Lecture: Bacterial Growth Curve and Aseptic Techniques

Part 1: Bacterial Growth Curve

Introduction to Bacterial Growth

 Bacterial Growth refers to the increase in the number of bacterial

cells through binary fission, where one bacterial cell divides into two
identical daughter cells. The process is asexual and leads to
exponential growth under optimal conditions.

 Unlike eukaryotic cells, bacteria do not grow in size but increase in

population. This growth can be represented graphically in a bacterial
growth curve, which shows the changes in the number of live
bacterial cells over time.

 Understanding bacterial growth is important for:

o Medical microbiology: To predict infection spread and apply

the correct antimicrobial treatment.

o Food microbiology: To control spoilage and pathogenic

bacteria.

o Industrial microbiology: For optimizing conditions for large-

scale production of microbial products (e.g., antibiotics,
enzymes).

Phases of the Bacterial Growth Curve

The bacterial growth curve consists of four distinct phases, each

characterized by different physiological states of the bacterial population.

 Lag Phase:

o During the lag phase, bacteria are adjusting to their new

environment after being inoculated into fresh media. While there
is no immediate increase in cell number, the cells are
metabolically active.

o Bacteria begin synthesizing essential enzymes, proteins, and

nucleic acids needed for replication. The length of the lag phase
can vary depending on factors like the age of the culture, the
composition of the growth medium, and the environmental
conditions.
 o Key Concept: This phase is crucial for cellular adaptation but
shows no visible growth in terms of cell number.

 Log (Exponential) Phase:

o In the log phase, cells divide at a constant rate through binary

fission, leading to exponential growth. Each bacterial cell divides
into two, resulting in rapid population growth.

o The rate of growth during this phase is influenced by nutrient

availability, temperature, and oxygen concentration.

o The log phase is important for studying bacterial metabolism, as

the cells are in their most active and healthy state during this
period.

o Doubling Time: The time it takes for the bacterial population to

double in number during this phase is referred to as the doubling
time, which varies between species and growth conditions (e.g.,
E. coli has a doubling time of 20 minutes under optimal
conditions).

o Key Concept: The log phase is the ideal phase for experiments
because the bacteria are uniform in growth and health.

 Stationary Phase:

o The stationary phase occurs when the rate of cell division

slows and the number of live cells remains constant. This
happens because essential nutrients are depleted, and waste
products accumulate in the medium.

o During this phase, some cells continue to divide while others die,
resulting in a dynamic equilibrium.

o Bacteria may alter their metabolism during this phase,

sometimes producing secondary metabolites like antibiotics,
pigments, or toxins. This phase is particularly important in
industrial microbiology for the production of antibiotics.

o Key Concept: In the stationary phase, bacterial growth is limited

by environmental stressors such as nutrient depletion and waste
accumulation.

 Death Phase:
 o In the death phase, the number of viable bacterial cells
declines due to the exhaustion of nutrients and the accumulation
of toxic byproducts.

o Some bacteria may undergo autolysis, where enzymes break

down their own cell walls. Others may form endospores (in the
case of Bacillus or Clostridium) to survive harsh conditions.

o The death phase can extend over a long period, and a small
fraction of the population may survive in a dormant state.

o Key Concept: This phase reflects the eventual decline of the

bacterial population when conditions become unfavorable for
survival.

Factors Influencing Bacterial Growth

 Nutrient Availability: Bacteria require macronutrients (carbon,

nitrogen, sulfur, etc.) and micronutrients (trace elements and vitamins)
for growth. Limitation of any essential nutrient can slow or stop growth.

 Temperature: Bacteria have optimal temperature ranges:

o Psychrophiles: Grow best at cold temperatures (0-20°C).

o Mesophiles: Grow best at moderate temperatures (20-45°C);

most human pathogens fall into this category.

o Thermophiles: Thrive in hot environments (above 45°C).

 pH: Most bacteria prefer neutral pH (around 7), but some are adapted
to acidic (acidophiles) or alkaline (alkaliphiles) conditions.

 Oxygen Availability: Bacteria can be classified based on their oxygen

requirements:

o Obligate aerobes: Require oxygen for growth.

o Obligate anaerobes: Grow in the absence of oxygen.

o Facultative anaerobes: Can grow with or without oxygen.

o Microaerophiles: Require low levels of oxygen.

