2. Introduction
• In this unit, we be focusing on population of
microbial cells and how their numbers
increase through growth and decrease or
disappear as a result of death.
3. Reproduction
• Microbial growth – an increase in a population
of microbes rather than an increase in size of
an individual
• Result of microbial growth is discrete colony –
an aggregation of cells arising from single
parent cell
• Reproduction results in growth
4. The Way Microorganisms Grow
• Most Bacteria elongate and
divide by binary fission
(cleavage near midpoint to
form two daughter cells of
approximately equal size).
• division exactly in half
• most common means of
bacterial reproduction
– forming two equal size
progeny
– genetically identical offspring
– cells divide in a geometric
progression doubling cell
number
5. Cont’d
• Some unicellular microorganisms, including a
few bacteria a few bacteria, replicate by
budding (forming a bubblelike growth that
enlarges and separates from the parent cell).
6. Phases of Growth
• Exponential growth can not continue for long.
The increasing number of cells uses nutrients and
produces waste at ever increase rates.
• Usually, essential nutrients runs out. Sometimes
toxic products stop further growth. Whatever the
cause, growth rate slows and eventually stops.
• Then the culture is said to pass from the
exponential phase (also called the logarithmic or
log phase) of growth to the stationary phase.
8. Stages in the normal growth curve
1. lag phase – “flat” period of adjustment,
enlargement; little growth
2. exponential or log phase – a period of
maximum growth will continue as long as
cells have adequate nutrients and a favorable
environment
3. stationary phase – rate of cell growth equals
rate of cell death caused by depleted nutrients
and O2, excretion of organic acids and
pollutants
4. death phase – as limiting factors intensify,
cells die exponentially in their own wastes
9. Factors Affecting the Growth of
Microorganisms
• There are several factors that affect the
growth of microorganisms, these are:
• Nutrients
• Temperature
• pH levels
• Moisture
• Oxygen and
• Osmotic Potential
10. Nutrients
• All microorganisms need food. The food sources can
vary, but the organisms primarily extract carbon and
nitrogen from substances such as proteins, fats and
carbohydrates. Some microorganisms seek out and
absorb such particles.
• Others may perform chemical reactions with
surrounding elements such as carbon dioxide to gain
what they need, while still others can produce their own
simple sugars through photosynthesis similar to plants.
• Nitrogen, which is used to synthesize proteins, can be
taken from the surrounding atmosphere or from other
organic matter.
11. Temperature
• In general, the higher the temperature, the more
easily microorganisms can grow up to a certain point.
Very high and very low temperatures both obstruct
the enzyme processes microorganisms depend on to
survive, but individual species of microorganisms
have grown to prefer different levels of temperature.
• Scientists usually divide them into three different
groups: psychrophiles, mesophiles and thermophiles.
Psychrophiles prefer temperatures from 0 to 5
degrees Celsius; mesophiles like it in the middle, 20-
45 degrees Celsius; and thermophiles like it hot,
thriving in temperatures around or above 55
degrees.
15. pH Levels
• Microorganisms also prefer a certain pH level in
the substance or environment in which they
grow--that is, they prefer to have particular
acidic qualities in their surroundings.
• Most microorganisms, including most human
pathogens, are neutriphils, organisms that
prefer a neutral pH level.
• Some like high pH levels, but most often, if
conditions are too acidic, then the organism's
enzymes break down.
16. Cont’d
• Acidophilies have their growth optimum
between pH 0-5.5; neutrophiles between pH
5.5-8.0; and alkaliphiles (alkalophiles),
between pH 8.0 – 11.5.
17.
18. pH
• Most bacteria prefer neutral pH (6.5-7.5)
• Molds and yeast grow in wider pH range, but
prefer pH between 5 and 6.
• Acidity inhibits most microbial growth and is used
frequently for food preservation (e.g. pickling).
• Alkalinity inhibits microbial growth, but not
commonly used for food preservation.
• Low pH also increases the effectiveness of heat
treatments. Acidic foods such as tomatoes and
fruits can be canned safely merely by boiling.
19. Moisture
• The free flow of water is vital to
microorganisms for their cells to exchange
materials and for their metabolic processes.
• All microorganisms require some level of
water, but a few can survive in low-moisture
conditions by conserving all the water they
find and by staying in a moisture-rich
environment.
• As a general rule, though, the more moisture,
the more microorganisms there will be found.
20. Oxygen
• In addition to water, microorganisms usually require
the presence of certain elements in the air--gases
that they absorb to produce needed nutrients.
Nitrogen is one necessary element, as is oxygen.
• There are many microorganisms that require an
oxygen-rich environment to survive (aerobe), but
others actually flourish in low-oxygen surroundings
(anaerobe). Between these two extremes is a wide
variety that may prefer more or less oxygen and that
will be able to flourish equally no matter how much
oxygen is present.
