Select the Physical Factors Below That Can Affect Microbial Growth.
ane. Temperature:
How does temperature affect optimal growth?
- pump in protons
- generate ammonia (+) by ammino acid degradation
| Bacteria | min pH | opt pH | max pH | |
| Thiobacillus | one.0 | 2-2.8 | iv-vi | |
| East. coli | iv.four | vi-vii | 9.0 | |
| C. sporogenes | 5.4 | six-vii.six | nine.0 | |
| P. aeruginosa | 5.6 | six.vi-7 | 8.0 | |
| Nitrobacter | 6.half-dozen | 6.6-8.six | ten.0 | |
three. Classification based on water activity/osmotic force per unit area (call up, depression water activeness = high osmotic pressure level)
- halophiles- require sodium >9%; extremes may live in 30%
- marine salt virtually 3%
- prison cell wall and membrane fall apart westward/o sodium
- nonhalophile - 0 to 1.5%
How do microbes adapt to low water activeness?
- Microbes tin can modify their internal osmotic surroundings
- E. coli in GI tract
- produce or import compounds that increase internal solutes
- uniform solutes - do non harm host
- ions, sugars, amino acids
4. Oxygen:
How does oxygen affect optimal growth?
- aerobes - require oxygen
- obligate/strict anaerobes - killed by oxygen
- aerotolerant anaerobes - does non require oxygen only not killed
- facultative anaerobe - grows in presence or absence of oxygen with different metabolic strategies
- microaerophiles - require reduced levels of oxygen
Why is oxygen toxic to some microbes?
- metabolic byproducts all organisms produce are toxic to cells
- hydrogen peroxide
- superoxide
- present in WBC to kill bacteria
- aerobes have catalase and/or peroxidase and superoxide dismutase (SOD)
- catalase: 2H202 -> 2H20 + 0two
- peroxidase: H202 + 2H+ -> 2H20
- SOD: xxii- + 2H+ -> 02 + Hii0two
- strict anaerobes lack these enzymes
- microaerophiles either reduced amounts of these enzymes or oxygen-sensitive forms of enzymes
- aerotolerant have these enzymes just non those used in aerobic metabolism
- Toxic forms of oxygen are broken down past several enzymes; one of these is catalase. This enzyme breaks down hydrogen peroxide to grade water and molecular oxygen. Our tissues as well as many microorganisms are catalase positive - thats why the bubbles when you cascade peroxide on a cut.
Why would our tissues have peroxide in them?
Some theories of cancer hypothesize that oxidants (like toxic forms of oxygen) damage DNA and thus cause the mutations which in turn cause cancer. These theories predict that antioxidants such as beta-carotene, vitamin E and vitamin C would thus take anti-mutation action.
commonly called vitamins, these are organic substances required for the growth of an organism only which the organism can not synthesize.
What is the deviation between a defined and an undefined medium?
WHAT FACTORS AFFECT GROWTH?
- Nutrients - Cell constituents and energy sources
- Physical factors - temp, pH, water, oxygen, force per unit area
I. Nutrients:
| Chemical element | Jail cell Role | ||||||
| C | backbone of organic cell components, energy | ||||||
| H | water, organic components, pH, hydrogen bonds, re-dox | ||||||
| O | h2o, organic components, respiration | ||||||
| N | amino acids, nucleotides, coenzymes, ATP | ||||||
| S | amino acids, coenzymes, enzymes | ||||||
| P | nucleic acids, phospholipids, coenzymes, ATP | ||||||
| Iron | cytochromes, enzymes | ||||||
| Na, Thousand, Ca, Cl, Mg, Mn | Trace elements: ship, ionic balance, cofactors (e- donor/acceptors) | ||||||
Ii. CLASSIFICATION OF ORGANISMS BASED ON carbon, energy, and electron sources
- Chemotrophs - Derives free energy from chemicals
- Phototrophs - Derives energy from sunlight
- Autotrophs - Use carbon dioxide for carbon
- Heterotrophs - Use organic substrates for carbon
- Lithotrophs - Inorganic compounds for electrons
Bacterial Growth
- Bacteria grow past binary fission. Starting with i organism how many organisms would you accept later 1,2,three,4,5,6,7,viii,9,10,xi,12 generations.
- Increase in jail cell numbers
- Binary fission
- Budding
- Fragmentation
- Deoxyribonucleic acid duplication
- Deoxyribonucleic acid repication
- single origin of replication, bidirectional
- theta intermediate
- Separation of Dna and cytoplasmic contents
- Cross wall germination
- fourth dimension required for a prison cell to divide or a population to double
- 1 to two or 100 to 200 or one meg to ii million
- Most mutual leaner have a generation fourth dimension 30-sixty min under opt. weather.
- Most common pathogens in the body, about five-10 hours.
- Why doesn't it grow immediately?
- What determines how fast it grows?
- Why does it cease growing?
- Lag stage - synthesis of new components or repair
- Log stage - reproduction at maximum rate (shortest generation fourth dimension)
- exponential growth one->2->4->8->16->32->64
- Stationary phase - no net increment, balance between jail cell partitioning, cell "expiry", maintenance
- Expiry phase - do they really die?
If y'all started with x organisms, how many would you lot accept after each of the above generations?
What is microbial growth?
| Mycobacterium tuberculosis (in lab) | 12 hrs |
| Clostridium botulinum (in lab) | 0.58 |
| Eastward. coli (in lab) | 0.30 |
| E. coli (in mouse) | 20 hrs |
What happens when you inoculate a unmarried bacterium from slant stored in refrigerator into nutritional medium and incubate?
