Bacterial Growth, Nutrition, metabolism and Genetics Flashcards
List the environmental effects on bacterial growth
Temperature
pH
Osmotic Pressure
Oxygen tension
any changes can stop the development of bacteria
How does temperature affect bacterial growth?
temp inc, reaction fast, temp is ideal human temp
Minimum temperature
o Temperature below which growth ceases, or lowest temperature at which microbes will grow
o They allow growth on a very small scale
Optimum temperature
o Temperature at which its growth rate is the fastest
o The optimum temperature for bacterial
growth is the normal body temperature
Maximum temperature
o Temperature above which growth ceases, or highest temperature at which microbes will grow
o Anything higher than this will cause the
bacteria to die that is why if you have to
sterilize you have to heat it very well
Ideal temperatures for Psychrophiles
0-20
Thrive at 4 deg
Polaromonas vacuolata
Ideal Temperatures for Mesophiles
20-45
warm-blooded animals
Thrives at 39°C
Bacillus stearothermophilus
Hyperthermophiles
Optima greater than 80°C
These organisms inhabit hot environments including boiling hot springs, as well as undersea hydrothermal vents that can
have temperatures in excess of 100°C
o Thrives at 88°C, Ex. Thermococcus celer
o Thrives at 106°C, Ex. Pyrolobus fumarii
but higher than that the growth stops
pH AND MICROBIAL GROWTH
acidophiles – optimum in pH
range 1-4 H. pylori, T. oxidans
alkalophiles – optimum in pH
range 8.5-11. V. cholera
Lactic acid bac. ideal pH
4-7
Thiobacillus thiooxidans pH
2.2 to 2.8
Fungi pH
4-6
How is internal pH regulated?
Buffers
What is the best pH for most organisms?
6-8
Pathogenic bacterium are
acidophile
neutrophile
Alkalophile
Neutrophile
OSMOTIC EFFECTS ON MICROBIAL GROWTH
• Osmotic pressure depends on the surrounding solute concentration and water availability
• Water availability is generally expressed in physical terms such as water activity (aw)
• Water activity is the ratio of the vapor pressure of the air in equilibrium with a substance or solution
to the vapor pressure of pure water (aw 1.00)
Osmophiles
organisms that thrive in high solute
Osmotolerant
organisms that tolerate high
solute
Halophiles –
organisms that thrive in high salt
Halotolerant –
organisms that tolerate high salt
Barophiles –
organisms that thrive in high
pressure
Barotolerant –
organisms that tolerate high
pressure
HALOPHILES
have evolved to grow best at
reduced water potential, and some (extreme halophiles, e.g. Halobacterium salinarium, Dunaliella) even require high levels of salt for growth
V. fischeri
HALOTOLERANT
can tolerate some reduction in
the water activity of their environment but generally grow best in the absence of the added solute.
o Ex. Staphylococcus aureus
XEROPHILES
are able to grow in very dry
environments
• Nonhalophile – Ex. Escherichia coli
AEROBES Obligate
require O2
Facultative aerobes
with or without o2
Microaerophiles
low levels of O2
Aerotolerant anaerobes –
can tolerate
oxygen but grow better without oxygen
Obligate –
do not require oxygen; killed by
oxygen
When treating infections specially in immunocompromised
patients, always think of a polymicrobial infection. You
always think that that infection has aerobic and anaerobic.
They can tolerate oxygen but grow better without oxygen,
these are anaerobes. That is why you will see anaerobic
infections in the back, in the gluteus maxims (butt), WHY?
Because you always lie in your back, that’s why there is low
oxygen tension there.
How to test for O2 requirements of Microorganisms?
Contains a reducing agent and provides aerobic and anaerobic conditions
a) Aerobic
b) Anaerobic
c) Facultative
d) Microaerophile
e) Aerotolerant
Carbon, Nitrogen weight per 100g of dry weight
50%, 12%
Most bacteria can use
Ammonia -NH3 and many
can also use NO3
Nitrogen fixers
can utilize atmospheric nitrogen
N2
NITROGEN REQUIREMENTS
• Although many biological components within living organisms contain N, and N2 is the most abundant component of air, very few organisms can “fix” or
utilize N2 by converting it to NH3
• N is often growth-limiting as organisms must find the source as NH4
+ for biosynthesis
• Photosynthetic organisms and many microbes can reduce NO3
to NH4
SIDEROPHORES
– iron-binding agents that cells
produce to obtain iron from various insoluble minerals
Anaerobes lack
superoxide dismutase and/or catalase
Anaerobes need high Chemica
Thioglycollate; pyrogallol + NaOH, H2
generator + catalyst
The candle jar
the technique used for anaerobic cultures
because if you do the candle jar you will consume all oxygen so you will have an anaerobic environment.
