Bacterial Population Growth Flashcards
Bacterial cell cycle/division
Understanding the bacterial growth curve is critical to control and prevention
Infectious disease caused by bacteria
Food spoilage
Pharmaceutical spoilage
Environmental microbial contamination
Step by step cell division:
- Cell elongates, enlarging its volume and DNA is replicated.
- Cell wall and plasma membrane begin to constrict
- Cross-wall forms, completely separating the two DNA copies
- Cell separate
What’s the purpose of cell division?
Increase in number of cells, not cell size
Procaryotes reproduce by Binary fission
Cell growth
4 Phases of bacterial population growth:
1) Lag phase
2) Log phase (or exponential)
3) Stationary phase
4) Death phase
1) Lag phase
Little or no cell division occurs
Intense metabolic activity. Individual cells increase in size
2) Log phase (or exponential)
Rapid and constant population growth (exponential manner)
Number of cells produced > Number of cells dying
3) Stationary phase
Population size begins to stabilize
Number of cells produced = Number of cells dying
- Death phase
Population size begins to decrease
Number of cells produced < Number of cells dying
What is the generation time of a cell cycle?
E. coli divides every 20 minutes.
Most bacteria divide every 1 to 3 hours.
Some bacteria (M. tubercolosis) require over 24 hours to divide.
Binary fission ______ the number of cells each generation
doubles
Biofilms - Bacteria population can grow in particular communities
Microbial communities
Form slime or hydrogels that adhere to surfaces
What does Biofilms do?
Bacteria communicate cell-to-cell
Share nutrients
Shelter bacteria from harmful environmental factors or microbicides
Where are Biofilms found?
ubiquitous in nature
found in the digestive system, dental plaqueand involved in infections. Also found in sewage treatment systems
What happens to a the number of cells?
Total number of cells = N0 x 2number of generations
where N0 is the initial cell number
x is generation
What are the The Requirements for bacterial growth?
Physical
- temp
- pH
-osmotic pressure
Chemical
- organic growth factors
- carbon source
- oxygen
- Ions, Trace, elements
- Nitrogen, sulphur and phosphate
Effects of temperature on Physical growth;
Each bacteria =
- Minimum growth temperature
- Optimum growth temperature
- Maximum growth temperature
Optimum growth temperature
Psychrophiles - “Cold-loving” (<15C)
Psychrotrophs - (20-30C)
Mesophiles - “Middle loving” (25-40oC)
Thermophiles - “Heat-loving” (50-60oC)
Hyperthermophiles - (>80oC)
How to regulate Preservation temperature?
Control of temp is ESSENTIAL for the storage of pharmaceutical products and food
pH - requirements for growth:
Most bacteria (neutrophiles) grow between pH 6.5 and 7.5
Some bacteria (acidophiles) grow in acidic environments (pH 0-5)
Rare bacteria (alkalophiles) prefer the pH range of 8.0 to 11.5
Acidic or Basic - microbial growth
Acidity inhibits most microbial growth > used for food preservation (e.g.: pickling)
Alkalinity (pH>8) inhibits microbial growth (not used for food preservation)
Osmotic pressure - physical requirements for growth
bacteria = similar reqs to human cells
Hypertonic environments (higher in solutes than inside the cell) cause plasmolysis due to high osmotic pressure (water moves from inside to outside)
Chemical reqs = Carbon
Structural backbone of all organic compounds
Obtained from organic molecules (Chemotrophs) or CO2 (Photoautotrophs)
Chemical reqs = Nitrogen, Sulfur, and Phosphorus
N > to form amino acids, DNA, and RNA
S > to form proteins and some vitamins (thiamin and biotin)
P > to form DNA, RNA, ATP, and phospholipids
Chemical reqs = Trace elements
(Fe, Cu, Zn in small amounts) used as enzyme cofactors
Chemical reqs = Organic growth factors
to form vitamins, amino acids, nitrogenous bases
Q; Salt has been used as a food preservative since ancient times prevent microbial contamination
Why might primitive civilisations have been used salt d s food preservation technique?
- ^ osmotic pressure
- HIGH conc of salt outside the cell means a HYPERtonic environment
- water moves from INSIDE to OUTSIDE the cell, causing PLASMOLYSIS
- only halophiles tolerate HIGH osmotic pressure
Chem reqs - Oxygen
why is oxygen important in structure?
