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Chapter 7

The Control of Microbial Growth


Control of Microbial Growth
• Sterilization = the complete removal or killing of all
microbes - they are unable to reproduce even in
most favorable conditions – no partial sterilization
– Endospores are one of the most resistant forms of life
• Their destruction is the goal of sterilization because any
process that kills endospores will kill the vegetative cell
– Used on inanimate objects or material ex. Liquid media
Control of Microbial Growth
• Disinfection = the reduction or removal
of vegetative pathogens but not
endospores by chemicals, UV radiation,
boiling H2O or steam
– Chemical agents used
• If object is inanimate (tabletop) = disinfectant
• If object is living (animal tissue) = antiseptic
– Antiseptics are regulated by FDA
Some definitions
• Sepsis = Greek for putrid
– Refers to growth of microbes in blood and
other tissues
– Is stem for septicemia (in blood), antiseptic,
aseptic
• Degerming = mechanical removal (not
killing) of microbes from surface of
object
– Ex. ROH w/ shot, wash hands
Some definitions
Physical Control of Microbes
• Thermal death point (TDP) = the lowest temp.
required to kill all microbes in a liquid culture
in 10 minutes
– For spore bearing organisms there are 2 TDPs – 1
for the vegetative cell and 1 for the spore form
• Thermal death time (TDT) = length of time to
kill all microbes in a liquid culture at a given
temperature
• TDP and TDT are important in the food industry
wh/ uses heat for preservation
Sterilization by Heat
• Heat is fast, reliable and cheap and does not
introduce toxic substances into material being
treated
• Moist heat
– Kills microbes by denaturing proteins – breaks H-
bonds thus chgs shape
– Penetrates material faster than dry heat so need less
time and lower temperatures
– Boiling H2O – not a sterilizing agent because of
spores and viruses
Sterilization by Heat
• Autoclave (pix –pg 191) = pressure is used to
raise temp of steam > 100 Deg. C
– Most rapid and efficient method of sterilization by heat
– As pressure > temp. rises
– At 15 psi pressure the temperature rises from 100 deg C
to 121 deg C
– 15 psi at 121 deg C 15 – 30 min depending on what
you are sterilizing
Sterilization by Heat
• Autoclave continued:
– Used to sterilize culture media, instruments,
dressings, glassware, equipment (transfusions)
• Steam can’t penetrate Al foil – use paper
– Sterilization check w/ Bacillus spores –
incubate to check for growth, tape
• Pasteurization – destroys Mycobacterium
tuberculosis (TB) and Coxiella burnetii (Q
fever)
Sterilization by Heat
• Pasteurization continued:
– Purpose is to reduce bacterial populations of
liquid (milk, juice) and to destroy organisms
that cause spoilage and human disease
without affecting flavor
– Does not sterilize – spores aren’t affected
– Methods
• Classic = heating at 63 deg C 30 min
• High temp short time tx – 72 deg C 15 sec. – kills
pathogens and lowers bacterial counts, >shelf life
Sterilization by Heat
• Pasteurization cont.
– Ultra – high temp tx
• – 140 deg. C for 1 sec
• Holding tube 3 sec.
• Vacuum chamber 1 sec to cool
• Can then store milk without refrigerating
– Actually sterilizes milk
Sterilization by Heat
• Dry heat – Direct flame – incinerates
microbes rapidly
– Bunsen burner – inoculation loop, culture
tube – organisms, dust, lint
– England 4 million cows in 2001 – infected with
hoof and mouth disease
– Used to burn diseased bodies centuries ago
– Cows and fields exposed to Anthrax (spores)
Sterilization by Heat
• Dry heat – hot air ovens
– Does not penetrate material easy so needs
long periods of time at high temps – 170 deg
C at least 2 hrs
– Used to sterilize dry powders, water free oily
substances, glassware
– Changes microbial proteins by oxidation rxs
Filtration

• Used in food industries, laboratories (medical,


environmental, industrial)
• A filter is a mechanical device used to remove
microbes from a solution or gas
• Several types of filters used in a laboratory
– Membrane filter – cellulose acetate or
polycarbonate – 0.22um, 0.45um, .01um – viruses, lg
protein molecules
Filtration

• Membrane filtration cont.


– Can use to sterilize media or any liquid
that can’t tolerate heat
• Serum, blood products, drugs, IV fluids, enzymes

– Can use for a bacterial count – traps


bacteria on filter which is place on media in a
petri dish and incubated
Membrane Filtration of Liquids Bacteria removal
is not quite
equivalent to
sterilization.

