Microbial growth Flashcards
an enzyme’s shape depends on….
pH, temperature, Osmolarity (saltiness) of outside enviornment
Microbial growth
increase in number of cells not size
Minimum growth temperature
the lowest possible temperature a bactrium can survive and reproduce in
Maximum growth temperature
the highest possible temperature a bactrium can survive and reproduce in
Optimum growth temperature
the best possible temperature a bactrium can survive and reproduce in
What are the growth requirements for bacteria?
good temperature good pH good osmolarity (osmotic pressure) Chemical building blocks (CHONPS) Trace elements
5 groups of bacteria based on their optimum growth temperature
Psychrophiles Psychrotrophs Mesophiles Thremophiles Hyperthermophiles
what is the temperature range for a psychrophile?
-10 to 20 degrees celcius
what is the temperature range for a psychrotroph?
0 to 30 degrees C
what is the temperature range for a mesophile?
10 to 50 degrees C
what is the temperature range for a thermophile?
40 to 70 degrees C
what is the temperature range for a hyperthermophile?
65 to 110 degrees C
what pH range do most bacteria grow in?
6.5 to 7.5 these are considered neutrophils
Acidophiles
bacteria that are very tolerant to acidity or thrive in it
Hypertonic environments (lots of salt or sugar) cause _______________.
Plasmolysis
plasmolysis
Plasmolysis is the process in which cells lose water in a hypertonic solution. The reverse process, cytolysis, can occur if the cell is in a hypotonic solution resulting in a lower external osmotic pressure and a net flow of water into the cell.
Obligate Halophiles
must be in a high salt enviornment
Facultative Halophiles
can live in a high salt environment but it is not necessary for their survival
CHONPS
main elements needed for life Carbon Hydrogen Oxygen Nitrogen Phosphate Sulfur
Nitrogen, Phosphorus, and sulfur are necessary for
found in amino acids and proteins proteins, DNA, and ATP
carbon composes up to _________ of cell’s dry weight
half
sulfur is necessary for
thiamine and biotin
phosphate ion
PO43-
Trace elements
K, Mg, Ca etc. often used as cofactors for enzymes and organic growth factors
What are the groups of bacteria based on O2 requirements?
Obligate Anaerobes Facultative Anaerobes Obligate Aerobes Aerotolerant Anaerobes Microaerophiles
A test tube with bacteria growth only at the surface of the thioglycolate medium indicates what type of bacteria?
Obligate Areobe
A test tube with heavy bacterial growth at the surface of the medium and some growth sprinkled through out the thioglycolate medium indicates what type of bacteria?
Facultative Aerobe
A test tube with bacteria growth only at the bottom of the thioglycolate medium indicates what type of bacteria?
Obligate anaerobe
A test tube with bacteria growth sprinkled evenly through out the thioglycolate medium indicates what type of bacteria?
Aerotolerant Anaerobes
A test tube with bacteria growth onlyin the middle of the thioglycolate medium indicates what type of bacteria?
Microaerophiles
Thioglycolate
Thioglycolate broth is a multi-purpose, enriched differential medium used primarily to determine the oxygen requirements of microorganisms
What are the toxic forms of oxygen?
O2- (superoxide free radicals) O22- (Peroxide anion) OH+ (hydroxyl radical)
superoxide dismutase
neutralizes superoxide radicals and makes hydrogen peroxide and oxygen
Catalase
neutralizes peroxide anions and makes 2 waters and oxygen
Peroxidase
Peroxidase (HRP) is a hemoprotein catalyzing the oxidation by hydrogen peroxide of a number of substrates such as ascorbate, ferrocyanide, cytochrome C and the leuco form of many dyes.
superoxide dismutase, catalase, peroxidase all …..
prevent damage from oxygen
planctonic bacteria
The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single-cells that may float or swim in a liquid medium.
Biofilms
microbial communities form slime or hydrogels. Starts with the attachment of plantonic bacterium to a surface structure
quorum sensing
Quorum sensing is a system of stimuli and response correlated to population density. Many species of bacteria use quorum sensing to coordinate gene expression according to the density of their local population. Autoinducers are the chemical regulators of quorum sensing. chemical communication between cells; it allows for symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation.
why are biofilms an advantage for bacteria?
because it protects them from desiccation, starvation, disinfection and other threats.
most nosocomial infections are thought to be caused by __________
biofilms
incubation
to provide the enviornment in which bacteria grow best (including correct temp, time, O2 and CO2 levels, and nutrients)
Culture medium
nutrients prepared for microbial growth. Must be sterile.
Inoculum
microbes introduced into a medium
Culture
Microbes growing in or on culture medium
Chemically defined medium
exact chemical composition is known (for research purposes only)
Complex media
extracts and digests of yeasts, meat, or plants e.g. nutrient broth, nutrient agar, blood agar.
Agar
a gelatin like complex polysaccharide that generally cannot be metabolized by bacteria. used as a solidifying agent for culture media in petri plates, slants, and deeps
at what temp does agar liquify
100 degrees C or 212 degrees F
At what temp does agar solidify?
