chapter 12

3) use dilution test

a 3rd way to evaluate chemical agents bacteria are grown until they are of a certain concentration 1 times 10 times 10 times 10 times 10 times 10 times 10 times 10 times 10 times 10 /ml ( 0.5 mcfarland standard )

antiseptic

a chemical agent that can be safely used externally on tissues to destroy microbes or to inhibit their growth some chemical agents are used as both but most disinfectants are too harsh to be used on the skin although some antibiotics are often used topically

disinfectant

a chemical agent used on an inanimate object to destroy microbes

3) pasteurization

a mild heating to destroy pathogens and other organisms that cause spoilage -louis pasteur it is used in 1) wine industry 2) milk 3) other dairy products 4) beer except draft beer the 2 two that we are worried about are salmonella and mycobacterium 2 methods 1) regular (holding) 62.9 degrees celcius for 30 mins 2)flash 71.6 degrees for 15 mins

2) bactericide

agent that kills bacteria(most do not kill spores)

4) freeze drying

also called lyophilization - the drying of material from the frozen state remember tasters choice coffee- also used in the long term storage of microorganisms atcc collection

refrigeration, freezing, drying, and freeze drying

although heat is much more effective in killing microorganisms cold can be used to retard the growth of microorganisms and prevent spoilage- recall the rule of 10

3) viricide

an agent that inactivates viruses

bactericidal agent

an agent that kills bacteria 5 different types

5) sporocide

an agent that kills bacterial endospores or fungal spores

4) fungicide

an agent that kills fungi

bacteriostatic agent

an agent that prevents further multiplication of bacteria (growth) without necessarily killing them

pathogen

any organism capable of causing disease in its host

2) organic material

as we have already stated, organic debris slows down the effectiveness of any chemical agent 3) temperature- increased temp speeds up the death rate of bacteria recall the rule of 10- any increase of 10 degrees centigrade roughly doubles the rate of chemical reactions, and thus, increases the potency of the chemical agent 4) pH acidic or alkaline pH agents can either increase or decrease the potency of the agent much of this relies upon the bacteria you are trying to kill since some prefer low or high pH

2) moist heat

better than dry heat- better penetration boiling kills some organisms but not all viruses and spores autoclaving- this procedure increases the temp of the water ( moisture) above 100 degrees Celsius it is this increase in temp that kills the microorganisms not the pressure- the presure allows water to be still liquid above 100 degrees celsius ..don't want to pack the autoclave or you will have to increase the time to allow for the stream penetration we check the efficiency of the autoclave with 1)specific tape 2) diacs- melt if okay 3) use cultures of heat resistant, spore forming bacteria, then culture to check for growth

advantages

cheap don't clog easily can filter large volumes rapidly can be autoclaved or purchased sterilized

mechanisms of action of chemical agents

chemical agents kill microorganisms 3 main ways and may be grouped accordingly 1) those that affect proteins, 2) those that affect cell membranes 3) those that affect other cell components there are those agents that inactivate viruses

1) reactions that affect proteins

denaturation- may be temp or permanent if temp- bacteriostatic if perm-bactericidal these agents hydrolyze proteins- denature them the hydrogen and disulfide bonds that hold the protein together in its 3 d shape (configuration) are broken without the appropriate structure there is no function some proteins, when denatured can regain ( renature) the appropriate configuration so as to properly function

exceptions to increased concentrations

ethyl and propyl alcohol - best at 70 % because water is needed to 1) denature proteins 2) increase the penetration of the alcohol into materials being disinfected

radiation

four types 1) uv light- light waves between 40 to 390 m 200 nm is best at killing-especially effective on viruses ( just nucleic acid and protein) PYRIMIDINE DIMERS dna can repair itself and then go on to make proteins so time is a factor in its effectiveness uv light does not penetrate very well ex-it doesn't penetrate glass, plastic, paper it also doesn't penetrate cloth well ex- white t shirt has an spf of 7 uv light doesn't go around corners/ number one cause of malignant cancer/ replacing chlorination in many sewage plants

4) strong visible light

has a bactericidal effect due to uv rays in sunlight

fomite

inanimate object

5) concentration

increased concentration of a chemical generally increases the potency of MOST chemical agents high concentrations may be bactericidal while lower concentrations may only be bacteriostatic

phenol coefficient

it is a very standardized method: for ex the organisms used are kept constant the 2 organisms normally used in the comparison are salmonella typhi (G-rod) and staphylococcus aureus ( G+ coccus) a phenol coefficient of 1.0 means that the disinfectant being compared to phenol has the SAME effectiveness as phenol a phenol coefficient of 2.0 means that the disinfectant being compared to phenol has twice the effectiveness as phenol a phenol coefficient of 0.5 means that the disinfectant being compared to phenol has half the effectiveness as phenol

