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Microbial
Growth
Objectives
• Define microbial growth
• Distinguish between binary fission and budding
• Explain growth cycle of Population
• Classify microbes into five groups on the basis of preferred temperature range
• Explain the importance of osmotic pressure to microbial growth
• Explain how microbes are classified on the basis of O2 needs
• Identify ways in which aerobes avoid damage by toxic forms of O2
• Explain microbial growth at low and high pH
• Provide a use for each of the four elements (C, N, S, P) needed in large amounts
for microbial growth
• 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
• Review some direct and indirect methods of measuring bacterial cell growth
10/16/2019 Microbiology 2
Definition
• Microbial Growth Defined:
1. Mother or parent cell doubles in size

2. Divides into two daughter cells
• Microbial growth is defined as the increase in the number of

cells, which occurs by cell division
• Binary fission (equal cell division): A cell duplicates its
components and divides into two cells
• Budding (unequal cell division): A small, new cell

develops from surface of exisiting cell and subsequently
separates from parent cell
• Septum: A partition that grows between two daughter

cells and they separate at this location
Binary Fission
Thin section of the bacterium Staphylococcus,
undergoing binary fission
Budding
Phases of Growth
• Consider a population of organisms introduced into a
fresh, nutrient medium
• Such organisms display four major phases of growth
1. The lag phase
2. The logarithmic phase
3. The stationary phase
4. The death phase
Bacterial Growth Curve
Foundation
Illustrates the dynamics of growth Fig 6.15
Phases of growth
•Lag phase
•Exponential or
logarithmic (log) phase
•Stationary phase
•Death phase (decline phase)
Compare growth in liquid and on solid media
Lag Phase
• Organisms do not increase significantly in number
• They are metabolically active
• Grow in size, synthesize enzymes, and incorporate
molecules from medium
• Produce large quantities of energy in the form of ATP
Log Phase
• Organisms have adapted to a growth medium
• Growth occurs at an exponential (log) rate
• The organisms divide at their most rapid rat
• a regular, genetically determined interval (generation
time)
• Stationary Phase:
1. Cell division decreases to a point that new cells are
produced at same rate as old cell die.
2. The number of live cells stays constant.
• Decline (Death) Phase:
1. Condition in the medium become less and less
supportive of cell division
2. Cell lose their ability to divide and thus die
3. Number of live cells decreases at a logarithmic rate
Microbial Growth
Microbial growth: Increase in cell number, not cell size!
Physical Requirements for Growth: Temperature (cardinal
temperature)
• Minimum growth temperature
• Optimum growth temperature
• Maximum growth temperature
Five groups based on optimum growth temperature
1. Psychrophiles
2. Psychrotrophs
3. Mesophiles
4. Thermophiles
5. Hyperthermophiles
Fig. 6 .3
Temperature
Physical Requirements for Growth:
pH and Osmotic Pressure
Most bacteria grow best between pH 6.5 and 7.5: Neutrophils
Some bacteria are very tolerant of acidity or thrive in it: Acidophiles
(preferred pH range 1 to 5)
Alkaliphiles grow between pH range 9 to 11
Molds and yeasts grow best between pH 5 and 6
Hypertonic environments (increased salt or sugar) cause plasmolysis
Obligate halophiles vs.
facultative halophiles
Fig 6.4
pH
Chemical Requirements for Growth: Oxygen
O2 requirements vary greatly
Table 6.1: The Effects of Oxygen on the Growth of Various Types of Bacteria
Toxic Forms of Oxygen
• Singlet oxygen: O2 boosted to a higher-energy state
• Superoxide free radicals: O2–
Peroxide anion: O22–
• Hydroxyl radical (OH)
Chemical Requirements for Growth: Carbon, N,
S, P, etc.
• Carbon
•  Half of dry weight
• Chemoheterotrophs use organic carbon sources
• Nitrogen, Sulfur, Phosphorus

• Needed for ?
• Found in amino acids and proteins (most bacteria decompose proteins)
• S in thiamine and biotin
• Phosphate ions (PO43–) Vit B1
• Also needed K, Mg, Ca, trace elements (as cofactors), and organic growth
factors
Vit B7
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 compo-
sition 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 metabo-
lized 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)
10/16/2019 Microbiology Fig 6.5 22

Capnophiles: Aerobic Bacteria Requiring High
CO2
• Low oxygen, high CO2 conditions
resemble those found in Candle jar
• intestinal tract Fig 6.7
• 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 colonies of different
microbes – e.g.  and 
hemolysis on blood agar;
MacConkey agar, EMB, mannitol
salt agar etc.
Enrichment Media/Culture
• Encourages growth of desired microbe
• 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
Preserving Bacterial Cultures
• Deep-freezing: Rapid cooling of pure culture in suspension liquid to –50°to –
95°C. Good for several years.
• Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a
vacuum. Good for many years.
The Growth of Bacterial Cultures
Binary fission – exponential growth
Budding
Generation time – time required for cell to divide (also

known as doubling time)
Ranges from 20 min (E. coli) to > 24h (M. tuberculosis)
Consider reproductive potential of E. coli
Fig 6.13
Figure 6.12b
g=t/n
Serial Dilution and Standard Plate Counts
• Standard plate count: One method of measuring

