dots on the Petri dish
are labeled colonies.
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Appendix 16
Step 1: The cell elongates and the D N A is replicated. The
drawing shows the rod-shaped bacterial cell with the cell wall,
plasma membrane, and tangle of D N A (nucleoid) labeled. Step
2: The cell wall and plasma membrane begin to constrict. The
drawing shows a pinching in of the cell wall and plasma
membrane at the center of the cell. D N A is in both poles of the
cell. Step 3: A cross-wall forms, completely separating the two D
N A copies. The drawing shows a cell wall completely separating
the two cells, and plasma membrane in both cells surrounds D N
A. Step 4: The cells separate. The drawing shows two cells,
each with cell wall, plasma membrane, and D N A.
Appendix 17
The micrograph (T E M) shows a rod shape surrounded by
a cell wall with a partially formed cross wall extending into
the middle of the cell from both the top and bottom. D N A
(nucleoid) is present in both ends of the cell. The scale is
0.3 micrometers, or about an eighth the length of the cell.
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Appendix 18
From left to right, the columns contain the following values: Numbers of Cells,
Numbers Expressed as a Power of 2 and Visual Representation of Numbers.
The row entries are as follows: Numbers of Cells: 1, Numbers Expressed as a
Power of 2: 2 to the power 0, Visual Representation of Numbers: 1 dot.
Numbers of Cells: 2, Numbers Expressed as a Power of 2: 2 to the power 1,
Visual Representation of Numbers: 2 dots. Numbers of Cells: 4, Numbers
Expressed as a Power of 2: 2 to the power 2, Visual Representation of
Numbers: 4 dots. Numbers of Cells: 8, Numbers Expressed as a Power of 2: 2
to the power 3, Visual Representation of Numbers: 8 dots. Numbers of Cells:
16, Numbers Expressed as a Power of 2: 2 to the power 4, Visual
Representation of Numbers: 16 dots. Numbers of Cells: 32, Numbers
Expressed as a Power of 2: 2 to the power 5, Visual Representation of
Numbers: 32 dots
Appendix 19
From left to right, the columns contain the following values: Generation Number,
Numbers of Cells and Log base 10 of Number of Cells. The row entries are as follows:
Generation Number: 0, Numbers of Cells: 2 to the power 0 = 1, Log base 10 of Number
of Cells: 0. Generation Number: 5, Numbers of Cells: 2 to the power 5 = 32, Log base 10
of Number of Cells: 1.51. Generation Number: 10, Numbers of Cells: 2 to the power 10 =
1,024, Log base 10 of Number of Cells: 3.01. Generation Number: 15, Numbers of Cells:
2 to the power 15 = 32,768, Log base 10 of Number of Cells: 4.52. Generation Number:
16, Numbers of Cells: 2 to the power 16 = 65,536, Log base 10 of Number of Cells: 4.82.
Generation Number: 17, Numbers of Cells: 2 to the power 17 = 131,072, Log base 10 of
Number of Cells: 5.12. Generation Number: 18, Numbers of Cells: 2 to the power 18 =
262,144, Log base 10 of Number of Cells: 5.42. Generation Number: 19, Numbers of
Cells: 2 to the power 19 = 524,288, Log base 10 of Number of Cells: 5.72. Generation
Number: 20, Numbers of Cells: 2 to the power 20 = 1,048,576, Log base 10 of Number of
Cells: 6.02
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Appendix 20
Generation number is on the x-axis from 0 to 20. Log 10 of
number of cells is on the left side y-axis from 0 to 6.0;
number of cells is on the right side y-axis from 0 to
1,000,000. The table below summarizes the data:
Generations, Log 10 of number of cells, Number of Cells. 0,
0, 0. 5, 1.51, 32. 10, 3.01, 1024. 15, 4.52, 32,768. 16, not
available, 65,536. 17, not available, 131,072. 18, not
available, 262,144. 19, not available, 524,288. 20, 6.02,
1,048,576.
Appendix 21
1. Lag Phase: Intense activity preparing for population growth, but no increase in
population. In the graph lag phase occurs for approximately the first two hours, and
shows no increase in the number of bacteria, represented by a horizontal line. 2. Log
Phase: Logarithmic, or exponential, increase in population. Log phase occurs
approximately between two and four hours. During this phase there is an exponential
increase in bacteria number, represented by a diagonal line. 3. Stationary Phase: Period
of equilibrium; microbial deaths balance production of new cells. This phase occurs
approximately between four and seven hours. Similar to the lag phase there is no
increase in bacteria number, also represented by a horizontal line. 4. Death Phase:
Population is decreasing at a logarithmic rate. In the graph death phase begins to occur
around seven hours. There is a rapid decrease in bacteria number shown by a diagonal
line with negative slope. At the end of the death phase bacteria number is zero. Key
concepts: Bacterial populations follow a sequential series of growth phases: the lag, log,
stationary, and death phases. Knowledge of the bacterial growth curve is critical to
understanding population dynamics and population control in the course of infectious
diseases, in food preservation and spoilage, and as well as in industrial microbiology
processes, such as ethanol production.
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Appendix 22
Each test tube corresponds to a Petri dish showing varying levels of bacterial growth.
One milliliter of broth was taken from the original inoculum and added to the first tube;
from there, 1 milliliter of broth is taken from each tube and added to its successor. A
milliliter of broth is then added to each plate. Dilutions and plating colonies are
summarized. 2 Calculation: The number of colonies on the plate times reciprocal of
dilution of the sample = the number of bacteria per milliliter. (For example, if 54 colonies
are on a plate of 1:1000 dilution, then the count is 54 times 1000 = 54,000 bacteria per
milliliter in the sample). Test tube, Dilution, Plate Colony, appearance on plate. 1, 1:10,
1:10 (10 to the negative 1 power), Dense growth covers the plate. 2, 1:100, 1:100 (10 to
the negative 2 power), Growth covers the plate, but is less dense than on the first plate.
3, 1:1000, 1:1000 (10 to the negative 3 power), Dots of growth scatter across the plate. 4,
1:10,000, 1:10,000 (10 to the negative 4 power), Dots of growth are sparse. 5, 1:100,000,
1:100,000 (10 to the negative 5 power), Only three dots of growth.
Appendix 23
Methods are as follows. a. The pour plate method. b. The spread plate
method. 1, a. Inoculate the empty plate with the bacterial dilution (1.0 or
0.1 milliliters). B. Inoculate the plate containing solid medium with the
bacterial dilution (0.1 milliliters). 2, a. Add melted nutrient agar to the
plate. B. Spread inoculum over surface of the medium evenly. A rod
