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chapter 6

Terms in this set (99)

Microbial Growth
Defined not in cell size but as the increase in the number of cells, cell division
Binary fission
A cell duplicates its components and divides into two cells
Mother or Parent cell
doubles in size and divides into two daughter cells
prokaryotic cells cycle
P cells have no cell cycles. DNA synthesis is continuous as cells continue to divide. Duplicated chromosome is attached to internal plasma membrane - each copy goes to new daughter cell
Describe Binary Fission
Chromosome duplicated
Septum begins to form (partition between daughter cells)
Cell division occurs
2 identical daughter cells produced (clones)
Generation Time
The time it takes for the population to double . time varies greatly depending on the organism
E. coli generation time
20 min
Mycobacterium tuberculosis generation time
12-24 hours
BActeria growth curve
Applies to bacteria freshly added to a nutrient liquid medium
List 4 major phases of growth
lag phase
logarithmic phase
stationary phase
death phase
Describe Lag Phase
cells are transferred to a nutritive medium. Cells are metabolically active. Synthesize enzymes , ribosomes, nucleic acid. Produce ATP and increase in cell size. Prepration for cell division, but no increase in bacterial number yet. Cells are still adjusting to new conditions
Describe Log Phase
Growth occurs at exponential (log) rate;organisms are dividing at their fastest rate. 2 n cells present after n doublings 2,4,8,16,32,.64 etc.
Log phase generation time
time required for each doubling
unique to each species
unique to the environmental conditions (nutrients , atmosphere)
Nonsynchronous Growth
appears as a smooth line, not as steps, on a graph. in log phase
Non-synchronous Growth( Log Phase)
All cells in a population are growing and dividing at different time intervals.Creates a smooth increase in cell numbers. Log phase = most rapid period of growth
Describe Stationary Phase
Cell division slows as the environment changes (fewer nutrients,pH and oxygen levels change) Some cells dying, some cells dividing: Total number oc cells remain about constant.
Describe Deline (death ) Phase
Conditions in the medium can no longer continue to support cell division
Cells can no longer divide, therefore , they die. Number of live cells decreases at a logarithmic rate
Continuous Culture methods;
microbes growing continuously in a chemostat.Fresh media is added continuously to support log phase growth
Bacteria Growth on Solid Surface
Colony "pile" of bacteria growing on solid surface
a. all cells descendents of original cell(clones)
b. Method used to obtain a Pure culture
All phases in the Growth curve evident in the colony
a. older cells in death phase in center: less access to nutrients
b. rapid growth occurring at edges
Culturing Bacteria
Identification of bacteria in the laboratory requires a pure culture.
Pure culture
a population of organisms descended from a single cell
Medium
a medium must be found that will support growth of the desired organism
Bacteria Indentification
made from the biochemical pattern exhibited by the organism
Methods of Culturing Bacteria
Allows for propagation of bacterial cells in culture.
Produces artificially high numbers of cells..Agar slant liquid broth, petri plate
Method of obtaining Pure Culture
Streak Plate Method
Pour Plate Method
Pour Plate method
Liquid medium at the end of a serial dilution is poured into a plate. Colonies form on agar surface.Colonies are embedded in agar> useful for isolation of microbes that cannot tolerate oxygen
Streak Plate Method
Note the greatly reduced numbers of growth colonies in each successive region ..Goal of the streak plate is isolated colonies
Why Pour Plate Method?
You have a bacterial culture and you want to know how many cells are in the culture. Can take 1ml of broth culture and add to 9ml of cooled liquid agar, mix and pour into petri plate.
Colonies Formed
Where a viable cell lands forms a colony. Therefore,numbers of colonies can be equated back to the numbers of bacterial cells in original sample
CFU
# of colonies =# of bacteria or colony forming units(CFU)
countable number of colonies
30-300
Serial Dilation
used for dense bacterial samples
10 -fold serial dilutions to dilute sample. Only living cells will produce colonies
Direct cell count (sd method
Cannot distinguish between live and dead cells
Turbidity using Spectrophotomer
Amount of turbidity is determined by a sample absorbance of light. Cannot distinguish between live and dead cells
Turbidity
A cloudy appearance in a culture tube indicates the presence of organism. Bateria in broth culture
Serial dilutions are perfomed
to obtain a plate that has countable colonies.Calculate number of viable cells of a dense culture.
Spectophotophotometer
Instrument can be used to measure bacterial growth by determining the degree of light transmission through the culture
Colony-forming unit represents
Wherever a single living bacterium is deposited on an agar plate, it will divide to form a colony.Each bacteria represents CFU
How to calculate CFU
CFU/(0.10)(Dilution factor)
Direct Microscope Counts
Bacteria growth can be measured by DMC
Direct Microscope Counts method
UsesPetroff-Hausser counting chambers device. Known volume of bacterial suspension counted in specific areas of the slide..Does not distinguish between live and dead cells.
How to calculate DMC
Calculate cells/ml using a mathematical formula
Other method Turbidity
