Chapter 6
Bacterial Growth
Objectives (Ch. 6)
3. Diagram the growth curve of a
bacterium and explain what is occurring during each phase.
4. Characterize the optimum growth conditions of bacteria, including effects of temperature, oxygen, pH, and complex growth factors.
5. Describe, compare and contrast the
different types of laboratory media used to
grow bacteria.
I. Mechanism and Stages of Bacterial Reproduction and Growth
A. Method
of reproduction - binary fission
1.
chromosome
replication and separation
2.
cell
membrane pinches in two
3.
cell
wall septum forms between two "daughter" cells
B. Generation time - time required for cell division to
occur. Range 20 min (E. coli) to
days to months (Mycobacterium leprae)
C. Bacterial growth curve
1. Lag phrase - resting (1-2 hours) no
division BUT high metabolic activity ADAPTATION to new environment
a.
store
nutrients
b.
synthesize
enzymes
c.
prepare
for division
2. Log phase - most rapid growth (from
end of lag to ~18-24h) maximum metabolism, cell # doubles with each generation
time until nutrients depleted/waste products reach toxic levels. A chemostat
gives a constrict flow of new media in/old out (including waste products)
maintains culture in log phase. With
infections, the disease symptoms appear in log phase as the organism grows in
the body.
3. Stationary phase - cell death and
cell division are equal. Nutrients almost
gone, toxic products building up (ex. pH changes). Sporeformers start to form
spores in late log and early stationary phase.
4.
Decline (death) phase - nutrients spent, more death than reproduction,
toxic byproducts leads to DEATH (victims of their own environment).
II. Effects
of Various Parameters (conditions) on Bacterial Growth
A. Temperature Effect on Growth- bacteria classified
according to their minimum VS optimum VS maximum growth temperatures (range)
1. Psychrophiles - 0-20°C optimum growth. No known
psychrophilic human pathogens.
2. Mesophiles - 20-40°C preferred range, optimum
35-37°C = 98°F body temp (most human
pathogens). This causes special
concerns with refrigerated foods
A special group
of mesophiles (Pathogenic Psychrotrophs) can grow (slower) at 5°C
a.
Staphyloccus aureus
b.
Salmonella sp.
c.
Proteus vulgaris
d.
Yersinia enterocolytica (blood bag contamination, endotoxic shock)
3. Thermophiles - 40-90°C growth range
4. Extreme thermophiles 80°C + range- hot springs, deep
sea thermal vents (Archae)
B. Osmotic pressure - salt tolerance
1.
facultative
halophiles can tolerate ~2=15% salt
2.
extreme
halophiles need up to 30% salt to grow
C. Oxygen requirements for growth
1.
Aerobic (obligate) bacteria – require oxygen for growth (room air)
2.
Anaerobic (obligate) bacteria - no oxygen (it is toxic to them). Anaerobes are cultivated using using sealed
jars/bags with a chemical reaction system that consumes the O2
inside or in special media containing reducing agents (Na thioglycollate or
pyrogallate) to consume the oxygen. Human pathogens that are obligate
anaerobes:
a.
Clostridium tetani – tetanus
b.
Clostridium botulinum – botulism
c.
Clostridium perfringens - gas gangrene and food poisoning
3.
Facultative anaerobic bacteria – grow with (preferred) or without oxygen. Most human pathogens are facultative
anaerobes
4.
Microaerophilic - needs small amount of air
Capnophile - requires extra CO2 (~3-5%) for growth (Neisseria), grow
in candle jar
5.
Aerotolerant anaerobes- (Lactobacillus) used in fermentation of
dairy products & pickles
D. pH requirements for growth- most bacteria (especially
pathogens) optimum is 6.8-7.2 (remember blood pH is 7.35- 7.45) Few grow below pH 4. Most molds (fungi) prefer a pH of 5-6.
Acid-loving (acidophile) bacteria important in formation of:
1.
sauerkraut
- mold-like (fungi - pH 5-6)
2.
dairy
products - cheese, yogurt, sour cream, buttermilk
E. Patterns of nutrition -
based on carbon source (1st
part) & energy source (2nd part of terminology)
1.