with a flattened edge is used to spread the inoculum. 3, a. Swirl the
plate to mix. B. Colonies grow only on the surface of the medium. A
drawing shows dots of growth on the medium; an exploded drawing
shows that the growth does not penetrate deeper than the surface of
the medium. 4, a. Colonies grow on and in the solidified medium. A
drawing shows dots of growth in the dish; an exploded drawing shows
that some growth is beneath the surface of the medium. B. not
available.
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Appendix 24
a. The bacterial populations in bodies of water can be determined by
passing a sample through a membrane filter. Here, the bacteria in a
100-milliliter water sample have been sieved out onto the surface of a
membrane filter. These bacteria from visible colonies when placed on
the surface of a suitable medium. The micrograph (S E M) shows
chains of fused green rod-shaped cells and dark spheres. The scale is
1.5 micrometers, or about the length of one rod-shaped cell. b. A
membrane filter with bacteria on its surface, as described in a, has
been placed on Endo agar. This medium is selective for gram-negative
bacteria. Lactose fermenters, such as the coliforms, form distinctive
colonies. There are 214 colonies visible, so we would record 214
bacteria per 100 milliliters in the water sample. Photograph is of a Petri-
dish containing a pink medium and spheres of growth.
Appendix 25
From left to right, the columns contain the following values: Nutrient
Medium Set (5 Tubes for Set), Inoculum Amount Added, and Number
of Positive Tubes in Each Set. The row entries are as follows: Nutrient
Medium Set (5 Tubes for Set): Set 1 (5 test tubes with brown liquid),
Inoculum Amount Added: 10 milliliters, Number of Positive Tubes in
Each Set: 5 (all 5 tubes with yellow liquid). Nutrient Medium Set (5
Tubes for Set): Set 2 (5 test tubes with brown liquid), Inoculum Amount
Added: 1 milliliter, Number of Positive Tubes in Each Set: 3 (3 tubes
with yellow liquid; 2 with brown). Nutrient Medium Set (5 Tubes for Set):
Set 3 (5 test tubes with brown liquid), Inoculum Amount Added: 0.1
milliliters, Number of Positive Tubes in Each Set: 1 (1 tube with yellow
liquid; 4 with brown)
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Appendix 26
From left to right, the columns contain the following values: Combination of Positives, M P N index per 100 milliliters,
95% Confidence Limits (Lower) and 95% Confidence Limits (Upper). The row entries are as follows: Combination of
Positives: 4 2 0, M P N index: 22, 95% Confidence Limits (Lower): 6.8, 95% Confidence Limits (Upper): 50.
Combination of Positives: 4 2 1, M P N index: 26, 95% Confidence Limits (Lower): 9.8, 95% Confidence Limits (Upper):
70. Combination of Positives: 4 3 0, M P N index: 27, 95% Confidence Limits (Lower): 9.9, 95% Confidence Limits
(Upper): 70. Combination of Positives: 4 3 1, M P N index: 33, 95% Confidence Limits (Lower): 10, 95% Confidence
Limits (Upper): 70. Combination of Positives: 4 4 0, M P N index: 34, 95% Confidence Limits (Lower): 14, 95%
Confidence Limits (Upper): 100. Combination of Positives: 5 0 0, M P N index: 23, 95% Confidence Limits (Lower):
blank, 95% Confidence Limits (Upper): 70. Combination of Positives: 5 0 1, M P N index: 31, 95% Confidence Limits
(Lower): 10, 95% Confidence Limits (Upper): 70. Combination of Positives: 5 0 2, M P N index: 43, 95% Confidence
Limits (Lower): 14, 95% Confidence Limits (Upper): 100. Combination of Positives: 5 1 0, M P N index: 33, 95%
Confidence Limits (Lower): 10, 95% Confidence Limits (Upper): 100. Combination of Positives: 5 1 1, M P N index: 46,
95% Confidence Limits (Lower): 14, 95% Confidence Limits (Upper): 120. Combination of Positives: 5 1 2, M P N index:
63, 95% Confidence Limits (Lower): 22, 95% Confidence Limits (Upper): 150. Combination of Positives: 5 2 0, M P N
index: 49, 95% Confidence Limits (Lower): 15, 95% Confidence Limits (Upper): 150. Combination of Positives: 5 2 1, M
P N index: 70, 95% Confidence Limits (Lower): 22, 95% Confidence Limits (Upper): 170. Combination of Positives: 5 2
2, M P N index: 94, 95% Confidence Limits (Lower): 34, 95% Confidence Limits (Upper): 230. Combination of Positives:
5 3 0, M P N index: 79, 95% Confidence Limits (Lower): 22, 95% Confidence Limits (Upper): 220. Combination of
Positives: 5 3 1, M P N index: 110, 95% Confidence Limits (Lower): 34, 95% Confidence Limits (Upper): 250 (this row is
highlighted). Combination of Positives: 5 3 2, M P N index: 140, 95% Confidence Limits (Lower): 52, 95% Confidence
Limits (Upper): 400. In the highlighted row, if we look up this combination in an M P N table, we find that the M P N
index per 100 milliliters is 110. Statistically, this means that 95% of the water samples that give this result contain 34 to
250 bacteria, with 110 being the most probable number.
Appendix 27
Step 1: The bacterial suspension is added here and fills the shallow volume over the
squares by capillary action. The drawing shows that the bacterial suspension is added
beneath the cover glass on a slide with parallel slots; a grid with 25 large squares is
visible beneath the suspension. Step 2: 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 times area). An exploded drawing
shows the cover glass over the bacterial suspension on the slide between slots; the
squares are between slots under the thinnest area of the film. Step 3: Microscopic count:
All cells in several large squares are counted, and the numbers are averaged. The large
square shown here has 14 bacterial cells. An exploded view of the grid shows one entire
large square with the surrounding squares (and their bacteria). Within the central large
square is a grid of 16 total small squares (arranged 4 times 4). There are 14 bacteria in
the large square. Step 4: The volume of fluid over the large square is 1 1 million 2
hundred fifty thousandth of a milliliter. If it contains 14 cells, as shown here, then there
are 14 times 1,250,000 = 17,500,000 cells in a milliliter.
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Appendix 28
The light reaches the light-sensitive detector on a
spectrophotometer; the inner number on the
spectrophotometer’s dial reads 100 (transmittance) and the
outer number reads 0 (absorbance). In a second drawing,
the light passes through a test tube containing a bacterial
suspension. Light scattered by the bacteria does not reach
the detector. Light not scattered by bacteria does reach the
detector on the spectrophotometer; the inner number on
the dial reads 20 (transmittance) and the outer number
reads 0.70 (absorbance).
47