~40 degrees C or 104 degrees F
gas pack
removes o2 in anaerobic chamber
reducing media
contains chemicals that will combine with O2 taking it out of the environment and making it part of its molecule (ex. thioglycollate).
Why are anaerobic cultures heated shortly?
to drive off O2
Oxyplate
no need for anaerobic jar because oxyrase is added to the growth media; usually done in clinical labs.
where are anaerobes grown in the lab
in an anaerobic jar
Capnophiles
anaerobic bacteria requiring High CO2
Why would a capnophile grow well in the instestinal, respiratorytract or other body tissues where pathogens grow?
because the low oxygen high carbon dioxide enviornment in these places is ideal for a capnophile.
an example of a capnophile
campylobacter jejuni
what is needed to grow a capnophile?
a candle jar CO2- generator packets CO2 incubators
Why is plasmolysis an issue how does it affect cell growth?
Plasmolysis is problematic since most of the chemical reactions within cells are aqueous. Without water to keep the enzymes and substrates dissolved, the molecules move very slowly and/or crystallize out of solution, hence slowing microbial growth.
What type of bacteria cause food spoilage?
Psychrotrophs cause food spoilage
Listeriosis
Listeriosis is a food-borne infection that causes about 2,500 people in the United States to become ill each year, and results in about 500 deaths. It is caused by food contaminated with the bacteria Listeria monocytogenes, a rod, coccobacilli shaped bacteria that can be arranged in chains or as single cells. The bacterium is motile at 20°-25°C, and is catalase positive. It is a psychrophile; therefore, it is neither killed nor does it grow slowly at cold temperatures. Listeria monocytogenes has been isolated from several sources including: ground beef, chicken and turkey, lunch meats, hot dogs, cheese, and poorly pasteurized milk. The disease usually affects pregnant women, newborns, and the immunocompromised. Fever• Muscle aches• Diarrhea• Nausea• Sore Throat
Selective medium
Additives (salts, dyes or other chemicals) suppress unwanted growth and encourage growth of desired microbes – e.g. EMB, mannitol salt agar etc
Differential medium
changed in recognizable manner by some bacteria Make it easy to distinguish colonies of different microbes – e.g. and hemolysis on blood agar; MacConkey agar, EMB, mannitol salt agar etc.
What are the three different types of media?
Selective, differential and enrichment media
Enrichment media
Encourages growth of desired microbe in a mixed culture.
What is an example of how enrichment media works?
Example: Assume soil sample contains a few phenol-degrading bacteria and thousands of other bacteria Inoculate phenol-containing culture medium with the soil and incubate Transfer 1 ml to another flask of the phenol medium and incubate Transfer 1 ml to another flask of the phenol medium and incubate Only phenol-metabolizing bacteria will be growing
Pure cultures
Contain only one species or strain.
Are patient and envoirnmental samples pure cultures?
No,Most patient specimens and environmental samples contain several different kinds of bacteria
What method is commonly used to culture bacteria on a plate?
Streak-plate method is commonly used
Colony forming unit/ Colony formation
A population of cells arising from a single cell or spore or from a group of attached cells
Only _____ of all bacteria can be sucessfuly cultured.
~ 1%
Examples of Capnophiles
- yeast
- fungi
- streptococcus
Why is differential media useful?
it ineracts with metabolic products from bacteria allowing us to distinguish bacteria based on the differences in their metabolic products.
A) Alpha hemolysis
B) Beta hemolysis
C) Gamma hemolysis
In a mannitol salt agar plate which ingredients are selective and which are differential, why?
- Salt is selsctive, because only organisms that can tolerate high salt concentrations will grow
- Mannitol is differential because the metabolic pathways that allow bacteria to break down mannitol will produce different protducts which can be used to tell bacteria apart.
Why is aseptic technique critical when growing bacteria?
To avoid introducing other microbes from the enviornment
Festidious organisms
picky; the organisms need special nutrients to grow,
How many bacteria are present in a colony?
- >106 bacteria per colony; 1 million
where do all the bacteria in one colony come from?
- they are all clones of the fist bacteria/bacterium to be introduced to the medium.
- The first bacteria to land on a petridish is the CFU or colony forming unit
What are the 2 primary ways bacterial cultures are preserved?
- Deep freezing
- Lyophilization
Deep freezing
Rapid cooling of pure culture in suspension liquid (glycerol) to –50°to –95°C. Good for several years.
Lyophilization
(freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum. Good for many years.
The bacteria are dehydrated into a powder.
How do bacterial cultures grow?
- Budding
- Binary fission
- aerial spore formation
- fragmentation
What is the difference between budding and binary fission?
They are both types of Asexual reproduction
In budding, the new organism is from the old organism, its kind of sprouting out. The cytoplasm is split unevenly. Mother and daughter cell can be distinguished easily.
Binary fission is when the organism seperate to form two new organisms. Here the cytoplasm is split evenly. exponential growth. Binnary fission is like a type of mitosis.
Generation time
- time required for cell to divide (also known as doubling time) or the population to double.
- Ranges from 20 min in E.Coli to > than 24 hrs in M. tuberculosis.
