Chapter 4 Flashcards
Microbial Growth
The growth of a population (increase in the number of cells not size) in a specific amount of time; lead by cell division
Binary Fission
A primitive form of cell division asexual reproduction) that does not use a spindle fiber apparatus:
- Bacterial cell doubles in size
- Replicates its chromosome (DNA)
- Two chromosomes attach to separates sites (opposite ends) on the plasma membrane
- A cell wall forms between the chromosomes and separates the cells, producing two daughter cells
Budding
A type of asexual reproduction in which a cell forms a bubble-like growth that enlarges and separates from the parent; a few bacteria and some eukaryotes (yeast) replicate this way
Phases of Growth (Closed System)
A microbial lab culture typically passes through 4 distinct, sequential phases of growth:
- Lag Phase
- Exponential Phase
- Stationary Phase
- Death Phase (phase of prolonged decline)
In the lab, cultures usually pass through all phases - not in nature
Lag Phase
Considerable metabolic activiity is occurring as the cells prepare to grow:
- Enzymes and proteins are synthesized to efficiently break down substances in the environment and to construct new macromolecules and ribosomes
- Length varies depending on species
- No perceivable change in numbers
Exponential (Logarithmic) Phase
Cell numbers increase exponentially:
- During each generation time, number of cells in a population increases by a factor of 2
- Slowly at first, but then extremely rapidly
- Lasts 4-10 hours; quickly use up most nutrients and metabolic byproducts accumulate to near toxic levels (oxygen depletion)
- Cells are very virulent, easily cause infection or disease, but are also most susceptible to antibiotics
Stationary Phase
The number of cells doesn’t increase, but changes within cells occur:
- Cells “realize” environmental conditions are turning detrimental to continued growth
- Cells become smaller and produce structures the provide resistance (glycocalyx, endospores, cytoplasmic inclusions)
- Signal to enter this phase may have to do with overcrowding (accumulation of metabolic byproducts, depletion of nutrients, etc.)
- Important phase when treating infections with antibiotics
Death Phase
Cells begin to die out:
- Occurs exponentially but at a low rate
- Cells have depleted intracellular ATP resources
- Not all necessarily die this way (phase of prolonged decline)
- More fit cells survive using nutrients from dying cells
Continuous Culture of Microbes
In nature, nutrients continuously enter the cell’s environment at low concentrations, and populations grow continually at a low but steady rate:
- Growth rate set by the concentration of the scarcest or limiting nutrient
- Other microbes use metabolic byproducts for their own metabolism
Colony Growth of Microbes
Liquid or agar:
- On agar, the position of the cell in the colony determines the environment it is in and what its phase is in the growth curve
- Exponential on the edges, stationary in the middle, death in the center
Generation Time
The length of time it takes one cell to divide into two cells; varies greatly between bacteria:
- Some bacteria can grow every 10 minutes if conditions are excellent
- Some can take 24 hours to finish one division
- The average can reproduce, under optimum conditions, about once every 30 minutes
Rate of Growth Formula
N+ = (No) x 2^n
- N+ = total population of cells in a given amount of time
- No = original number of cells
- n= number of cell divisions in a given amount of time
Reasons Microbes Exist in Many Environments
- Small size
- Easily dispersed
- Need only small quantities of nutrients
- Diverse in their nutritional requirements
Moisture’s Influence on Bacterial Growth
Since they are free-living, independent cells get all their nutrients by diffusion from the surrounding environment, and therefore, need water in order to grow; very resistant to desiccation; dehydration preserves food (may not kill)
Minimum Temperature
Lowest temperature that permits growth and metabolism, but usually at a very slow pace; very cold temperatures do not usually denature proteins or destroy microorganisms
Maximum Temperature
Highest temperature that permits growth and metabolism, usually at depressed rates; going above, enzymes will become denatured and metabolism will stop, destroying the cell
Optimum Temperature
Typically covers a small range in which organism’s metabolic processes and growth are fastest; why fevers (even slight) can be helpful in fighting off bacterial infections
Psychophiles (Cold-Loving)
Bacteria that will grow in temperatures from 5’ C to 20’ C:
- Will not cause infections in humans
- Responsible for the spoiling of refrigerated, and some frozen, food (e.g., blood)
- Perishable items must be frozen far below freezing to retard growth
Mesophiles (Middle-Loving)
Bacteria that will grow in temperatures from 20’ C to 50’ C:
- Optimum temperatures from human pathogens are around 37’ C
- Pathogenic bacteria
- Different organisms will grow best in different regions of the body (e.g., Mycobacterium leprae grows best on the extremities - leprosy)
Thermophiles (Heat-Loving)
Bacteria that will grow in temperatures from 50’ C to 80’ C:
- Found in natural hot springs, volcanic vents, etc.