Culture media
supply the nutritional needs of
microorganisms (C, N, Phosphorus, trace elements,
etc.). The media that are used in microbiology
laboratories to culture bacteria are referred to as
synthetic or artificial media, because they do not
occur naturally; rather they are prepared in the
laboratory; is used when we grow bacteria
Defined medium:
precise amounts of
highly purified chemicals; is one in which
all the ingredients are known
Complex medium or (undefined):
highly
nutritious substances.
Complex medium or (undefined):
highly nutritious substances.
Selective:
contains compounds that
selectively inhibit; is used to discourage the growth of certain organisms without
inhibiting the growth of the microorganism being sought
Differential
contains indicator; allows one to
readily differentiate among the various types of organisms that are growing in the medium.
Media can be classified on three primary levels:
- Physical State
- Chemical Composition
- Functional Type
LIQUID MEDIA
- Water-based solutions
- Do not solidify at temperatures above freezing or tend to be free flowing
- Includes broths, milks, and infusions
- Measure turbidity
- Example: Nutrient Broth, Methylene Blue Milk, Thioglycollate Broth
SEMI-SOLID MEDIA
- Exhibits a clot-like consistency at ordinary room temperature
- Determines motility
- Used to localize a reaction at a specific site.
- Example: Sulfide Indole Motility (SIM) for hydrogen sulfide production and indole reaction and motility test.
SOLID MEDIA
• Firm surface for discrete colony growth; for
morphology
• Advantageous for isolating and culturing
• Two Types
o Liquefiable (Reversible) - Reversible to liquid phase, contains a solidifying agent that
changes its physical properties in response
to change in temperature.
o Non-liquefiable - Non-reversible, less
versatile applications than agar as they do
not melt.
• Examples: Gelatin and Agar (Liquefiable)
Cooked Meat Media,
Potato Slices (Non-liquefiable)
SYNTHETIC MEDIA
- Chemically defined
- Contain pure organic and inorganic compounds
- Exact formula (little variation)
COMPLEX OR NON-SYNTHETIC MEDIA
- Contains at least one ingredient that is not chemically definable (extracts from plants and animals)
- No exact formula; tend to be general and grow a wide variety of organisms
SELECTIVE MEDIA
- Contains one or more agents that inhibit the growth of a certain microbe and thereby encourages, or selects, a specific microbe.
- Example: Mannitol Salt Agar [MSA] encourages the growth of S. aureus. MSA contain 7.5% NaCl which inhibits the growth of other Gram + bacteria
DIFFERENTIAL MEDIA
• Differential shows up as visible changes or variations in colony size or color, in media color changes, or in the formation of gas bubbles and
precipitates.
• Example: Spirit Blue Agar to detect the digestion of fats by lipase enzyme. Positive digestion (hydrolysis) is indicated by the dark blue color that
develops in the colonies. Blood agar for hemolysis
(α, β, and γ hemolysis), EMB, MacConkey Agar, etc.
ENRICHMENT MEDIA
- Is used to encourage the growth of a particular micro-organism in a mixed culture; adding extra nutrients to a medium called nutrient agar.
- Examples: Manitol Salt Agar for S. aureus, blood agar , chocolate agar, Slenite F broth
Generation time
is the time it takes for a single
cell to grow and divide
Number of total bacteria
2n x number of initial bacteria
2 stands for the doubling of the number of the bacteria
n stands for the number of generations ( to get this, convert 3 hours to minutes divided by 30 minutes or how long the bacteria divides)
26 x 100 =6, 400 cells
the lag phase
cells are recovering from a period
of no growth and are making macromolecules in
preparation for growth; The lag phase represents a
period during which cells, depleted of metabolites and
enzymes as the result of the unfavorable conditions
that existed at the end of their previous culture history
log phase
cultures are growing maximally;
the bacteria multiply so rapidly that the number of
organisms doubles with each generation time (i.e.,
the number of bacteria increases exponentially).