 Water Activity and Osmotic Pressure: Bacteria need water for

growth, and changes in osmotic pressure (e.g., high salt
concentrations) can inhibit growth.
Applications of the Bacterial Growth Curve

 Medical Microbiology: The growth curve helps in understanding the

progression of bacterial infections. Antibiotic treatment is often most
effective during the log phase when bacteria are actively dividing.

 Food Microbiology: The bacterial growth curve is essential for

predicting spoilage rates and food safety. Refrigeration and
preservatives slow bacterial growth by keeping bacteria in the lag or
stationary phase.

 Industrial Microbiology: Industries use the bacterial growth curve to

optimize the production of microbial products. For example, antibiotic
production often occurs during the stationary phase.

Part 2: Aseptic Techniques

Introduction to Aseptic Technique

 Aseptic technique refers to a set of practices used in microbiology to

prevent contamination of cultures and the environment. It is essential
for ensuring accurate results in experiments, preventing contamination
of bacterial cultures, and protecting lab personnel from pathogenic
microbes.

 Aseptic techniques are widely used in:

o Clinical microbiology (e.g., handling of patient samples and

culture of pathogens).

o Laboratory research (e.g., maintaining pure bacterial cultures).

o Industrial microbiology (e.g., ensuring sterile conditions for

fermentation processes).

Principles of Aseptic Technique

 Sterilization:

o Heat sterilization:

 Autoclaving: Uses moist heat (steam under pressure) to

sterilize media, instruments, and waste (typically at 121°C
for 15 minutes).
  Dry heat: Used for sterilizing glassware in a hot air oven
(at 160-170°C for 2 hours).

o Chemical sterilization: Chemicals like ethanol, bleach, or

hydrogen peroxide are used to sterilize surfaces and equipment
that cannot withstand heat.

o Filtration: Filters with pore sizes of 0.22 µm are used to remove

bacteria from heat-sensitive solutions (e.g., vitamins, antibiotics).

 Disinfection:

o Alcohol: 70% ethanol or isopropanol is commonly used for

surface disinfection in the lab.

o Phenolics and Quaternary Ammonium Compounds: These

are used for disinfecting lab benches and equipment.

o Proper Surface Cleaning: Disinfection of work surfaces is

crucial before and after working with microbial cultures to reduce
the risk of contamination.

 Personal Protective Equipment (PPE):

o Lab coats, gloves, and sometimes face masks are essential to

protect both the experimenter and the microbial cultures from
contamination.

o Handwashing before and after experiments minimizes the

transfer of contaminants.

Aseptic Techniques in Practice

 Handling Cultures:

o Use sterilized equipment such as inoculating loops, needles, and

pipettes. Loops and needles should be sterilized by passing them
through a Bunsen burner flame before and after use.

o Avoid direct contact with the culture medium or sterilized

instruments.

 Inoculation Techniques:

o Streak Plate Method: This technique is used to isolate pure

colonies from a mixed bacterial culture by streaking the sample
across the surface of an agar plate.
 o Pour Plate Method: Involves mixing a sample with molten agar
and pouring it into a Petri dish, allowing colonies to grow within
the medium.

o Spread Plate Method: A small volume of diluted bacterial

culture is spread evenly across the surface of an agar plate using
a sterile spreader.

 Minimizing Exposure to Air:

o To avoid airborne contaminants, culture tubes and plates should

be opened only briefly and near a flame (to create an aseptic
zone). The mouth of tubes should be flamed before closing them
to sterilize the air inside.

Common Errors in Aseptic Technique and How to Avoid Them

 Touching sterile tools: Contaminating loops, pipettes, or culture

plates by touching them with non-sterile surfaces (gloves, hands) is a
common error.

o Always flame the inoculating loop before use and keep tools
covered when not in use.

 Prolonged air exposure: Leaving culture plates open for too long can
lead to contamination by airborne microorganisms.

o Minimize the time that plates and tubes are open, and perform
procedures near a flame if possible.

 Improper sterilization of loops: Loops that are not flamed until red-
hot can still carry contaminants.

o Ensure the entire loop or needle is passed through the flame to

sterilize it completely.