1) germicide

kills certain microorganisms

disadvantages

many ( especially large pore sizes) allow viruses to pass they may absorb large amounts of filtrate they may introduce metallic ions into the filtrate these filters are used to make and sterilize vaccines drugs vitamins sera, and draft beer they are also widely used in air sampling water sampling

3rd principle of sterilization

microorganisms differ in their susceptibility to antimicrobial agents its important to clear away organic debris to allow the antimicrobial agent to work organic debris slows down the effectiveness of any antimicrobial agent

2) freezing

normal freezer temp -4 degrees f..does not sterilize slows down chemical reaction rates in microbes, so growth is slow and foods do not go bad foods should not be thawed and refrozen microorganisms can be preserved by freezing but at much lower temps -78 degrees c (dry ice solid co2) or 180 degrees c with liquid nitrogen

3) microwave radiation

not used to sterilize/ bacterial endospores are not destroyed/ cooking only heats unevenly and may not cook all the way it is necessary to rotate food in order to kill bacteria and parasites that may be in the food

2) reactions that affect cell membranes

of course cell membranes contains proteins and can be altered by being denatured as we have just discussed cell membranes also contain lipids and these can be disrupted by substances that dissolve lipids ex- surfactants such as soaps and detergents a surfactant is a compound that reduces the surface tension of its solvent also alcohols, phenols, and quarternary ammonium compounds ex-zephrin ( benzalkonium chloride) used as antiseptic on skin-work better if mixed with alcohol than water detergents act as wetting agents are are often used in conjunction with other chemical agents ex- iodophors- iodine ( a halogen) mixed with an organic solvent lysol- detergent breaks surface tension, phenol dissolves lipids and denatures proteins

1) phenol coefficient

phenol is also known as carbolic acid its been around since lister first used it in the OR and it has become the standard to which all other disinfectants are compared the result of the comparison is called phenol coefficient

ultra high temp UHT processing

raises temp from 74 degrees celsius to 140 degrees celsius and drops it back to 74 degrees celsius in less than 5 seconds some small containers of coffee creamer are treated this way ex-parmalat 284 degrees f for 3 seconds it sterilizes the milk

disinfection

reducing the number of pathogenic organisms on objects or in materials so that they pose no threat of disease

3) reactions that affect other cell components

such as nucleic acids or metabolic pathways examples- a) alkylating agents replace h on amino or alcohol groups b)certain dyes ( crystal violet) used to treat some protozoan and fungal infections interfere with cell wall formation c) lactic acid often used in food preservation d) propionic acid often used in food preservation the last 2 inhibit fermentation and prevent energy production

pathogenicity

the capacity to produce disease

sterilization

the killing or removal of all microorganisms and infectious agents in a material or on an object

filtration

the passage of a material through a filter the pores must be small enough to prevent bacteria and viruses from passing through to sterilze membrane filters are used to sterilize material that may be heat sensitive these filters are usually made of nitrocellulose these filters can be made with different pore sizes depending on what you want to filter out of the product the pore sizes are altered by changing the proportions of the chemicals that make them up

chemical anti microbial agents

the potency (or power) of chemical agents is affected by many things: 1) time- the amount of time that organisms are exposed to the chemical agent the agent is not a nuclear weapon it does not kill instantly so time is necessary for the agent to work

sterility

the state in which there are no living organisms or in fectious agents in materials so that they pose no threat of disease there are no degrees of sterility- either it's sterile or its not

disinfection selection

there are several criteria that you should consider when choosing a disinfectant no disinfectant meets ALL criteria- choose the one with the greatest number of criteria also some are more effective in certain locations or applications than in others in addition, some can be used in dilute concentrations on your skin and in stronger concentrations on inanimate objects (fomites)

evaluation of effectiveness of chemical agents

there are various ways of evaluating just how effective a chemical agent is no one way is perfectly satisfactory but we do need some method or methods to compare the effectiveness of chemical agents- especially when evaluating new ones

Draw/ demonstrate

this broth culture is used to coat small stainless steel cylinders and allowed to dry each cylinder is dipped into tubes with chemical agents that have been serially diluted for 10 minutes each cylinder is removed after 10 mins, rinsed with water, and placed into a tube of broth these tubes are then incubated after incubation, the tubes are inspected for turbidity ( cloudiness) the chemicals agents that prevent growth ( turbidity) at the greatest dilutions are the most effective this is probably a more meaningful measurement that phenol coefficient

affect proteins...