Stationary phase
occurs when nutrients are
depleted and wastes accumulate (Growth rate = death rate); eventually, the exhaustion of nutrients or the accumulation of toxic products causes growth to
cease completely
death phase
death phase, the death rate is greater than
the growth rate; As overcrowding occurs, the
concentration of toxic waste products continues to increase and the nutrient supply decreases. The microorganisms then die at a rapid rate
METHODS USED TO MEASURE MICROBIAL
GROWTH
- Count colonies on plate or filter (counts live cells)
- Microscopic counts
- Flow cytometry (FACS)
- Turbidity
Viable Counts
o Each colony on plate or filter arises from
single live cell
o Only counting live cells); is typically
considered the measure of cell concentration.
For this, a 1-mL volume is removed from a
bacterial suspension and serially diluted 10- fold followed by plating 0.1-mL aliquots
(portions) on an agar medium. Each single
invisible bacterium (or clump of bacteria) will grow into a visible colony that can be counted
DIRECT COUNT:
POUR PLATE, SPREAD OR STREAK PLATE
MICROSCOPIC COUNT
- Need a microscope, special slides, high power objective lens
- Typically only counting total microbe numbers, but differential counts can also be done
streak plate vs spread plate
www.differencebetween.com
purpose of streak plate
isolate or purify specific species
Streak plate inoculating tool
inoculation loop or cotton swab
is a micropippete necessary for Streak plate?
No
inoculum quantity streak plate
one loopful
Sterilization of inoculum in streak plate
flamed until red hot
Method of inoculation streak plate
Zig-zag spread
purpose of spread plate
enumerate bacteria colonies in a sample
inoculating tool spread plate
Sterile spreader
is a micropipette necessary for spread plate
yes
Sterilization of inoculum spread plate
95% alc and flamed
Method of inoculation spread plate
spread evenly in fresh medium
MICROSCOPIC COUNT
Need a microscope, special slides, high power objective lens
Typically only counting total microbe numbers, but differential counts can also be done
FLOW CYTOMETRY
Flow cytometry is a laser-based method used for the
analysis of cells and selected cell components. One of the
most popular applications of flow cytometry is
immunophenotyping of cell populations. In this method,
single-cell suspensions are stream through a flow cell in
which the cells pass through a laser beam for sensing. As
the cells pass through the laser, they scatter light
TURBIDITY
• Cells act like large particles that scatter visible light
• A spectrophotometer sends a beam of visible light
through a culture and measures how much light is scattered
Scales read in either absorbance or % transmission
• Measures both live and dead cells
Linear pathways –
glycolysis,
Spiral or cyclic
Krebs cycle
ENZYME ACTIVITY
• Enzyme activity may change due to inhibitor or activator molecules called effectors. • Inhibitors can be competitive (bind at substrate active site) • Non-competitive inhibitors and activators bind to allosteric (regulatory) sites; separate from the active site; • These effectors change the shape of the protein and its activity as a catalyst.
Feedback Inhibition:
o Rate limiting enzyme is first in pathway
and is allosteric
o End-product is a negative effector
(inhibitor) of first enzyme
Feed Forward Activation
o Rate limiting enzyme of a branch point is allosteric
o Earlier-substrate is a positive effector
(activator) of a forward reaction enzyme
REVERSE METABOLIC PATHWAYS
Amphibolic pathways:
o Catabolic direction
o Anabolic direction
LUCOSE CATABOLISM
• ATP as the cellular energy storage unit can be
formed during respiration (R) or fermentation (F)
• Both contain the Glycolysis pathway; which produces ATP, the electron carrier molecule NADH, and pyruvate from glucose
• Aerobic Respiration will proceed via Krebs cycle and an ETC if there is oxygen to react as a terminal electron acceptor
• Oxygen is not the only possible terminal electron
acceptor in some bacteria (e.g. NO3 or SO4 can be used); called Anaerobic Respiration
products of Pyruvate from Clostridium
Butyrate and Acetoacetate
Pyruvate of Gluconobacter
Acetate,
Pyruvate of Streptococcus and lactobacillus
lactic acid
pyruvate of acetobacterium
Acetyl CoA
HYDROLYSIS OF MAJOR BIOMOLECULES
Enzymes of Hydrolysis:
• Proteins by proteases
• Polysaccharide and other carbohydrates by glycosidase
• Nucleic acids (DNA or RNA) by nucleases
• Lipids by lipases
AMPHIBOLIC NATURE OF METABOLISM
Most catabolic pathways have anabolic counterparts, so not
all compounds are used to generate ATP, but rather shunted
to make new cell biomass.
