2. Bacterial Cultures
Bacterial growth = refers to an increase in the number
of bacterial cells (population) not in individual cell size
Colony = visible bacterial growths; arose from single cell
Isolated colonies (visibly separated from each other)
represent pure cultures = genetically identical clones
Streak plate method = most common laboratory method
of obtaining pure cultures
3. Generation Time
Generation time = the time it takes for a cell to
divide (and thus for its population to double)
binary fission = most common method of
reproduction by bacteria
• Cell divides to produce 2 identical daughter cells
GT varies according to the organism and
environmental conditions
Ex) E. coli GT is 20 mins in optimal conditions
• After 20 generations (7 hrs) 1 million cells
• After 30 generations (10 hrs) 1 billion cells
4. Generation Time
Large magnitude of population numbers requires us
to use logarithmic scales when graphing growth
Can more clearly show us population changes when
dealing with extremely large values at the end of a curve
5. Phases of Bacterial Growth
A bacterial growth curve can be plotted showing
growth over time of a pure culture inoculated from a
small amount of bacteria
Four basic stages of growth:
Lag = little/no cell division, adjusting to new medium
Log = exponential growth, appears as a straight line
when plotted logarithmically
Stationary = growth slows, new cells = dying cells
Death = dying cells > new cells formed
7. Control of Microbial Growth
Sterilization = elimination of ALL microbial life
• High heat is most effective
• Endospores most resistant
Commercial sterilization = use of heat to a level that
does not destroy the food, but kills Clostridium
botulinum endospores (produce toxin)
• Thermophilic bacterial endospores survive
• Won’t grow at room temp, though
Clostridium
botulinum
8. Control of Microbial Growth
Degerming = removing microbes from a limited area
Biocide/germicide = killing microbes
Bacteriostasis = inhibiting, not killing, microbes
Sepsis = refers to microbial contamination
Asepsis = the absence of significant contamination
• Aseptic surgery techniques prevent microbial
contamination of wounds
9. Control of Microbial Growth
Disinfection = destruction of vegetative pathogens
• Usually achieved with chemicals
• Sometimes UV radiation or moderate heat (boiling
water or steam)
Antisepsis = a gentler version of disinfection for
destruction of pathogens on living tissue
• Chemicals used = antiseptics
Sanitation = measures of microbial control to protect
public health. May include antisepsis, disinfection, or
sterilization
10. Actions of Microbial Agents
1. Alter membrane permeability
• Damage to membrane lipids or proteins
diminished cell growth
2. Damage proteins
• Proteins can be denatured by heat & chemicals
3. Damage nucleic acids
• Nucleic acids (DNA & RNA) damaged by heat,
chemicals, or UV
- Hinders replication and protein synthesis
11. Microbial control:
Physical methods
Various physical (non-chemical) methods can be
used to kill or inhibit growth of microorganisms:
• Heat
• Filtration
• Cold
• High pressure
• Dessication
• Osmotic pressure
• Radiation
12. Heat
Thermal death point (TDP) = lowest temp at which the
microbes in a liquid suspension will be killed in 10 mins
Thermal death time (TDT) = minimum length of time for all
microbes in a liquid culture to be killed at a given temp
• TDP and TDP indicate severity of treatment (temp and
time) required to kill a given population of bacteria
Decimal reduction time (DRT, D-value) = time (mins) in which
90% of a bacterial population will be killed at a given temp
- important in canning industry
13. Moist heat sterilization:
boiling & unpressurized steam
• kills organisms by denaturing their
enzymes
• Boiling water destroys most
bacteria, viruses, fungi and
spores within 10 mins
• Unpressurized steam works in the
same way (it’s the same temp as
boiling water)
• Bacterial endospores, hepatitis
viruses survive 30 mins to 20
hours of boiling/steam
14. Moist heat sterilization:
autoclaving
Autoclave = machine utilizing pressurized steam to
produce even hotter temps than boiling/free-flow steam
• higher pressure = higher temp
• Effective if material to be sterilized isn’t damaged
by high heat or moisture
• An autoclave at 15 psi (121oC) kills ALL microbes and
their endospores in 15 mins
• Used in laboratories and medical settings to sterilize
culture media, solutions, instruments, etc.