• Almost all multicellular organisms are completely
dependent on atmospheric oxygen for growth – that
is, they are obligate aerobes.
21. Cont’d
• Microaerophiles are damaged by the normal
atmospheric level of oxygen (20%) and require
oxygen levels in the range of 2 to 10% for growth.
• Facultative anaerobes do not require oxygen for
growth but grow better in its presence.
• Aerotolerant anaerobes grow equally well
whether oxygen is present or not. In contrast,
strict or obligate anaerobes are usually kiled in
the presence of oxygen.
22. Osmotic Potential
• This is the concentration of solutes in the environment.
Because a selectively permeable membrane separates
microorganisms from their environment, they can be
affected by changes in the osmotic concentration.
• If the solute potential of the environment is greater the
water will move out the cell and the cell will die
(hypertonic). This will cause the cell membrane to
shrink. Used to control spoilage and microbial growth.
Sugar in jelly and salt on meat.
• If the reverse occurs, water will enter the cell and
cause it to burst (hypotonic).
23. Measuring Growth
• Viable Cell Count – Counting viable cells depends
on the live cell’s ability to grow and to form a
colony or develop into a turbid culture.
• Plate counts and filtration counts depend on the
colony formation.
• Most probable number counts depends on the
development into a turbid culture.
• The above mentioned counts are examples of
viable cell count methods.
24. Measuring Growth
• Direct Methods of Measurement
• 1. Plate Count:
Most frequently used method of measuring bacterial
populations
Inoculate plate with a sample and count number of
colonies
Assumptions:
- Each colony originates from a single bacterial
count.
- Original inoculum is homogenous
- No cell aggregates are present
25. Measuring Microbial Growth
• Advantages:
- Measure viable cells (live cell)
• Disadvantages:
– Takes 24 hours or more for visible colonies to
appear.
– Only counts between 25 and 250 colonies are
accurate.
– Must perform serial dilution to get appropriate
numbers/plate.
26. Serial Dilutions are used with the Plate
Count Method to Measure Numbers of
Bacteria
27. Measuring Microbial Growth
• Plate Count (continued):
• A. Pour plate:
– Introduce a 1.0 or 0.1 ml inoculum into an empty
Petri dish.
– Add liquid nutrient medium kept at 50oC.
– Gently mix, allow to solidify, and incubate.
Disadvantages:
- Not useful for heat sensitive organisms
- Colonies appear under agar surface.
28. Measuring Microbial Growth
• B. Spread Plate:
– Introduce a 0.1 ml inoculum onto the surface of
Petri dish.
– Spread with a sterile glass rod.
Advantages: Colonies will be on surface and not
exposed to melted agar.
30. Measuring Microbial Growth
• Direct Methods of Measurement
• 2. Filtration:
– Used to measure small quantities of bacteria.
– Example: fecal bacteria in a lake or in ocean water.
– A measured volume (usually 1 to 100 ml) of
sample is filtered through a membrane filter
(typically with a 0.45 μm pore size)
– The filter is placed on a nutrient agar medium
and incubated
– Colonies grow on the filter instead of plates and
can be counted
31. Measuring Microbial Growth
• Direct Methods of Measurement
• 3. Most Probable Number (MPN):
– Used mainly to measure bacteria that will grow on
a solid medium.
– Dilute a sample repeatedly and inoculate several
broth tubes for each dilution point.
– Count the number of positive tubes (cloudy) in
each set.
– Statistical Method: Determines 95% probability
that a bacterial population falls within a certain
range.
32. Measuring Microbial Growth
• 4. Direct Microscopic Count Using a Petroff Hauser:
– A specific volume of a bacterial suspension (0.01 ml) is
placed on a microscope slide with a special grid.
– Stain is added to visualize bacteria.
– Cells are counted and multiplied by a factor to obtain
concentration.
Advantages:
- No incubation time required
Disadvantages:
- Cannot always distinguish between live and dead bacteria.
- Motile bacteria are difficult to count.
- Requires a high Concentration of bacteria (10 million/ml)
33.
34. Example
• 1 ml or 1 cm3 of a sample was placed on
microscope slide with a special grid and the
chamber calibrated to 0.01 ml. We then view
the sample on the grid and count the number
cells present in the 0.01 ml to calculate the
amount present in 1 ml.
• If 12 cells were counted on the grid calibrated
at 0.01ml, how much cell would be in 1 ml?
35. Measuring Microbial Growth
• Indirect Methods of Measurement
• 1. Turbidity:
– AS bacteria multiply in media, it becomes turbid (cloudy).