Phases in bacterial growth in batch civilisation (see above picture)
Mathematics of growth- generation time
Growth equation:
n = log 10 Nf - log 10 Ni
0.301
cells/ml. Calculate n = (5 - four)/0.iii = 1/0.3 = three.33 generations.
Generation fourth dimension: Total time = 6 hours = 360 minutes/3.33 generations = 108 minutes/generation
Conclude: generation fourth dimension = 108 minutes
Note: be able to calculate one thousand.t. Pay attending to units!
Graphical measurement of growth
· Plotting # of cells vs. time gives a curved line.
· Plotting log # of cells vs. time gives a direct line --- easier to interpolate, use.
· Plotting # of cells vs fourth dimension on semilog paper also gives a straight line --- easiest way in
practise to work with growth measurements.
· Annotation: frequently what is plotted on the Y-centrality of semilog paper is not # of cells, just something
more easily measurable, such as Absorbance (see below).
.
CULTURE MEDIA
A civilisation medium is any textile prepared for growth of an organism in a laboratory setting. Microbes that tin can be cultured on a petri-plate or in a test-tube containing media are said to grow under in vitro conditions ("within-glass".)
It was not until the era of Robert Koch and his coworkers that Agar was introduced as a a common medium for bacterial growth. Agar is a complex polysaccharide derived from a marine sea weed. Few leaner possess enzymes capable of digesting agar and therefore information technology is useful as a solidifying agent and for isolating microbes in pure culture. Prior to the advent of agar, gelatin was used as a growth medium. Unfortunately, many bacteria possess enzymes that liquify gelatin and therefore this medium is non useful for isolating pure cultures. Nevertheless, gelatin liquefaction is one among a series of biochemical tests that helps differentiate species of bacteria.
What is a PURE CULTURE?
- A pure civilisation represents a single species (clonal in nature) of microorganisms
- A clone is a genetically identical population of microbes that accept descended from a unmarried parent cell
- Colonies are visible clones that accept grown on solid media and stand for millions of bacterial cells
- Distinctive characteristics of colonies should be noted such as:
- pigmentation
- olfactory property
- elevation
- margin (border of the colony)
- consistency, such as mucoid, irridescence, filamentous, etc.
a) Chemically divers media: exact chemical composition is known. Such media is often
commercially prepared.
b) Selective media. Comprise chemicals which encourage growth of certain types of microbes
but inhibits the growth of others.
c) Differential media allows different microbes to exist distinguished on the basis of various
biochemical reactions. Fermentation reactions involving the catabolism of diverse sugars are
particularly useful biochemical tests
- Note: Many media are both selective and differential, such as MacConkey (Mac) agar and Mannitol Salt agar (MSA).
d) Enrichment media contains a rich supply of nutrients to encourage the encourage
growth of microorganisms. A commonly used enrichment medium is claret agar. This
medium is as well differential and it permits detection of differnt patterns of hemolysis.
Measurement of growth
a) Full Cell count
· Petroff-Hausser sleeping accommodation slide -- needs large conc. (tenseven cells/ml minimum).
· Coulter Counter (for larger microbes; fungi, yeasts, protozoa, etc.) --- uses electric charge
difference in passing through small hole. Not so useful with bacteria, become errors due to
clumping, droppings, unable to differentiate bewteen live and dead cells, etc.
b) Viable count
CFU (colony forming units) assay
1. carry out dilution series
ii. plate known volumes on plates
3. count only plates with 30 - 300 colonies (best statistical accurateness)
4. extrapolate to undiluted cell conc.
Measures colony forming units (CFU), may or may not be aforementioned as number of cells --- accurate, but requires time for incubation.
Two means to carry out viable count:
i. Spread plate: bacteria are spread on the surface of agar using some sterile spreading
device. Advantages: if properly carried out, all colonies should be hands counted.
Disadvantages: takes some fourth dimension, not always reliable in inexperienced hands, cells with low
tolerance to oxygen will not grow. If "spreaders" are present may overgrow plate surface.
two. Cascade plate: leaner are mixed with melted agar and cooled; colonies grow throughout the
agar. Advantages: almost neglect-proof technique, colonies well separated. Tin allow growth
of organisms with lower oxygen tolerance in agar. Disadvantages: colonies variable size,
harder to encounter similarity in colony morphology between those on surface and in agar.
Counting may exist more difficult. Oestrus may impale some cells before agar cools and gels.
c) Light techniques
Oftentimes, can gauge jail cell numbers accurately by measuring visible turbidity. Calorie-free scattered is proportional to number of cells. This only works above cell densities of x7 in pure cultures. Eyeball method. This is not a precise measurement, but shoud allow estimation within an order of magnitude.
� no turbidity ways less than 10vii cells/ml
� Slight turbidity = 10seven - 108 cells/ml
� high turbidity= ten8 - 10nine cells/ml
� Very. high turbidity = greater than 109 cells/ml (cultures rarely become as high as x10 cells/ml)
Absorbance (ordinarily at wavelengths around 400-600 mn). Accurate measure of cells when concentration non too high. Piece of cake and quick to measure (can sample in less than a infinitesimal).
d) Batch vs. Continuous culture methods
· Batch method: put small-scale inoculum of pure civilization into sterile medium, let grow. Common lab
procedure, only not typical of many existent environments.
� Continuous culture (see text section 6.xi): employ chemostat or turbidostat. Trickle fresh
medium into culture at wearisome but steady rate, displace = volume of culture as overflow.
� Cells remain in exponential (just suboptimal) state, growing at known rate. Good simulation for
study of many natural environments.
Source: https://www2.nau.edu/~fpm/bio205/chapter6.html
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