Microbial growth requires certain physical, chemical, and energy requirements to be met. Understanding these growth requirements allows us to control microbes, especially pathogens. Key growth factors include temperature, pH, oxygen levels, and nutrients. Different microbes have varying optimal temperature, pH, and oxygen ranges. Using specialized culture media that provide appropriate conditions enables isolation and growth of microbes from clinical specimens in the laboratory, facilitating identification of pathogens.

1. Microbial growth

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

17. A chemically defined medium (synthetic medium)

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)

20. An Anaerobic
Chamber

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

25.  Plating serial
dilutions of the
specimen
 Pour plate method
 Spread plate
method

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

30. Beta-hemolysis

31. Enriched, selective, and differential media help
establish the presence of pathogens
 Many selective media are also differential media

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

38. Direct Method
Counting Bacteria
by Membrane
Filtration

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