Microbiology Lecture-Bacterial growth
Microbes grow almost anywhere-adapted to even the most extreme conditions, hot springs, 300 meters below surface of ground, dead sea, Antarctica.
Growth-increase in total mass. Difficult to measure mass increase of a single bacterium. Measure bacterial growth by increase in numbers of population. Growth in microbes usually refers to population. Other organisms, growth usually refers to individual organism.
Bacterial cells divide rapidly (about 20 mins). Billions of cells in short time. Important to research and industry. Growth is exponential.
Time required for a cell to divide or a population to double is the generation time. This is a measure of the rate of growth. Ranges from 5-10 mins. to days. Mycobacterium leprae is 10-30 days. Salmonella 20 mins (avg is 30-60 mins. )Knowing generation time allows a comparison between growth rate of different bacterial populations.
Size of a population over time
nf=number of cells at some point in growth phase
ni=# of cells at starting time
n=# of generations (total growth time in minutes/generation time)
How many Staph in an egg salad sandwich after 4 hrs if you start with 10 cells (assume a generation time of 20 minutes)
n=240 mins/20 min (avg for Staph dividing)=12
nf=10 X 212
nf=10 x 4096=40,960 cells.
Exponential growth follows a very specific pattern.
log scale-constant rate=straight line, changing is curved
Bacterial growth curve:
Growth curve is divided into 7 phases:
1. Lag phase- no increase in #'s. Cells adapting to new conditions (synth. new enzymes). If cells are old or have been dormant, essential cell components have been depleted that must be replaced now.
2. Phase of posit. acceleration- some cells begin to divide. All cells don't reach this point at once. Gradual shift from no cells dividing to few to all. Culture not synchronized.
3. Log or exponential phase- All cells growing at maximum rate (biotic potential). Culture synchronized.Max. rate determined by genetic factors, physical environment. At this stage cells most susceptible to inhibitors. This stage continues until conditions become less than ideal ((carrying capacity reached). Best time to do motility studies, staining.
4. Phase of neg. acceleration- Slows begin to slow down, but not all at same time (culture not synchronized).
5. Stationary phase- all cells eventually enter this. No population increase at this point. Depletion of nutrients, accumulation of toxins. Here, less susceptible to inhibitors. Most cells switch to low levels of metabolism "suspended state". No longer dividing but can return to active state if conditions improve. Spore form. begins here. Don't incubate cultures beyond this phase. refrigerate before they get to this.
6. Death phase- after extended stationary, nutrient conc. so low and toxins so high that cells can't survive, even if metabolism is low. Most of cells die. Cells won't even recover if put into fresh media. Spore formation definitely here.
7. Survival phase (some species). Some cells may survive. Nutrient conc. doesn't drop much more nor are many new toxins made (most of the cells dead). Some cells can use nutrients from dead cells. Persistors.
When you compare generation time of different populations, you need to make sure they are in the same phase of growth. (usually exponential).
Counting microorganisms: (necessary to establish population size and to generate a growth curve).
Concentrate on advantages, disadvantages, and applications of each procedure.
1. Direct cell count. Fast (no wait for incubation). Tedious. Equipment relatively cheap in some cases. Lots of room for error. Doesn't distinguish living from dead.
a) Petroff-Hauser counting chamber. Accepts known vol. of liquid. Slide ruled into grid. Count a random sample of grids, get an avg. per grid. Multiply by # of grids. Gives estimation of # of cells per slide (get conc. in cells per ml. if slide has a know volume).
Problems with motility, seeing cells, can't tell living from dead.
b) Coulter counter-more expensive but faster, less tedious, and less error. Electron device to count particles in the culture (can be confused by debris). Flow cytometer is a similar device that can also measure cell mass and differentiate between living and dead.
2. Cultural methods (standard plate count)- Slow but differentiates living from dead.
Do serial dilutions of sample. Plate aliquots and incubate. Count colonies. 30-300 considered significant. Assumes each colony came from a separate cell (not always true). Errors in pipetting. Calc. Initial concentration of an unknown sample by the following formula: CiX df= Cf (df= mls. diluate/mls. diluate+ diluant).
CI=cf (# colonies)/df
CI=# colonies X 1/df
Some samples too dilute to do this way. Concentrate these with a millipore filter and trap bacteria on a sterile pad with media. Membrane filter technique for water analysis.
Either situation can be coupled with differential or selective media.
3. Cell mass-
Most common way to measure this is by turbidity. Turbidity measured with spec. Greater light absorbance more turbidity, more cells. Need to have std. curve to turn OD reading into a conc. Make standard curve by determining counts by one of other counting methods for several bacterial suspensions of different absorbencies. Have to make a new std. curve for each type of bacteria and each set of environmental conditions. Common technique in research and industry.
4. Cell activity-measure cultures ability to perform a particular function. Need a standard curve to quantitate results.
a) oxygen consumption (or some other substrate)
b) product formation (CO2)
5. Measurement of disease production-clinically important
Technique called 50% end point analysis is often used to determine titer (measure of relative conc. of a substance (pathogens). Requires that measured organism be able to produce specific clinical symptoms in a test animal.
Take sample and do several dilutions. Inject each dilution into a series of test animals. Greatest dilution that causes symptoms in at least 50% of test animals is 50% end point dilution. Titer is -log of 50% end point dilution (if dil is 10 minus 2) then titer is 2 (ID 50 or LD 50).> titer number, more infectious organisms.
Factors affecting population growth:
Nutritional- many have simple needs (water, energy, nitrogen, carbon, sulfur, phosphorous, growth factors, oxygen. Simple media provides these for most microbes. Some organisms (fastidious) have special needs (vitamins, cofactors, aa's)
Physical factors:influence enzymes tremendously
1. Temp. minimum, maximum, optimum (some pathogens have such a narrow temp range that they only live certain parts of the body-leprosy bacteria requires cooler temps-33-35 C -extremities). Others (S aureus)-6-46 C.
a. psychrophiles (0 min-20 max C)-optimum below 15. Deep ocean, polar ice. Facultative psychrophiles-optimum above 20 but grow slowly in the cold (even refrigerator).
b. mesophiles (optimum 20-40 C) (thermoduric-tolerate high temps for a short time). Majority of pathogens.
c. thermophiles (optimum > 45 C, most not higher than 80 but some up to 110 or higher). Many of these are being sought by biotechnologists (PCR works best at 65-72 C, need a DNA polymerase that works at this high temp).
Each related to enzyme temp. opt. Thermophiles have evolved ways to protect enzymes. Thermophiles found in undersea vents. Archebacteria. Evolution of living forms much faster under these hotter conditions.
2. pH- optimum related to enzymes. Fungi prefer more acidic than neutral bacteria. Thermoplasma-grow in hot coal piles of pH 1-2, lyse at 7.
b. facultative anaerobes (less growth in absence than presence of oxygen)
d. obligate anaerobes (aerotolerant-won't grow in o2 but won't be killed, strict anaerobe-killed by O2 (no enzyme to break down H2O2 or superoxide which are both made in presence of O2)
4. Solute concentration- too many solutes cause water to be pulled out of cells. Halophiles (min 15%, opt 25%-many are Archaebacteria). These can pump salt into the cytoplasm so that it is isotonic witht the environment. Saccrophiles (molds that grow in sugary jams). High salt tolerance of some species (Staph) allows them to grow where most other bacteria can't (salty skin surface)