2. 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
3. The Requirements for Growth
Physical requirements include
Temperature
pH
Osmotic pressure
Microorganisms have physical, chemical, and
energy requirements for growth
4. 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
5. Categories of
microbes based
on temperature
ranges for
growth
Human pathogens are mesophiles
(Optimum growth temperature is ~ 37C)
Effect of temperature
6. 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
7. 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
8. 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.
9. 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)
10. 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
11. Trace elements or
micronutrients are
minerals essential for
the function of
certain enzymes
Include
copper
zinc
manganese
molybdenum
Trace elements and
growth factors
12. Oxygen Requirements
Capnophiles are microbes that require higher concentration of
carbon dioxide (3-10%) in addition to low oxygen levels
13. 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
14. 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
15. 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
16. 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
18. A complex medium
Nutrient broth is the liquid version of the medium - without
agar (another example is TSB/TSA: Trypticase soy broth/agar)
19. 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)
21. 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)
22. 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!!!!
23. Health care
professionals collect
specimens according to the
CDC - Standard precautions
Sterile swabs
Needle aspiration
Intubation
Catheter
Clean catch method
Sputum
(coughing/catheter)
Biopsy
24. 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
26. Characteristics of bacterial colonies can help in the
process of identification
*
*
*
*
*
*
*
*
Mixed
culture
Pure
culture
27. 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
28. 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
29. 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
31. Enriched, selective, and differential media help
establish the presence of pathogens
Many selective media are also differential media
32.
33. 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
34. 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
35. Typical microbial growth curve
Stationary phase
Death
(decline)
phase
Log
(exponential)
phase
Lag phase
Time
Numberoflivecells(log)
When bacteria are grown in a broth, the bacterial growth curve
has four distinct phases
36. 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!!!!
37. Direct Method: Viable Plate Counts
After incubation, count colonies on plates
25 to 250 colonies - CFUs: colony-forming units
Serial dilutions of the specimen
39. 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
40. 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
41. 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