Autotrophs (self-feeder) - can synthesize their own food from (CO2 fixation)
simple carbon sources. Energy can come
from light (photoautotrophs) or from chemicals (chemoautotrophs).
2.
Heterotrophs (feed on others) - use preformed organic food sources. Energy source as above, can come from light (photoheterotroph) or a chemoheterotroph
(energy and carbon source are usually same organic molecule. Most pathogens are
chemoheterotrophs
F. Specific Nutritional requirements (special growth factors
required?): Special Carbon source
(many prefer glucose), Special nitrogen source (often proteins), phosphorous,
some inorganic compounds like magnesium, zinc, and vitamins
1.
Fastidious (dysgonic) - picky eaters.
Requires enriched media with special growth factors.
2.
Non-fastidious (eugonic) - will grow in simple media
III. Bacterial
Cultivation in the Laboratory
A. Most bacteria can be grown (cultivated) using type(s) of prepared
artificial media (rather than living tissue) Agar- Complex polysaccharide
derived form brown algae, whose physical/chemical properties make it an ideal
solidifying agent for bacteria media (melts ~95°C, solidifies~45°C).
1.
Complex (natural) Media- ex. Nutrient broth/agar or Tryptic Soy broth/agar.
Commercially available.
2.
Chemically defined (synthetic) Media- No plant or animal extracts; each chemical
component known. (ex. glucose, NH3, SO4, KPO4,
Mg, SO4, NaCl). Some
fastidious bacteria can't grow on defined media- they need complex organic
growth factors (ex. vitamins, amino acids, purines, pyrimidines).
3.
Enriched media- 5% sheep blood; or other special enrichments (lab atlas p.41)
a. Blood agar, (to differentiate hemolytic
groups of pathogenic streptococci)
b. Chocolate agar (heated blood; turns brown)
(Neisseria gonorrhoeae, N. meningitidis)
4. Selective media - contains growth inhibitors
for some groups of bacteria, but will allow others groups to grow (lab atlas p.
10-20)
a. Salmonella-Shigella agar; Hektoen agar-
only gram negatives can grow
b. Columbia CNA agar; Phenylethyl alcohol
agar- only gram positives can grow
5. Differential media – usually contains
a substrate (lactose, mannitol, citrate, starch, urea, etc.) and an indicator
(phenol red, neutral red, bromthymol blue, hemoglobin, eosin, etc.). The indicator molecule gives a visual
difference in the actual growth or in
the area of the media immediately surrounding the growth, that distinguishes
between bacteria based on whether they can utilize (break down) the substrate.
a. Blood agar- differentiates based on
hemolysis reactions (p. 41)
b. Milk agar- differentiates based on casease
production
c. Spirit Blue agar- differentiates based on
lipase production
d. MRVP broth- differentiates based on type
of lactose fermentation (mixed acid/neutral)
Many bacteriologic media
contain a combination of enrichment &/or selective &/or differential
agents
(for example):
|
Media |
Enrichment Agent |
Selective Agent |
Differential Agent |
pH ind. |
|
Blood Agar |
RBCs |
- |
Hemoglobin |
- |
|
Eosin Methylene Blue Agar |
- |
eosin & methylene blue |
lactose |
- |
|
MacConkey Agar |
- |
crystal
violet & bile salts |
lactose |
neutral red |
|
Mannitol Salt Agar |
- |
7.5% NaCl |
mannitol |
phenol red |
B. Cell culture – some pathogenic bacteria, most protozoal
& helminth pathogens and all viruses cannot be grown on artificial
laboratory media, and must be grown either in
living, cultured host cells (tissue culture) or in laboratory animal
(animal model).
1.
Rickettsia, Chlamydia, & viruses- cell culture or animals
2.
Mycohacterium leprae culture in animals (nude mouse foott pads or
armadillos)
IV. Relationships
Between Microbe and Living Host
A. Normal flora -
organisms normally present in the absence of disease; they normally prevent the
overgrowth of harmful organisms by microbial antagonism (by affecting
pH, O2 availability, bacteriocin production, etc.) (Table 14.1 in
text)
1.