this could happen if the agent (or heat) is applied for 1) too short a time 2) the agent is too weak most antimicrobial agents are used in strong enough concentrations and for long enough time to prevent this perm denaturation of proteins kills the microorganisms if its perm the agent is bactericidal if its temp the agent is bacteriostatic

3) drying

used to preserve foods endospores can survive but don't produce toxins used in preserving fruits, raisins, beans, peas, etc hanging clothes in the sunshine and in the dryer kills pathogen/ uv light as well

4) reactions that affect viruses

viruses consist primarily of nucleic acids and proteins, therefore, those agents that affect these things will generally work on viruses examples a) alkylating agents- such as ethylene oxide, nitrous acid, hydroxylamine alter dna or rna b) detergents c) alcohols -both detergents and alcohols denature proteins d) certain dyes-such as acridine orange and methlyne blue affect viruses by making them susceptible to visible light some viruses remain infective even after denaturation of their proteins

principles of sterilization and disinfection

we saw that not only did bacteria grow exponentially (log rate) during the log phase but we also saw that bacteria die during the decline or death phase. the point is that organisms treated with antimicrobial agents obey the same laws regarding death rates as those declining in numbers from natural causes ex- if 20% of the organisms die in the first minute then 20 % of those remaining will die in the 2nd minute and so on

2) ionizing radiation

x ray and gamma radiation- these two cause atoms to loose electrons (forming ions) a) kills organism and viruses b) damages dna c) produces peroxides which are toxic (compounds containing the ion o22 d) in humans can kill cells or cause mutations widely used in sterilizing plastic lab and medical equipment used to prevent spoilage of seafood meats and vegetables- no danger of radiation by eating these

2) filter paper method:OVERHEAD

you will use this method in the lab its simpler than determining the phenol coefficient filter paper disks are soaked, each with a different disinfectant and placed on an agar plate that has been streaked for confluent growth DESCRIBE -after incubation, zones of inhibition are measured and compared with each of the other disinfectants the agent with the largest zone of inhibitions is the best of those tested and for the organisms used in the test

the principle is

1) a definite proportion of the organism die in a given time interval can you see that if we start with a small number of organisms, we will achieve a sterile condition in less time than if we were to start with a large number of organisms 20% kill rate-1000 cells (28 mins to achieve sterility) 100 cells (18 minutes to achieve sterility)

physical antimicrobial agents

1) dry heat - penetrates more slowly than most heat used on glassware and metal objects also the only appropriate means for oils and powders we use dry heat when we use the bunsen burner to sterilize inoculating loops and the mouths of tubes we may cause ashes and aerosols ( droplet nuclei) to be released into the air..may spread infection

some examples of protein affecting agents

1)acids- like boric acid and strong alkalis -hydrolyze proteins 2)oxidizing agents-(electron acceptors); hydrolyze s-s (disulfide bonds) & SH (sulfhydryl groups) in proteins ex-h2o2 kmno4 3)halogens- may act as oxidizing agents ex- cl, fl,br, I 4) heavy metals- attach to -SH groups ;alter them ex-mercury, silver, selenium, copper 5) alkylating agents- these donate ch3 or similar groups to proteins ex- formaldehyde- used to inactivate viruses without destroying antigenic properties, thus, used to make some vaccines some dyes..glutaraldehyde- used to sterilize equipment ethylene oxide- used on fomites that would be harmed by high temps

problems

1.some disinfectants are drippy 2.some are thick 3.some RUN when placed on the agar 4. some are volatile and evaporate quickly but it is a way to compare disinfectants

second principle of sterilization

2) smaller the number of organisms present, the shorter the time needed to achieve sterility different antimicrobial agents have different effects on various species of bacteria and their endospores also any given species may be more susceptible to an antimicrobial agent at one growth phase than at another the most susceptible phase for bacteria is the log phase-because many enzymes are being synthesized and interfering with these enzymes may kill the organism

1) refrigeration

41 degrees f-good for a few days..some bacteria can continue to grow at this temp..even clostridium botulinum has been known to grow in deep containers where anaerobic conditions may exist listeria monocytogenes too a) gram + bacteria b) food borne transmission is the most common c) improperly processed milk, cheese, meat vegetables, is the most common source d) sometime a zoonosis especially in immunocompromised e) now a leading cause of infection in kidney transplant patients f) its transplacental and cause abortion, still birth, and neonatal death g) often appears several weeks after birth with the onset of meningitis we now have a bacteriophage product (5 diff phages) that will kill listeria strains