– Based on the diffraction or “scattering” of light by bacteria in a broth
culture
– Light scattering is measured as optical absorbance in a
spectrophotometer
– Optical absorbance is directly proportional to the concentration of
bacteria in the suspension
Advantages:
- No incubation time required.
Disadvantages:
- Cannot distinguish between live and dead bacteria
- Requires a high concentration of bacteria (10 – 100 million
cells/ml).
36. Cont’d
• So basically, we look at how cloudy the
medium is by passing light through the
medium and measure the light before and
after with goes through the medium.
• The more microorganisms present the more
light will be absorbed, turbidity will increase,
because less light is transmitted through the
culture, and the reading on the
spectrophotometer is higher.
38. Measuring Microbial Growth
• 2. Metabolic Activity:
- As bacteria multiply in media, they
produce certain products, such as:
- Carbon dioxide (gas)
- Acids
- Measure the rate of formation of
metabolic products.
- Expensive
39.
40. Measuring Microbial Growth
• 3. Mass determination :
- Cells are removed from a broth culture by
centrifugation (using centrifuge to rotate vessels
called centrifuge tubes at a high rate of speed,
generating a centrifugal force that pushes small
particles, including microbial cells, to the bottom
of the tube) and weighed to determine the “wet
mass.”
- The cells can be dried out and weighed to
determine the “dry mass.”
- Doesn’t distinguish live and dead cells.
41. Control of Microorganism
• 1. Sterilization:
- is the process by which all living cells are
destroyed or removed from an object or
habitat.
- A sterile object is totally free of viable
microorganisms, spores and other infectious
agents. When sterilization is achieved by a
chemical agent, the chemical is called
sterilant.
42. Cont’d
• Heat sterilization can be dry or moist. Both kill
by destroying proteins.
• But dry heat kills largely by speeding up
oxidations, which inactivate proteins.
• Moist heat on the other hand, denatures
(alter) proteins by breaking the bonds that
confer secondary and higher protein structure.
43. Cont’d
• A. Dry Heat
• In microbiological laboratory, dry-heat
sterilization is commonly used in the form of
flaming and hot-air ovens.
• Direct flaming is the process of heating an
instrument until its get red hot or running it
quickly through a flame (depending on the
material of the instrument).
• A hot-air oven may be used were severe
treatments – 171oC for 1 hour, 160oC for 2 hours,
or 121oC for 16 hours. Example sterilization of
empty glass instruments.
44. Cont’d
• B. Moist Heat Sterilization
• Moist heat in the microbiological lab is applied
either by boiling or in autoclaves.
• Moist heat is effective at lower temperatures
than dry heat, and it penetrates more quickly.
• Boiling water kills most bacterial and fungal cells
and inactivates many viruses in just a few
minutes. Endospores will survive boiling
• Autoclave is more effective than boiling because
it uses pressure to raise the temperature
considerably above that of boiling water. At a
pressure of 15 pounds per square inch, the
temperature in an autoclave reaches temperature
of 121oC (endospores are killed).
45. Control of Microorganism
• 2. Disinfection
• - Disinfection is the killing, inhibition, or
removal of microorganisms that may cause
disease; therefore disinfection is a treatment
that reduces the number of pathogens to the
level at which they pose no danger of disease.
• Disinfectants are agents, usually chemical, used
to carry out disinfection and normally used
only on inanimate (non-living) objects. A
disinfectant does not necessarily sterilize an
object because viable spores and few
microorganisms may remain.
46. Control of Microorganism
• 3. Sanitization
• This is closely related to disinfection.
• In sanitization, the microbial population is
reduced to levels that are considered safe by
health standards.
• The inanimate object is usually cleaned as well
as partially disinfected. For example, sanitizers
are used to clean eating utensils in
restaurants.
47. Control of Microorganism
• 4. Antiseptics
• It also is frequently necessary to control
microorganisms on or in living tissue with
chemical agents.
• Antisepsis is the destruction or inhibition of
microorganisms on a living tissue; it is the
prevention of infection or sepsis.
• Antiseptics are chemical agents applied to
tissue to prevent infection by killing or
inhibiting pathogen growth; they also reduce
the total microbial population.
48. Control of Microorganism
• 5. Chemotherapy
• Is the use of chemical agents to kill or inhibit
the growth of microorganisms within host
tissue.
49. Control of Microorganism
• 6. Filtration
• Microorganisms other than viruses can be removed
from liquids by filtration. Thus filtration does not, in
a strict sense, sterilize.
• But it is used to remove cellular microorganisms
from certain media, vitamin solutions, and
antibiotics that are heat sensitive.
• Rather than directly destroying contaminating
microorganisms, the filter simply removes them.
• There are two types of filters, these are; depth and
membrane filters (read up on both).