- The autoclave is used to isolate them in a culture
Hyperthermophiles
Bacteria that will grow in temperatures about 80’ C; found in compost heaps and in boiling hot springs
Acidophiles
Bacteria that live in very acidic environments; optimal pH is below 5.5, but some can live in environments close to 0 (e.g., Helicobacter pylori, which cause ulcers in the stomach; Lactobacillus, which ferments millk)
Neutrophiles
Bacteria that grow best in pHs of 6-8; most human pathogenic bacteria have an optimum pH near 7.3
Alkalinophiles
Bacteria that live in very alkaline environments; optimal pH is above 8.5, but some can live in environments of pHs close to 10 (bleach)
Antioxidants
Enzymes used by organisms to detoxify oxygen
Superoxide Dismutase (SOD)
Enzyme that detoxifies superoxide ions (O2-)
Catalase
Enzyme that detoxifies peroxides by converting them to water and oxygen; cause hydrogen peroxide to bubble (release oxygen) when poured into a wound
Catalase Test
A clinical test used to determine if catalase is present; allows for a quick determination of an organisms capability of surviving in oxygen
Obligate Aerobes
Bacteria that typically have both superoxide dismutase and catalase and, therefore, can tolerate an oxygen rich environment:
- Conduct aerobic respiration and are totally dependent upon oxygenfor their metabolism
- Without oxygen, the microorganism will not grow, and will die
- e.g., most fungi, protozoans, some bacteria (Bacillus, Pseudomonas)
Facultative Anaerobes
Bacteria that typically have superoxide dismutase and catalase:
- Can conduct aerobic or anaerobic pathways to generate ATP
- Grow best with oxygen present but can grow and reproduce without it (slower)
- e.g., E. coli, Staphylococcus, Saccharomyces (yeast)
Obligate Anaerobes
Bacteria that usually lack both enzymes necessary to live in an oxygen rich environment:
- Conduct anaerobic pathways only (glycolysis, fermentation) to generate ATP used for growth
- Can not grow if oxygen is present
- Usually live in places that totally lack oxygen like deep mud, lakes, oceans, and the bodies of animals (the human body has many places relatively fee of oxygen - e.g., dental cavities, large intestine)
- e.g., Clostridium
Microaerophile
Bacteria that contain a small amount of catalase and superoxide dismutase, and so, can tolerate only small concentrations of oxygen (2 - 10%)
- Large amounts of oxygen are inhibitory
- Uses oxygen in catabolic pathways
- Found within the mucous lining of the hollow organs
- e.g., Helicobacter pylori (causes stomach ulcers)
Aerotolerant Anaerobe (Obligate Fermenter)
Bacteria that don’t use oxygen, but it doesn’t harm them
Hypertonic
Higher concentration of solute outside of a cell; high salt/sugar environments draw water out of the cell causing in to dry out
Hypotonic
Lower concentration of solute outside of a cell; water will move into the cell
- Bacteria have cell walls that resist osmotic pressure; pump in K+ or produce amino acids that will keep the environment within the cell hypertonic as compared to the environment
Halotolerant
Bacteria that can tolerate moderate concentrations of salt up to 10% NaCl; Stahylococcus on salty skin
Halophiles (Obligate Halophile)
Specialized bacteria that require high levels of NaCl to live; 15-30% NaCl to live, grow, and maintain their cell walls; inhabit oceans, salt lakes
Osmophiles
Bacteria that grow in environments where sugar concentrations are high
Hydrostatic Pressure
Pressure exerted by standing water (lakes, oceans, etc.):
- Some bacteria can only survive in these types of environments (ocean valleys in excess of 7000 meters)
- Necessary to keep their enzymes in the proper 3-dimensional shape; if lose shape and denature, the cell dies
Radiation
UV rays and gamma rays can cause mutations in DNA:
- Rays may damage or kill microorganisms
- Some bacteria have enzyme systems that can repair some mutations
Nutritional (Biochemical) Growth Factors
Nutrients needed by microorganisms include:
- Carbon
- Nitrogen
- Sulfur
- Phosphorus
- Vitamins
- Certain trace elements: ex. copper, iron, zinc, sodium, chloride, potassium, calcium, etc.