CH 06 Microbial Growth

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Serratia Marcescens Bacteria

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The Requirements for Growth (2 of 3)

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Figure 6-1 Typical Growth Rates of Different Types

For Long description, see slide 92: Appendix 1.

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Figure 6-2 Food Preservation Temperatures

For Long description, see slide 93: Appendix 2.

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For Long description, see slide 94: Appendix 3.

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Figure 6-4 Plasmolysis

For Long description, see slide 95: Appendix 4.

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The Requirements for Growth (3 of 3)

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– Some bacteria use NH4 or NO3  from organic

– A few bacteria use N2 in nitrogen fixation

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– Some bacteria use SO4 2 or H2S

– PO4 is a source of phosphorus

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Table 6-1 the Effect of Oxygen on the

For Long description, see slide 96: Appendix 5.

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state and is reactive

• Peroxide anion: O22–

Organic Growth Factors

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Figure 6-5 Biofilms

For Long description, see slide 97: Appendix 6.

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For Long description, see slide 98:

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Table 6-2 A Chemically Defined Medium for

For Long description, see slide 99: Appendix 8.

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Table 6-3 Defined Culture Medium for

For Long description, see slide 100: Appendix 9.

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Table 6-3 Defined Culture Medium for

For Long description, see slide 101: Appendix 10.

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Table 6-4 Composition of Nutrient Agar, a Complex

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Anaerobic Growth Media and Methods

For Long description, see slide 102: Appendix 11.

Figure 6-7 An Anaerobic Chamber