Structural backbone of all organic compounds
Obligate Aerobes
Facultative anaerobes
Obligate anaerobes
Obligate Aerobes
require oxygen to live. E.g. Pseudomonas, causing infections in humans, mostly in hospital patients
Facultative anaerobes
can grow via fermentation or anaerobic respiration when oxygen is not available. Grow best in aerobic conditions. E.g. E.coli
Obligate anaerobes
do not tolerate oxygen and are harmed by it. E.g. Clostridium bacteria that cause tetanus and botulism
Bacteria culture
Define culture?
Microbes growing in/on culture medium at appropriate conditions (physical requirements)
Culture medium
Nutrients prepared for microbial growth in a laboratory (chemical requirements)
HAVE to be sterile, meaning?
not contain living microbes) and contain nutrients and incubate
Inoculum
introduction of microbes into a medium
Agar
Complex polysaccharide
Used as a solidifying agent for culture media in Petri plates
(liquefies at 100C and Solidifies at ~40C)
Generally not metabolized by microbes
What are the Specific culture Media types?
selective + differential
Selective media
Suppress unwanted microbes and encourage desired microbes
E.g. Saboraud’s Agar: 5.6pH discourages bacterial growth. Used to isolate fungi.
Differential media
Allow distinguishing of colonies of different microbes on the same plate
E.g. Blood Agar: to distinguish bacteria that destroy red blood cells (hemolysis).
Enrichment Culture
Encourages the growth of a desired microbe by increasing very small numbers of a desired organisms to detectable levels (without suppressing other microbes).
What is Pure culture?
isolating microorganisms
(individual organisms must be isolated:
Streak-plate method is commonly used)
What is Aseptic technique critical?
Procedures under suitably controlled conditions to maintain the sterility, free from external sources of contamination
Two steps to achieve pure culture; isolating micro-organisms?
(a) streaking techinque,
(b) Colony formation
Step by Step - isolating microorganisms (8 steps)
1) loop is sterilised
2) loop is inoculated
3) first set of streaks made
4) loop is sterilised
5) second set of streaks made
6) loop is sterilised
7) final set of streaks made
8) isolated colonies develop after incubation
(a) streaking techinque
sterile loop is inserted into a sample and streaked onto a plate in a pattern (e.g. 3 sectors), to obtain individual colonies.
(b) Colony formation
A population of cells arising from a single cell (also referred to as CFU, colony forming unit).
Measuring Microbial Growth is important to…
1) Diagnose bacterial infections from patient specimens (blood, urine, etc)
2) Food safety and hygiene
3) Microbiologic sampling of environmental sources
4) Assessing microbial contamination of sterile pharmaceutical products - test for sterility for Quality Assurance (YEAR 3)
5) Microbiological assessment of non-sterile pharmaceutical products (e.g. topical use preparation, herbal remedies)
Direct measurements–count microbial cells
Plate count
Filtration
Direct microscopic count
Indirect measurements–count microbial cells
Turbidity (mass)
Metabolic activity
Cell mass - Dry weight
How to complete the process of Plate count?
Count colonies (CFUs) of bacteria sample poured or spread on the surface of an agar plate
How do you ensure the right number of colonies (countable)?
The ORIGINAL inoculum MUST be DILUTED via serial dilution (sequential dilutions in a stepwise manner)
Serial dilution
he process of stepwise dilution of a solution with an associated dilution factor
e.g. 54 colonies on plate
1:1000 dilution
How many bacteria were on the original plate?
54 x 1000 = 54 000 bacteria/ml in sample
Membrane filtration
- membrane filter on a filter support
- water sample filtered through membrane filter (0.45 um)
- membrane filter removed and placed in plate containing the appropriate medium
- incubation for 24hrs
- typical colonies
Direct microscopic count is to
Rely on light microscopy and a cell counter
Placing a small amount of samples on a microscope slide with a special grid
why is stain added?
to visualise bacteria
Cells are counted and multiplied by a factor to obtain concentration.
equation of number of bacteria =
Number of cells counted / Volume of area counted
disadvantage of direct microscopic count
Difficult to distinguish live/dead bacteria
Often laborious
Only suitable with high counts
INDIRECT methods
- Turbidity/Cell mass
- Metabolic activity
- Cell mass / Dry Weight
Turbidity/Cell mass
measurement of cloudiness/optical density (linked to the cell mass) of liquid media by a spectrophotometer
Metabolic activity
amount of metabolic product is proportional to the population size
Cell mass / Dry Weight
bacteria are filtered, dried, and weighed; used for filamentous organisms
