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
Eosin Methylene Blue Agar
eosin & methylene blue
crystal violet & bile salts
Mannitol Salt Agar
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)