Media
A liquid or solid material used to grow bacteria
Liquid Media (broth)
Media good for growing large numbers of bacteria in a short amount of time
Agar
Solid media; a complex polysaccharide extracted from seaweed:
- First used by Robert Koch
- Primarily used to provide a solid surface for bacteria to grow on (isolated colonies)
- Added to a liquid media at a 1.5% concentration
Characteristics of Agar
- Not a nutrient for most bacteria; gelatin (a protein), which can be used as a nutrient, was used prior
- Melts at 100’ C and solidifies at 45’ C; bacteria can be inoculated in the liquid as it cools, before it solidifies, without killing the cells
- Can be sterilized; heat at 121’ C for at least 15 minutes at 15psi; steam pressure kills cells and endospores
- used in motility studies; added to a liquid media at a .4% concentration
Defined (Synthetic) Medium
Prepared in the lab from materials of precise or reasonably well defined composition
Complex Medium
Contains certain reasonably familiar materials but varies slightly in chemical composition from batch to batch (contains extract from beef, yeast, blood); ex. nutrient agar, nutrient broth
Selective Medium
Encourages the growth of some bacteria but suppresses the growth of others
Differential Medium
Has an ingredient that causes an observable change when a particular biochemical reaction occurs (ex. a color or pH change)
Pure Culture
Culture that contains only one species of an organism
The Streak Plate Method
- Bacteria are picked up on a sterile wire loop
- Wire is moved lightly along the agar surface, depositing streaks of bacteria
- The loop is flamed and a few bacteria are picked from the region already deposited and streaked into a new region
- Fewer and fewer bacteria are deposited as the streaking continues
- Individual organisms (cells) are deposited in the region streaked last
Indirect Measurements
Measure a property of the mass of cells and then estimate the number of microbes
Turbidity
Hold tube up to the light and look for cloudiness as evidence of growth (difficult to detect slight growth); not sensitive in terms of numbers of bacterial cells and not useful for detecting minor contaminations
Spectrophotometer
A device that can measure how much light a solution of microbial cells transmits; the greater the mass of cells in the culture, the greater its turbidity (cloudiness) and the less light that will be transmitted
Indirect Measurements Using Metabolic Activity
- The rate of formation of metabolic products, such as gases or acids, that a culture produces
- The rate of utilization of a substrate, such as oxygen, glucose, or ATP
- The rate of reduction of certain dyes (ex. methylene blue becomes colorless when reduced)
Direct Measurements
Give more accurate measurements of numbers of microbes than indirect methods
Direct Counts
Methods that determine total numbers of cells; quick and give total count - dead and living cells
Direct Microscope Count
Organisms are counted in a special glass slide under the light compound microscope
Coulter Counter
Electronic counter; rapid and accurate only if bacterial cells are the only particles present in the solution; solutions must be diluted
Viable Cell Count
Determine total number of organisms that can grow under certain conditions
Standard Plate Count
Bacterial colonies are viewed through the magnifying glass against a colony-counting grid (Quebec colony counter):
- Use serial dilutions to decrease cell numbers before counting
- Ideal number of colonies on final plate is 30-300 colonies
- Multiply colonies by serial dilutions
Filtration
A known volume of liquid or air is drawn through a membrane filter by vacuum; the pores in the filter do not allow microbial cells to pass through; the filter is placed on a solid medium and incubated; the colonies that develop are the number of viable microbial cells in the volume of liquid that was filtered; great technique for concentrating a sample
Candle Jars
Inoculated tube or plate is placed in a jar and a candle is lit before the jar is sealed
Thioglycollate Medium
Oxygen-binding agent added to the medium to prevent oxygen from exerting toxic effects on anaerobes; media is usually dispensed in sealed screw-cap tubes
Anaerobic Chamber (Brewer Jar)
A catalyst is added to a reservoir in the lid of the jar; water is added to the gas-pak; water is converted to hydrogen gas and carbon dioxide; the hydrogen gas can then bind with any oxygen in the jar to form water
