Chapter 7 micro

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


• 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


• 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


• 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)


• 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


• 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


• 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)


• 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

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Bacteria removal is easier than removal of viruses.

Bacteria removal is not quite equivalent to sterilization.

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

HE

PA

Filt

ratio

n of

Air


• 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


• 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 Microbestables 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


• 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


• 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


• 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

Iodi

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Iodo

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es


• 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


• 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

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Chapter 7 micro