measure turbidity with a spectrophotometer. Detects how much light penetrates a turbid culture. Amount of light absorbed correlates with concentration of bacteria . Does not distinguish between live or dead cells
Factors Affecting Bacterial Growth
Bacteria growth depends on a variety of environmental conditions . Physical factors pH, Temperature ,oxygen concentration,osmotic pressure, Hydrostatic pressure,moisture, radiation.
Factors Affecting Bacterial Growth Nutritional factors
Available of carbon, nitrogen , sulfur, phosphorus, trace minerals and in some cases vitamins....
pH
Acidity or Alkalinity of a medium is expressed in terms of pH
Optimum pH
The pH at which the microorganism grows best(most pH 6-5 -7.5)
Acidophiles, Neutrophiles, or Alkaliphiles
According to their tolerance for acidity/alkalinity bacteria are classified as.
Acidophiles (acid loving)
Grow best T pH 0.1-5.4
Neutrophiles
Grow best at pH 5.4 to 8
Alkaliniphiles(base loving)
Grow best at pH 7.0-11.5
How are bacteria classified accoding to their Growth Temperature Range
Bacteria can be classified as
Psychrophiles 15-20 C usually enviromental
Mesophile 25-40 C most pathogens
Thermophiles 50-60 C extreme enviromental
Thermophile
Thermophilic sulfur metabolizing bacteria can live and grow in the runoff waters from such geysers despite the near-boiling tempatures
Aerobes
require energy to grow
Anaerobes
Can't grow in presence of oxygen
To grow bacteria in culture
Must provide the appropriate oxygen level in the environment
Oxygen requirement
is dependent on the type of metabolism the bacteria perform
Obligate aerobes
must have free oxygen for aerobic respiration(e.g. Pseudomonas)
Obligate anaerobes
Killed by free oxygen (clostridium sp.)
Facultative anaerobes
Carry on aerobic metabolism when oxygen is present, but shift to anaerobic metabolism, when oxygen is absent (escherichia)
Microaerphile
Grow best in lower levels of oxygen but not anaerobic conditions(Camphlobacter)
Aerotolerant anaerobes
Can survive in the presence of oxygen but ferment(metabolize without oxygen at all times)(Lactobacillus)
What makes an Anaerobe vs. Aerobe
o2 reacts easily with electrons which produces toxic derivatives (superoxide o2-) These derivatives are toxic intermediates and may be free radicals. Free radicals steal electrons from other compounds in the cell.
Aerobes many have enzymes to degrade these toxic intermediates
Ananerobes usually lack these enzymes: therefor oxygen may be toxic
Enzymes that Degrade Toxic Products
Catalase, Peroxidase and Superoxide dismutase
Candle Jar for culture of anaerobe microaerophiles
Burning candle uses up froo oxygen until self-extinguishes
Anaerobe Chamber
A large chamber with a generator that creates a low/no oxygen enviroment
Anaerobe Jar
To culture obligate anaerobes, all molecular oxygen must be removed
A Gas Pak
Contains chemical substances that binds free oxygen and generates carbon dioxide and water. An indicator strip shows whether all oxygen has been voided
Osmotic Pressure
Environments contain dissolved substances and can exert osmotic pressure- Tendency to draw water out of cells.
Hyper osmotic (hypertonic) Enviroment
Cells lose water and undergo plasmolysis(shrinking of cells)
Preserving mechanism of jams and jellies
Hypoosmotic (hypotoic) Enviroment
Cells gain water and swell and burst--- can happen when cells lose their cell walls
Nuttitional Factors
carbon source, Nitrogen sources, Sulfur and phosphurs, Trace elements,(copper,iron, zinc and cobalt)Vitamins (folic acid, vitamin B-12, vitamin K)
Types of Culture Media
Synthetic medium or chemically defined medium each ingredient composition is well defined.
Complex medium: contains material which are general ingredients-ingredients not defined- examples peptone a product of enzyme digestion of proteins, beef extract
Chemically defined media
All ingredients known
Complex media
Exact composition of all ingredients no known
Selective Medium
encourages growth of some organisms but suppresses growht of others-Mannitol Salt Agar, Eosin Methylene Blue
Differential medium
contains a constituent that causes an observable change in the colony and or agar-
MacConkey agar, CHROMagar
Selective Agar:Mannitol Salt Agar
Allows for the growth of gram positive while inhibiting the growth of gram negative bacteria. Some selective agars may be also differential.
Different Candida species
Give a differing color reaction when grown on CHROMagar
Crucial in identifying C. albicans (medically important) from other Candida yeast
Obligate
Means the organism must have the specific enviromental conditions.
How does bacteria grow in a colony
Pile of bacteria growing on solid surface . All cells decedents of original cells
Identify areas of rapid-growing cells and dying cells
Older cells in death phase in center. Less access to nutrients
Rapid growth/ edges
Describe two techniques of isolating bacteria to obtain pure cultures. (red)
Streak Plate and Pour Plate.
Useful for isolation of microbes that cannot tolerate oxygen
List and describe methods of measuring bacteria
1. Serial dilution=used for dense bacteria samples
-10 fold serial dilutions to dilute sample
Only living cells will produce
2.Direct cell count= Cannot distinguish between live and dead cells
3. Turbidity using Spectrophotomer= Amount or turbidity is determined by a sample's absorbance of light
What are methods using solid media
Look up
How many colonies can be counted directly on a plate
30 to 300
Understand how a spectophotometer works to measure growth
lookup
What factors can effect bacterial growth
Depends on variety of environmental conditions. Physical factors.

