3 bio265 microbial growth instructor dr di bonaventura
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Transcript of 3 bio265 microbial growth instructor dr di bonaventura
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Microbial growth
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Microbial Growth
Increase in number of cells rather than an increase in size
Understanding the
requirements for microbial
growth
Allow us determine how to
control the growth of
microbes
Specifically, of those
microbes that cause
disease and food
spoilage
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The Requirements for Growth
Physical requirements include Temperature pH Osmotic pressure
Microorganisms have physical, chemical, and
energy requirements for growth
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Effect of temperature
Minimum growth temperature - microbe is able to conduct metabolism
Maximum growth temperature – microbe continues to metabolize
Optimum growth temperature – highest growth rate
Growth rate plotted against temperature
Growth of Escherichia coli on nutrient agar at three different temperature
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Categories of microbes based on temperature ranges for growth
Human pathogens are mesophiles (Optimum growth temperature is ~ 37C)
Effect of temperature
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Treponema pallidum (the
causative agent of
syphilis) likes lower
temperatures
Lesions are first seen on
exterior parts of the body
including lips, tongue, and
genitalia
Variable temperature requirements are seen in certain diseases
Chancroids
Temperature and bacterial growth
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Temperature and bacterial growth
Variable temperature requirements are seen in certain diseases
Mycobacterium leprae (the
causative agent of leprosy)
also likes lower temperatures
Leprosy is initially seen on
the extremities of the body,
like face, ears, hands, feet,
and fingers
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Effect of pH
Neutrophiles Grow best in a narrow range
around neutral pH (pH 6.5-7.5) Acidophiles
Grow best in acidic habitats Alkalinophiles
Live in alkaline soils and water
Most pathogens are neutrophiles
Helicobacter pylori (causative agent of gastric ulcers) is not an acidophile but an acid-tolerant (secretes bicarbonate and urease)
Vibrio cholerae, the cause of cholera, can thrive at a pH as high as 9.0.
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Effect of Osmotic Pressure
Isotonic Hypertonic (plasmolysis)
Osmotic pressure is the pressure exerted on bacterial cells by their environment
Hypotonic: the bacterial cell gains water and swells to the limit of its cell wall
Some opportunistic pathogens are facultative halophiles Staphylococcus aureus - colonizes the surface of the skin (salt)
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Chemical Requirements Microorganisms use a variety of chemicals (nutrients) as a source
of energy to build organic molecules and cell structures
Several core chemicals are required for bacterial growth
Chemoheterotrophs, which include pathogenic bacteria, use organic molecules as a source of carbon and energy
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Trace elements or micronutrients are minerals essential for the function of certain enzymes
Include copper zinc manganese molybdenum
Trace elements and growth factors
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Oxygen Requirements
Capnophiles are microbes that require higher concentration of carbon dioxide (3-10%) in addition to low oxygen levels
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Superoxide dismutase (SOD) converts superoxide radicals (O2-) to
molecular oxygen and hydrogen peroxide, which is also toxic
Catalase converts hydrogen peroxide (H2O2) to water and oxygen
Catalase test Phagocytic cells use toxic forms of oxygen to kill ingested pathogens
Hydrogen peroxide can be used as an antimicrobial agent
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Many of the bacteria that form our normal flora and many pathogens are facultative anaerobes
Some pathogens can be obligate anaerobes
Gas gangrene is caused by Clostridium perfringens
Exposure of this organism to air is a lethal event for the bacterium
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How do we culture microbes?
To cultivate (or culture) microorganisms
A sample (inoculum) is placed into/on broths (liquid media) and solid media
Microorganisms that grow from an inoculum are called a culture Cultures visible on solid
media as discrete units are called colonies
Petri plate
Deeps
Slants
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What criteria must a culture medium meet?
All nutrients required by bacteria in the specimen including growth factors
Sufficient moisture, properly adjusted pH of the medium, oxygen requirements
Proper temperature of incubation for growth
Sterilization and aseptic techniques are designed to minimize contamination of the specimen
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A chemically defined medium (synthetic medium)
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A complex medium
Nutrient broth is the liquid version of the medium - without agar (another example is TSB/TSA: Trypticase soy broth/agar)
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Anaerobic microbial cultures, media, and systems
Stab cultures
Reducing media are used to culture anaerobes
Contain chemicals such as thioglycollate that combines with oxygen and removes it from the medium
Anaerobic culture system (anaerobic jar or GasPakTM jar)
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An Anaerobic Chamber
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Handling and culturing clinical specimens
Clinical specimens are collected to identify a suspected
pathogen
Specimens often include microorganisms associated with
the normal microbiota
Suspected pathogen in the clinical specimen must be
isolated from the normal microbiota in culture
Several techniques can be used to isolate organisms in pure
cultures (axenic cultures)
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Handling and culturing clinical specimens
Properly collected and labeled
placed in sterile containers
promptly transported to a clinical laboratory to
avoid death of the pathogen
minimize the growth of members of the normal microbiota
transport media are often used to move specimens from one
location to another
If clinical specimens are not handled or cultured properly
Pathogenic bacteria may be missed or may not survive
leading to wrong diagnoses!!!!