- Cell elongates and DNA is replicated
- Cell wall and plasma membrane begin to constrict
- Cross wall forms, completely separating the 2 DNA copies.
- Cells separate
What process is pictured here?
Binary fission
What process is shown in this picture?
Budding
Eukaryotes have ______________ and Prokaryotes have a __________________.
- linear chromosomes
- single circular chromosome
What is the formula that represents the numer bacteria in each generation time?
2n= number of bacteria present at generation n.
Bacterial generations are counted logrithmically, why?
- Recording bacterial growth logrithmicaly produces straight lines on graphs which allows us to see trends in bacterial growth.
What is this and what does it do?
Bacterial growth curve; it illustrates the changes that occur during bacterial growth.
- Lag phase
- Log phase
- Stationary phase
- Death phase
Lag phase
intense activity preparing for population growth, but no increase in population
Log phase
Logarithmic, or exponential growth in population
The logarithmic growth in the log phase is due to reproduction by binary fission (bacteria) or mitosis (yeast).
Stationary phase
Period of equilibrium; microbial deaths balance production of new cells
Death phase
Population is decreasing at a logarithmic rate
How is bacterial growth in a liquid and solid medium different?
growth in liquid is more efficent because nutrients float freely and bacteria have more access to nutrients. Growth in solid media is slower because the bacteria only have access to nutrients near them.
What are the direct methods of counting bacteria in a sample?
- Plate counts
- Filtration
- Direct microscopic count
Plate count
The plate count method means diluting bacteria with a diluent solution (e.g. sterile saline) until the bacteria are dilute enough to count accurately when spread on a plate. The assumption is that each viable bacterial cell will develop into a single colony. Bacterial cell numbers need to be reduced by dilution, because more than 200 colonies on a standard 9 cm plate are likely to produce colonies too close to each other to be distinguished as distinct colony-forming units (CFUs).
How do you calculate the original amount of bacteria in a sample.
- You dilute the innoculum until 30-300 colonies are present on a petriplate
- Count the number of colonies
- Number of colonies x reciprocal of dilution sample = number of bacteria per ml.
- 54 colonies on a plate of 1:1000 dilution; then the count is 54 x 1000 = 54,000 bacteria per ml in a sample.
what is a countable plate?
30-300 colonies; more or less colonies gives you an unreliable statistic.
TNTC
to numerous to count
TFTC
to few to count
spread plate method
- innoculate plate containing solid medium with bacterial dilution
- Spread innoculum over surface evenly
- Colonies grow only on the surface of the medium
Pour plate method
- inoculate empty plate
- add melted nutrient agar
- swirl to mix
- colonies grow on and in solidified medium
Why is the filtration method of counting used?
it is the method of choice for low counts. at least 10 ml of water is passed through the membrane and the filter is transfered to a petridish containing a nutrient medium.
Direct microscopic count
microbes in a measured volume of bacterial suspension are counted with the use of a specifically designed slide
What are some ways to estimate bacterial numbers by indirect methods?
- Turbidity- measurement of cloudiness with a spectrophotometer
- Metabolic activity- amount of metabolic product is proportional to the number of bacteria; acid, gas or ATP production.
- Dry weight- bacteria are filtered, dried, and weighted; used for filamentous organisms
Spectrophotometer
used to determine turbidity by measuring the amount of light that passes through a suspension of cells.
How are pure cultures usually obtained
by employing the streak plate method
what happens to most microbes in a hypertonic solution?
they undergo plasmolysis; except for halophiles
All organisms require a __________ source
- Carbon; because they are carbon based lifeforms
- chemoheterotrophs use an organic molecule
- autotrophs usually use carbondioxide
Why do bacteria need nitrogen? Where can they get it?
for protein and nucleic acid synthesis. Nitrogen can be obtained from the decomposition of proteins from ammonium NH4+ or nitrate NO3 - A few bacteria are capable of nitrogen gas fixation N2
Which classes of bacteria must have the enzymes superoxide dismutase, catalase or peroxidase?
- Aerobes
- Facultative anaerobes
- areotolerant anaerobes
where do biofilms form?
on solid surfaces in contact with water
do most bacteria live in planktonic form or in biofilms
biofilms
microbes in biofilms are ____________ to antiboitics than planktonic bacteria.
more resistant
What is a culture medium?
any material prepared for the growth of bacteria in a laboratory
Petriplates containing anaerobes can be incubated in _____________________________.
- Anaerobic jars
- Anaerobic chambers
- Oxyplates
Some parasitic and fastidious bacteria must be cultured in ___________________.
in living animals or cell cultures
Why are CO2 incubators or candle jars used?
to grow bacteria that need an increased CO2 concentration.
Procedures and equipment to minimize the exposure to _____________ are designated as ___________________.
- pathogenic microbes
- biosafety levels 1-4
Colony
a visable mass of microbial cells that theoretically arose from one cell
Heterotrophic plate count
reflects the number of viable microbes and assumes that each bacterium grows into a single colony; plare counts are reported as number of colony forming units (CFUs)
MPN
the most probable number method can be used for microbes that will grow in liquid medium; it is a statistical estimation.