pH , temperature, oxygen concentration, osmotic pressure, Hydrostatic pressure, moisture, radiation.

Nutritional factors
Availability of carbon, nitrogen, sulfur, phosphorus, trace elements and Vitamins
What is optimum pH
The pH that microorganisms grow best
How are bacteria classified according to their growth temperature range and where do you find each type
Psychrophiles 15-20 C found =Environmental
Mesophiles 20-40 C Pathogens
Thermophiles 50-60 C Extreme enviroments
Which are mostly pathogenic
Mesophile
How are bacteria classified according to their oxygen needs ( RED)
Aerobes require oxygen
Anaerobes can't grow in the presense of oxygen
What are the fundamental differences between aerobes and anaerobes (red)
OXYGEN
Which enzymes are present in Aerobes (red)
Catalase, Peroxides, superoxide
What are the patterns of Oxygen use that each will exhibit (red)
Look up
What techniques are use to culture anaerobes in a lab (red)
Candle Jar for culture of anaerobes and microaerophiles
Burning candle uses up free oxygen until self-exstingishes
Anaerobe Chamber- a large chamber with a generator that creates a low/no oxygen environment
Anaerobe jar To culture obligate anaerobes, all molecular oxygen must be removed
A-"Gas Pak" contains chemical substances that binds free oxygen and generates carbon dioxide and water An indicator strip shows whether all oxygen has been voided
What nutrients are needed for bacterial growth
Carbon sources
Nitrogen sources
Sulfur and Phosphorus
Trace elements ( copper, iron, zinc and cobalt)
Vitamins(folic acid, vitamins B-12, vitamin K)
Compare and contrast selective media and differential media Give examples of each (red)
Selective Media : encourages growth of some organisms, but suppresses growth of others
-Mannitol Salt Agar, Eosin Methylene Blue

Allows for the growth of gram positive while inhibiting the growth of gram negative

Differential Medium: contains a constituent that causes an observable change in the colony and or agar
-MacConkey agar, CHROMagar

Different Candida species give a differing color reaction when grown on CHROMagar
Crucial in identifying C. albicans (medically important) from other Candid yeast
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