Bacteria removal
is easier than
removal of
viruses.
Filtration
• HEPA filters (high efficiency particulate
air) – removes microbes from air
– Removes 99 % of particles > 0.3um
– Used in hospitals – whole rooms, labs –
hoods, home air filters
HEPA Filtration of Air
Control of Microbial Growth
• Low temperatures – refrig, freezer
– Slows growth but does not kill
– < enzyme activity, lowers rate of chemical rxs in
cytoplasm
• Desiccation = drying
– Viruses, endospores resistant
• Lyophilization = freeze drying
– Coffee, fruit additives for cereals, used to
preserve microbes in laboratory
Control of Microbial Growth
• Osmotic pressure
– High salt, sugar or spice external environments
(hypertonic environment) – higher water
concentration in cell than outside cell causing
H2O to flow from the cytoplasm thru cell
membrane out to the environment
– Causes cell to shrivel and die – cell membrane
pulls away from cell wall = plasmolysis
– Preserves foods – ex. Ham, bacon
Radiation
• Ionizing radiation = high energy radiation that
induces electrons to jump out of molecules they
strike and create ions = atoms or molecules that
lack 1 or more electrons
– ex. X-rays, gamma rays, high energy electron beams
– X-rays and gamma rays enter cells and break bonds in
DNA causing cell mutations and death
Radiation
• Ionizing radiation cont.
– Some foods have been irradiated for more than 50
years
• Radiation is used to kill bacterial pathogens, insects,
worms and to inhibit the sprouting of potatoes ( > shelf
life of food)
– Used to sterilize heat sensitive vitamins,
hormones, Abs, plastics, suture material
• Used to sterilize mail after Anthrax scare
• Used to preserve food
• Bone, skin and heart valves for grafting - sterilized
Radiation
• Non-ionizing radiation - has a longer
wavelength and less energy
– Ultraviolet Light (UV) – has a wavelength of 100 - 400
nm
• Used for airborne or surface contamination in hospital rooms,
food industry, toilets
• Bacteria are destroyed at approx. 260 nm – causes thymine
dimers (bonds form between adjacent bases on the same
DNA strand) – bacteria can no longer produce proteins or
reproduce DRAW
– Disadvantage = does not penetrate liquids or solids
Chemical control of Microbes
tables pg 207-08
• Most chemical methods are unreliable for
sterilization but are effective as
disinfectants and preservatives
• Evaluating a disinfectant
– Phenol coefficient test – compare the
activity of a disinfectant with that of
phenol
– American official Analytical Chemist’s use –
dilution test – current standard
Evaluating a Disinfectant
• Use – dilution test
– Use Salmonella, Staphylococcus, and
Pseudomonas as test microbes
– A series of tubes containing concentrations of
the test disinfectants are inoculated and
incubated
– The more the chemical can be diluted and
still be effective the higher its rating
Evaluating a Disinfectant
• Disk-diffusion method – pg 199
– Filter paper disk is soaked with a chemical
and placed on an agar plate that has been
inoculated with a test organism and then
incubated
– After incubation if the chemical is effective will
see a clear zone of inhibition around the
disk
• Also used to evaluate microbial susceptibility to
Abs
Types of Disinfectants
• Phenol and phenolics (phenol derivatives)-
disrupt plasma membranes resulting in
leakage of cellular contents, denature proteins
• 1860’s – Joseph Lister established the
principles of aseptic surgery by using carbolic
acid (phenol) on wounds and instruments
• Phenol is caustic to skin and has been replaced
by phenol derivatives wh/ are better germicidals
and have lower toxicity
Types of Disinfectants
• Phenol derivatives
– Cresols – derived from coal tar
• O-phenylphenol used in Lysol, Amphyll
– Bisphenols = 2 phenol molecules –
prominent in modern disinfection
• Hexachlorophene = pHisoHex – combined with
detergent to bath infants to retard Staph infections
of scalp and umbilical cord – found could be
absorbed through the skin and cause neurological
damage – removed from OTC products
Types of Disinfectants
• Bisphenols cont.
– Triclosan – broad spectrum antimicrobial agent – destroys plasma
membrane by blocking lipid synthesis
• Found in soap
• Halogens oxidize proteins, disrupts membranes
– Chlorine – as gas or in combination with other chemicals
• Germicidal action caused by hypochlorous acid (HOCL) that forms when
chlorine is added to H2O
• Sodium hypochlorite 5% - Clorox bleach, disinfectant in dairies, food
processing plants, hemodialysis systems
– In emergency CDC recommend 2 drops/liter (clear H2O) for 30 min.
• Chloramines (Cl + ammonia) release free chlorine slower than hypochlorite
solution and are more stable
– Used for disinfectants, antiseptics, sanitizing agents, tx of H2O supplies
Types of Disinfectants
• Iodine – more germicidal than chlorine,
found in marine seaweed
– Tincture of iodine (iodine dissolved in ROH)
– used as antiseptic for wounds
– Iodophors = iodine-detergent complexes
that release iodine over a long period of
time ex. Betadine – for local wounds
Iodine & Iodophores
Types of Disinfectants
• Alcohols – denature proteins and
disrupt membranes
• ETOH and isopropyl ROH are effective
in killing vegetative bacteria and fungi
but not endospores or nonenveloped
viruses
– Used to disinfect thermometer, medical
instruments, mechanically remove bacteria
from skin before drawing blood or giving a
shot
Heavy Metals
• Hg, Ag, Cu – denatures proteins
• Small amts of heavy metal (Ag, Cu) have
antimicrobial effects = oligodynamic action
– Demonstrated when place a coin on petri dish w/ bacteria
and growth is inhibited from diffusion of metals into media
– 1% silver nitrate solution used to be placed in eyes of
newborns to guard against Neisseria gonorrhoeae
• Can cause blindness if contracted while going thru birth canal
• Can cause eye irritation so now use antibiotics
Heavy Metals
• Hg used in the form of mercuric chloride used
for skin disease but toxic to skin
– Mercury compound – thimerosal – vaccines, 1999
CDC advised removal
• Copper as Cu sulfate is used as an algicide to
control algal and cyanobacteria growth in
swimming pools, fish tanks, and H2O supplies
• Zinc chloride used in mouth washes, Zn oxide
is used in paints as an anti-fungal
Types of Disinfectants
• Soaps and detergents are surfactants that
emulsify particles and reduce surface
tension, good degerming agents
• Aldehydes – denature proteins
– Formaldehyde
• Gas at high temps used to sterilize hospital
gowns, medical instruments
• MC as formalin a 37% aqueous solution used to
preserve biological specimens, embalming fluid,
inactivate bacteria and viruses in vaccines
Aldehydes
• Glutaraldehyde – disinfectant that can be
considered as a sterilizing agent
– Kills bacteria and viruses in 10 min. and
spores in 3 -10 hrs
– Used to sterilize endoscopes, respiratory
therapy equipment
– Also used for embalming