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Health care professionals collect specimens according to the CDC - Standard precautions
Sterile swabs Needle aspiration
Intubation
Catheter Clean catch method Sputum
(coughing/catheter) Biopsy
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Streak-plate technique of isolation
The method of serial dilutions
Techniques to isolate microorganisms in pure cultures or axenic cultures
Pour-plate/spread-plate techniques of isolation
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Plating serial dilutions of the specimen Pour plate method Spread plate
method
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Characteristics of bacterial colonies can help in the process of identification
*
*
*
*
* *
*
*
Mixed culture
Pure culture
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Clinical implications of bacterial growth and culture media
Bacteria can be “fastidious” in a laboratory setting, Nesseria gonorrhoeae or Haemophilus influenzae
Some cannot be grown on culture media: Mycobacterium leprae (armadillos) or Treponema pallidum (rabbits)
Some others are obligate intracellular parasites (chlamydias and richettsias) and require cultures of living cells
Chocolate agar used to culture H. influenzae and N. gonorrhoeae
Enriched medium
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Enriched, selective, and differential media help establish the presence of pathogens
A selective medium contains ingredients that inhibit the growth of some organisms while encouraging the growth of others
Sabouraud dextrose agar selects for the growth of fungi while inhibits the growth of bacteria
Nutrient agar - pH 7.3 Sabouraud agar - pH 5.6
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Enriched, selective, and differential media help establish the presence of pathogens
Blood agar plate (BAP) is an enriched and differential medium, which is usually used to detect hemolytic activity
No hemolysis (gamma-hemolysis)
Alpha-hemolysis
Beta-hemolysis
Streptococcus pyogenes Streptococcus pneumoniae Enterococcus faecalis
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Beta-hemolysis
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Enriched, selective, and differential media help establish the presence of pathogens
Many selective media are also differential media
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Enriched, selective, and differential media help establish the presence of pathogens
Many selective media are also differential media
MSA (Mannitol salt agar)
High salt concentration (7.5%) to select for Staphylococcus species while inhibiting the growth of other species
Mannitol sugar in MSA helps differentiate Staphylococcus species
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Bacterial growth by binary fission – asexual reproduction
Generation time is the time required for a bacterial cell to grow and divide
Under optimal conditions, E. coli or S. aureus have a generation time of ~ 20 min
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Typical microbial growth curve
Stationary phase
Death (decline) phase
Log (exponential) phase
Lag phase
Time
Nu
mb
er
of
live
ce
lls (
log)
When bacteria are grown in a broth, the bacterial growth curve has four distinct phases
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How do we measure microbial growth?
Direct Methods
• Plate counts*
• Filtration*
• MPN
• Direct microscopic count
Indirect Methods
• Turbidity*
• Metabolic activity
• Dry weight
Working with clinical specimens can involve quantitative analysis such as assessing a significant bacteriuria - urine samples
Assessing effectiveness of disinfectants, antibiotics …… requires quantitative analysis!!!!
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Direct Method: Viable Plate Counts
After incubation, count colonies on plates 25 to 250 colonies - CFUs: colony-forming units
Serial dilutions of the specimen
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Direct Method Counting Bacteria by Membrane Filtration
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Direct Method: Counting Bacteria by Membrane Filtration
Bacteria are filtered out and retained on the surface of the filter
The filter is transferred to a culture medium
Colonies arise from the bacterial cells on the surface of the filter
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Indirect Methods Turbidity
This method uses an instrument called spectrophotometer
The amount of light hitting the detector is inversely proportional to the number of bacteria
The less light transmitted, the more bacteria in the sample
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Preserving Bacterial Cultures
Bacterial cultures are stored by slowing the cell’s metabolism Prevent exhaustion of all nutrients and excessive accumulation
of waste products
Storage for short period of time Refrigeration (weeks to months)
Long-term storage
Deep-freezing (years) Lyophilization (freeze-drying) (decades)
Involves removing water from a frozen culture using an intense vacuum. Lyophilized cultures are restored by adding them to liquid media