Skin- Staphylococcus epidermidis & S. aureus,
Propionibacterium, Corynebacterium,Pityosporum & Candida (fungi)
2.
Upper Respiratory Tract- Staphylococcus
epidermidis & S. aureus, Streptococcus pneumoniae,
Hemophilus,Neisseria, diptheroids
3.
Mouth-
Streptococcus, Lactobacillus, Corynebacterium Actinomycetes, Treponema,
Bacteroides, Fusobacterium, Candida
4.
Large Intestine- Gram – enterics (E. coli, Enterobacter, Citrobacter, Proteus,
Klebsiella, Shigella), Enterococcus, Lactobacillus, Bacteroides, Fusobacterium,
Clostridium, Candida
5.
Urethra- S.epidermidis,
Micrococcus, Enterococcus, Lactobacillus, Pseudomonas, Klebsiella, Proteus, diptheroids
6.
Vagina-
Lactobacillus, Streptococcus, Staphylococcus, Bacteroides,
Clostridium, Candida, Trichomonas vaginalis (protozoan)
7.
Male external genitalia- Mycobacterium smegmatis, other skin flora
B. Symbiosis - Two populations living together in a close and
permanent association. Different types:
1. Mutualism - both
populations benefit. Examples include:
a. Rhizobium - bacteria in legume root
nodules fix Atmospheric N2 for plain legumes, the soybean, alfalfa
root nodule supply carbon and protected environment for bacteria.
b. E. coli & others in gut- make vitamin K & some B vitamins, and
they get nutrients from our food
2. Commensalism - one population
benefits while the other is neither helped nor harmed. Examples include: Corynebacterium in
eye conjunctiva, Mycobacterium in external ear & genitals
3. Synergism - two populations live together to accomplish
what neither could accomplish alone.
Example: Trench mouth (acute
necrotizing ulcerative gingivitis)- 2 or more bacterial species needed for oral
infection.
4. Parasitism - beneficial to one
population (parasite), but harmful to the other (host). Example: Any disease
causing bacteria, fungi, protozoa, and viruses.
V. Pure cultures - obtain with streak on pour plates
preservation: 1) refrigeration
2) freeze in glycerol: H2O
3) flash freezing in liquid
N2
4) lyophilization - freeze
dry (vacuum)
VI. Measuring
Bacterial Growth
A. Direct Methods:
The first 3 methods below only count live cells
1. Plate counts- using serial dilutions. colony forming units (cfu) = colony count_____________
dilution factor X amount plated
a. spread plates - surface colonies
b. pour plates - surface AND sub-surface colonies
2. Filtration - for dilute food/H2O
samples – use 0.22 - 0.45 um pore size, filter up to 100 ml. Place filter on solid growth median-
colonies grow on filter.
3. Most Probable Number (MPN) – 3 series
of tubes of liquid media are inoculated with 10-fold dilutions of food or water
sample, then tubes with growth (or gas) are counted and a statistical table is
used to estimate bacterial concentration in the original sample.
4. Direct Microscopic count-
a. Petroff Hauser cell counter (similar to
hemacytometer)- slide with well of known vol. and cross hatch pattern (count
known portion of well & use to calculate # bacteria/ ml or / ul)
b. Coulter counters - automatic cell counting
B. Indirect Methods
1. Turbidometric (use spectrophotometer)
– shine light through sample, optical density (O.D.) measured by photoelectric
cell. During log phase O.D. versus time plot gives a straight line
on semi-log paper to allow calculation of Generation (doubling) time of a bacterium - must do control colony counts
first time for each different bacterial and each media and incubation
conditions used.
2. Measure acid or CO2 (respirometer) produced
3. Dry Weight Measurement- at regular
intervals, withdraw small sample volume, dry, and weight cells. Especially good for filamentous organisms
(fungi, Actinomycetes/Streptomycetes)