bacterial growth
• Agar plate: A petri dish containing a nutrient

medium solidified with agar
• Serial dilutions are used to dilute the original

bacterial culture before you transfer known
volume of culture onto agar plate
Serial Dilution
Calculation of the number of bacteria per
milliliter of culture using serial dilution
• Pour plate: made by
first adding 1.0ml of
diluted culture to 9ml
of molten agar
• Spread plate: made
by adding 0.1ml of
diluted culture to
surface of solid
medium
Direct Measurements of Microbial Growth
Viable cell counts: Plate counts: Serial dilutions

put on plates CFUs form colonies
Fig 6.16
Fig 6.17
Figure 6.15, step 1
Additional Direct Measurements
1. Filtration method of choice for low counts
2. Direct microscopic count: Counting chambers
(slides) for microscope
Fig 6.20
Direct Microscopic Counts
• Another way to measure bacterial growth
• Petroff-Hausser counting chamber
• Bacterial suspension is introduced onto chamber with a calibrated pipette
• Microorganisms are counted in specific calibrated areas
• Number per unit volume is calculated using an appropriate formula
The Petroff-Hausser Counting Chamber
Most Probable Number (MPN)
• Method to estimate number of cells
• Used when samples contain too few organisms to give reliable

measures of population size by standard plate count
• Series of progressively greater dilutions
• Typical MPN test consists of five tubes of each of three volumes (e.g.
10, 1, and 0.1ml)
A MPN test: those tubes in which gas bubbles are
visible (labeled +) contain organisms
Positive carbohydrate fermentation test
+ Gas/+Acid + Acid - No Acid or Gas

Turbidity, or a cloudy appearance, is an indicator of
bacterial growth in urine in the tube on the left
Estimating Bacterial Numbers by Indirect
Methods
Spectrophotometry to measure turbidity

OD is function of cell number
Bacterial colonies viewed through the magnifying glass
against a colony-counting grid
A Spectrophotometer: This instrument can be used to
measure bacterial growth by determining the degree of light
transmission through the culture
Measuring Microbial Growth - Overview
Direct Methods Indirect Methods

•Plate counts •Turbidity
•Filtration •Metabolic activity
•MPN •Dry weight
•Direct microscopic count
Culturing Bacteria
• Culturing of bacteria in the laboratory presents two problems:
1. A pure culture of a single species is needed to study an organism’s

characteristics
2. A medium must be found that will support growth of the desired
organism
• Pure culture: a culture that contains only a single species of

organism
• Chemostat
Continuous culture, the
experimenter can control growth
rate and population density
independently of each other
• Batch culture
Closed system culture, fixed volume
The Streak Plate Method uses agar plates to
prepare pure cultures
A Streak Plate of Serratia marcescens. Note the greatly reduced
numbers of growth /colonies in each successive region
Types of Culture Media
• Natural Media: In nature, many species of microorganisms grow together in
oceans, lakes, and soil and on living or dead organic matter
• Synthetic medium: A medium prepared in the laboratory from material of

precise or reasonably well-defined composition
• Complex medium: contains reasonably familiar material but varies slightly in

chemical composition from batch to batch (e.g. peptone, a product of enzyme
digestion of proteins)
Commonly Used Media
• Yeast Extract
• Casein Hydrolysate
• Serum
• Blood agar
• Chocolate agar
Selective, Differential, and Enrichment
Media
• Selective medium: encourages growth of some
organisms but suppresses growth of others
(e.g. antibiotics)
• Differential medium: contains a constituent that

causes an observable change (e.g. MacConkey agar)
• Enrichment medium: contains special nutrients that

allow growth of a particular organism that might not
otherwise be present in sufficient numbers to allow it
to be isolated and identified
Preserved Cultures
• To avoid risk of contamination and to reduce mutation rate, stock culture
organisms should be kept in a preserved culture, a culture in which organisms
are maintained in a dormant state
1. Lyophilization
2. Frozen at -70oC
3. Refrigeration
• Reference culture (type culture): a preserved culture that maintains the

organisms with characteristics as originally defined

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Microbial

• Microbial Growth Defined:

1. Mother or parent cell doubles in size

• Microbial growth is defined as the increase in the number of

• Budding (unequal cell division): A small, new cell

• Septum: A partition that grows between two daughter

O2 requirements vary greatly

Peroxide anion: O22–

• Hydroxyl radical (OH)

• Nitrogen, Sulfur, Phosphorus

• Are heated shortly before use to drive off O2

• Use anaerobic jar

• Novel method in clinical labs:

10/16/2019 Microbiology Fig 6.5 22

• E.g: Campylobacter jejuni

Differential medium: changed

Generation time – time required for cell to divide (also

Consider reproductive potential of E. coli

• Standard plate count: One method of measuring

• Agar plate: A petri dish containing a nutrient

• Serial dilutions are used to dilute the original

Viable cell counts: Plate counts: Serial dilutions

• Petroff-Hausser counting chamber

• Bacterial suspension is introduced onto chamber with a calibrated pipette

• Microorganisms are counted in specific calibrated areas

• Number per unit volume is calculated using an appropriate formula

• Used when samples contain too few organisms to give reliable

• Series of progressively greater dilutions

+ Gas/+Acid + Acid - No Acid or Gas

Spectrophotometry to measure turbidity

Direct Methods Indirect Methods

1. A pure culture of a single species is needed to study an organism’s

• Pure culture: a culture that contains only a single species of

• Synthetic medium: A medium prepared in the laboratory from material of

• Complex medium: contains reasonably familiar material but varies slightly in

• Differential medium: contains a constituent that

• Enrichment medium: contains special nutrients that

• Reference culture (type culture): a preserved culture that maintains the

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