16. Moist heat sterilization:
Pasteurization
Louis Pasteur founded a method of mild heating to
prevent spoilage of foods
• Later, method was applied to milk:
Pasteurization = elimination of pathogens and reduction
of overall microbes in milk through mild heating
• Can be used for other foods as well: beer is one
Phosphatase test = properly pasteurized milk will have
inactive phosphatases (enzymes naturally found in milk)
17. Moist heat sterilization:
Pasteurization
High-temperature short-time pasteurization (HTST) =
heat (72oC) is applied to milk for 15 seconds as it
flows past a heat exchanger
Ultra high temperature (UHT) sterilization = can be
used to sterilize milk for storage at room temperature
by exposing it to 140oC for 4 seconds followed by
rapid cooling and storage in an airtight container
18. Dry heat sterilization
• Kills microbes by oxidation
Flaming = using an open flame for
sterilizing inoculating loops and
other lab tools
Hot-air sterilization = use of an
oven at about 170oC for 2 hours for
sterilization of various materials
19. Filtration
Filtration = passage of liquid/gas through a screen-like
material with pores too small for microbes to pass
• Vacuum creates a negative air pressure in a
receiving flask forces the liquid to pass through
the filter into the flask
• Used on heat-sensitive liquids
High efficiency particulate air (HEPA) filters = remove
airborne microbes larger than 0.3 μm in diameter
Membrane filters = filters with pores as small as 0.1
μm for industrial and lab use to filter bacteria
21. Cold
• Inhibits microbial reproduction and toxin synthesis
• Effect varies depending on the microbe and how cold
the applied temperature is
Psychrotrophs = grow at slightly colder temperatures
(like in the fridge) and alter taste & appearance of food
• Freezing: some bacteria can grow below freezing
• Slow freezing is more harmful than rapid freezing;
ice crystals form in the cell and disrupt cell structure
22. High Pressure
Can inactivate bacterial cells by denaturing
proteins within the cell
• Bacterial endospores relatively resistant
• Favored for some food products
• Preserves flavor, color and nutrient value
• Commonly used to treat fruit juices
23. Dessication
Microbes cannot grow or reproduce under conditions
of dessication (absence of water)
• Can remain viable, but dormant for years
• When water is available, growth & division resumes
• Bacterial endospores can lie dormant for centuries
Freeze-drying = preserving cells
through dessication
- occasionally to preserve food
24. Osmotic Pressure
Highly concentrated salts and sugars can preserve
food because of osmotic pressure
Hypertonic environment = high concentration of
solute outside the cell causes water to leave the cell
by diffusion ( plasmolysis)
• Similar situation to dessication
• High salt commonly used to preserve meat
• High sugar used to preserve fruits
25. Radiation
Radiation can kill microbes depending on wavelength,
intensity and duration
Ionizing radiation = shorter wavelength (high energy)
• Gamma rays
• X-rays
Produce hydroxyl (-OH) radicals from the ionization of
water that react with cell components like DNA
Non-ionizing radiation = longer wavelength (low energy)
• UV light
Damages DNA by pyrimidine dimers interfere with
proper replication
26. Microbial Control:
Chemicals
• Chemicals can reduce bacterial populations
or remove vegetative (non-endospore) forms
• Generally don’t achieve sterility
• Different disinfectants are effective for
different microbial groups
27. Evaluation a disinfectant:
Disk-diffusion method
Paper disk is soaked in a chemical and placed on
an agar plate inoculated with test organism
Zone of inhibition = clear area where
chemical inhibited microbial growth
28. Types of Disinfectants:
Phenol
• Disrupt plasma membrane lipids
Phenol = has antiseptic and disinfectant properties
but is irritating to skin and has bad odor
Phenolics = derivatives of phenol altered to reduce
irritating qualities
• Stable – not easily inactivated
• After application, continue to disinfect for
long periods of time
O-phenylphenol = a phenolic of the group cresols,
main ingredient in disinfectants like lysol
29. Types of Disinfectants:
Phenol
Bisphenols = derivatives of phenol containing two
connected phenolic groups
Triclosan = a bisphenol used in antibacterial soaps and
some toothpastes
• Inhibits fatty acid (lipid) synthesis plasma
membrane integrity
• Especially effective against gram (+) bacteria
• Can be used on gram (-) bacteria and yeast too
• Widespread use resistant bacteria
31. Types of Disinfectants:
Biguanides
Biguanides = effective against gram (+) and gram (-
) bacteria by disruption of plasma membranes
• Not sporicidal
• Some activity against enveloped viruses
Chlorohexidine = biguanide used on skin and
mucous membranes
• Combined with detergent or alcohol for pre-op
skin preparation and surgical hand scrubs
32. Types of Disinfectants:
Halogens
Halogens = Five non-metallic elements in group 17 of
the periodic table
• Fluorine
• Chlorine
• Bromine
• Iodine
• Astatine
• Highly electronegative (attract other electrons)
thus highly reactive with atoms of other elements
Hydrogen halides = the halides form compounds with
hydrogen strong acids (HF, HCl, HBr…)
34. Types of Disinfectants:
Halogens
Iodine = One of oldest and most effective antiseptics
• Effective against all kinds of bacteria, their
endospores, viruses, and fungi
• Complexes with amino acids and fatty acids to
impair protein synthesis and alter cell
membranes
Betadine = an iodophor (combo of iodine and an
organic molecule) that consists of iodine and povidone
for the slow-release dispersion of iodine
35. Types of Disinfectants:
Halogens
Chlorine = elemental chlorine (Cl2) gas forms
hypochlorous acid (HOCl) when added to water
• HOCl diffuses through cell wall as fast as water
• Inactivates cellular enzymes
Sodium Hypochlorite (NaOCl) = chlorine compound
used in bleach for disinfection
Chloramines = Chlorine in combination with ammonia;
stable compounds for slow release of chlorine
• Used for glassware and eating utensil sanitation
36. Types of Disinfectants:
Alcohols
Alcohols = kill bacteria and fungi, but not endospores
and non-enveloped viruses
• Denaturation of proteins
• Disruption of lipid membranes
• Evaporate to leave no residue
Ethanol = Aqueous solutions (~70%) most effective for
denaturation
Isopropanol = aka rubbing alcohol; less volatile
38. Types of Disinfectants:
Heavy Metals
Heavy metal ions = combine with sulfhydryl groups on
cellular proteins denaturation
• Silver
• Mercury
• Copper
• Very small amounts can kill or inhibit growth
Silver in antimicrobials:
• Plastic food containers with silver nanoparticles
• Silver-sulfadiazine cream for use on burns
• Surfacine surface disinfectant with silver iodide
39. Types of Disinfectants:
Heavy Metals
Mercury in antimicrobials:
Mercuric chloride = toxic and corrosive compound;
broad spectrum and bacteriostatic (inhibits growth)
Copper in antimicrobials:
Copper sulfate = used to destroy green algae in
pools, ponds and fish tanks at 1 ppm
Zinc in antimicrobials:
Zinc chloride = used in mouthwashes
Zinc pyrithione = used in antidandruff shampoo
40. Heavy Metal Ions and
antimicrobial activity
Charm and dime
contain silver, penny
contains copper
41. Types of Disinfectants:
Surfactants
Surfactants = aka surface acting agents; decrease
surface tension of molecules making up a liquid
• Soaps
• Detergents
• Don’t kill or inhibit growth of microbes,
mechanically remove them through scrubbing
• Breaks the film of oil, dead skin cells, sweat and
microbes into droplets emulsification
• Rinsing with water lifts and carries away the
emulsified droplets
42. Types of Disinfectants:
Surfactants
Quaternary Ammonium Compounds = most widely
used surfactants; modifications of the (+) charged
ammonium ion
• positive charge gives “quats” their cleansing ability
• Kill gram (+) bacteria, fungi, amoebas, and
enveloped viruses
• Don’t kill endospores or mycobacteria some can
actually grow in the disinfecting solution
• Likely affects plasma membrane
44. Types of Disinfectants:
Food Preservatives
Chemical food preservatives = added to food to prevent
spoilage; simple organic acids or salts of organic acids
• Sulfur dioxide (SO2)
• Sodium benzoate
• Sorbic acid
• Calcium propionate
• Regarded as safe to eat
• More acidic foods (pH lower than 5.5) are susceptible to
mold spoilage
• Calcium propionate = prevents growth of surface molds
45. Types of Disinfectants:
Food Preservatives
Sodium nitrite = preserves meat like ham, bacon, hot
dogs & sausage by preventing botulism endospore growth
• Inhibits iron-containing enzymes of C. botulinum
• Nitrosamines = carcinogens thought to be produced by
reaction of nitrates with amino acids
• Nitrates are still used because of their value in
reducing botulism
• Formed in the body by other sources, so risk of
cancer by nitrates in food is thought to be low
46. Types of Disinfectants:
Food Preservatives
Antibiotics = limited use in food preservation
Nisin = antibiotic added to cheese to inhibit growth
of endospore-forming bacteria
• Tasteless
• Readily digested
• Non-toxic
Natamycin = aka pimaricin; antifungal antibiotic
commonly used in cheese
48. Figure 7.10 A comparison of the effectiveness of various antiseptics.
49. Types of Disinfectants:
Aldehydes
Aldehydes = some of the most effective antimicrobials
• Inactivate proteins by cross-linking with functional
groups (–NH2, –OH, –COOH, –SH)
Formaldehyde = usually in the form of formalin, a 37%
aqueous solution of formaldehyde gas
Glutaraldehyde = less irritating and more effective;
commonly in the form of a 2% aqueous solution (Cidex)
• Bactericidal, tuberculocidal, and viricidal within
10 mins and sporicidal in 3-10 hours
50. Chemical Sterilization
Ethylene oxide = gaseous chemosterilant
• replaces proteins’ hydrogen atoms with a
chemical radical denaturation
• Applied in a closed chamber over several hours
• Toxic and explosive in pure form usually
mixed with a non-flammable gas such as CO2
Chlorine dioxide = used to fumigate enclosed
areas contaminated with anthrax endospores
51. Types of Disinfectants:
Peroxygens
• oxidizing agents used to disinfect inanimate objects
Hydrogen peroxide = broken down to water and oxygen
by catalase in aerobic cells
• Sporicidal at high concentrations
Peracetic acid = one of most effective liquid chemical
sporicides; can be used as a sterilant
• Kills endospores and viruses in 30 mins
• Leaves no toxic residues
• Can be used for washing fruits and vegetables