Genetics is the study of heredity which is concerned with how
○ information in nucleic acids is expressed ○ nucleic acids are duplicated and transmitted to progeny ○ these processes account for the characteristics of progeny ○ structure & function of genetic material ○ transmission of biological traits from parent to offspring.
Genome
– sum total of genetic material of an
organism (chromosomes +
mitochondria/chloroplasts and/or plasmids)
GENOMES VARY IN SIZES
● smallest virus –4-5 genes
● E. coli–single chromosome containing 4,288 genes; 1 mm; 1,000X longer than cell
● Human cell –46 chromosomes containing 31,000 genes; 6 feet; 180,000X longer than cell
Read DNA and RNA functions
page 16 of trans
CENTRAL DOGMA THEORY
The central dogma theory of molecular biology is
represented by a simple pathway: DNA ® RNA ®
protein, which demonstrates the flow of genetic
information in a living cell
Major processes of CDT theory
replication, transcription, and translation
DNA polymerase enzyme
replicates all the DNA in the nuclear genome in a semi-conservative manner, meaning that the double stranded DNA is separated into two and a
template is made by DNA polymerase.
transcription.
The process in which DNA is copied into RNA by RNA Polymerase
REVERSE TRANSCRIPTASE
● Another process in this pathway is reverse transcription, which involves copying RNA information into DNA using reverse transcriptase
● Recently, this process has been defined and may expand the central dogma
● For example, retroviruses use the enzyme “reverse transcriptase” to transcribe DNA from an RNA
template
● The viral DNA then integrates into the nucleus of the
host cell. Then it is transcribed, and further translated into proteins
● This biological process effectively adds another pathway to the central dogma of molecular biology
DNA REPLICATION IN BACTERIA
● Bacteria contain 1 chromosome
● Many contain plasmids
● When bacterial chromosomes replicate both strands are duplicated. Each strand functions as a template
STEPS OF DNA REPLICATION
- DNA unwound with enzyme (replication fork)
- Complementary bases added to template
(parent strand) using enzyme - Replication fork moves down strand
- Newly replicated DNA rewinds
- Process called Semiconservative Replication
Replication speed
1000 nucleo/sec
Direction of copying in Replication
5’ to 3’
RNA SYNTHESIS IN BACTERIA
1) RNA polymerase binds to DNA at a
promoter site near the gene to be
transcribed.
2) RNA polymerase travels the length of the DNA using it as a template to duplicate.
3) The RNA polymerase continues until it
reaches a termination site at which time the transcription is complete.
STAGES OF PROTEIN SYNTHESIS IN BACTERIA
Protein synthesis is continuous and takes place in three stages:
- Initiation
- Elongation
- Termination
CHAIN INITIATION
● The beginning of protein synthesis starts methionine which is the start codon. ● Start codon is known as formyl methionine (fmet). ● It is coded as AUG.
CHAIN ELONGATION
By a complex that begins with f-met, amino acids attach to form a chain (amino acids joined repeatedly to form proteins)
CHAIN TERMINATION
● Ends when the synthesis comes to a termination codon ● Termination codons are codes as UAA, UAG, and UGA
Rifamycin
binds to RNA polymerase
Actinomycin D
○ binds to DNA & halts mRNA chain
elongation
Erythromycin & Spectinomycin
○ interfere with attachment of mRNA to
ribosomes
Chloramphenicol, lincomycin & tetracycline
○ bind to ribosome and block elongation
Streptomycin
○ inhibits peptide initiation & elongation
DIFFERENCE BETWEEN
EUKARYOTIC TRANSCRIPTION AND TRANSLATION
FROM PROKARYOTIC
● Do not occur simultaneously. Transcription occurs
in the nucleus and translation occurs in the cytoplasm
● Eukaryotic start codon is AUG, but it does not use formyl-methionine
● Eukaryotic mRNA encodes a single protein, unlike bacterial mRNA which encodes many
● Eukaryotic DNA contains introns - intervening sequences of noncoding DNA-which have to be
spliced out of the final mRNA transcript
Codons
A codon is a group of three nucleotides in DNA which acts as a code in the placing of an amino acid in a protein molecule ● AUG begins protein synthesis ● UAA, UAG, UGA are termination codons ○ When this codon is reached, the ribosome falls off and the last tRNA is removed from the polypeptide.
