Microbial Growth Control

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

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

Generation TimeLarge 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

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

Phases of Bacterial GrowthLo

g of

# o

f bac

teria

Time

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

Control of Microbial Growth

Degerming = removing microbes from a limited areaBiocide/germicide = killing microbesBacteriostasis = inhibiting, not killing, microbes

Sepsis = refers to microbial contaminationAsepsis = the absence of significant contamination• Aseptic surgery techniques prevent microbial

contamination of wounds

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

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

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

Heat

Thermal death point (TDP) = lowest temp at which the microbes in a liquid suspension will be killed in 10 minsThermal 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

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

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.

An autoclave!

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)

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

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

FiltrationFiltration = 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

Filtration

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

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

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

centuriesFreeze-drying = preserving cells through dessication- occasionally to preserve food

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

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

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

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

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

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

Structure of phenol, the phenolics and bisphenols

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

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…)

The Periodic Table

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

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

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

Summary: Phenols, Biguanines, Halogens, Alcohols

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

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 mouthwashesZinc pyrithione = used in antidandruff shampoo

Heavy Metal Ions and antimicrobial

activity

Charm and dime contain silver, penny contains copper

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

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

Types of Disinfectants:Surfactants

Quaternary Ammonium Compounds:

Zephiran = benzalkonium chloride • Surface disinfectants• Skin antiseptics

Cepacol = cetylpyridinium chloride• mouthwashes• sore throat sprays• Toothpastes (prevents plaque and

gingivitis)

- Both are colorless, odorless, tasteless- Stable, easily diluted, non-toxic

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

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

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

Summary: Heavy metals, Surfactants, chemical food preservatives

Figure 7.10 A comparison of the effectiveness of various antiseptics.

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

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

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

Summary: Aldehydes, chemical sterilization, peroxygens

Figure 7.11 Decreasing order of resistance of microorganisms to chemical biocides.