How to control bacterial growth ? Lab number 6 How to control bacterial growth ?

How to control bacterial growth? We usually control bacterial growth by sterilization and disinfection. Sterilization: the process by which all viable organisms including spores are removed or killed. Disinfection: the process by which most and not all viable organisms are removed or killed.

Sterilization A. Physical methods : Dry heat. B. Chemical methods: Moist heat. Filtration. Radiation. B. Chemical methods: Chemicals. Gases. The efficacy of these methods depends on Processing time. Material being treated. Presence of organic material.

Physical methods Dry heat: A- Flaming to red hot: Mechanism: Mainly by oxidation; Dry heat coagulates the proteins in any organism, causes oxidative free radical damage, which causes drying of cells and can even burn them to ashes, as in incineration. A- Flaming to red hot: Used for metal instruments such as loops, dental mirrors and scalpels. First put the metal in alcohol and then burn it off.

B. Hot air oven: They are electrical devices used in sterilization. There is a digitally controlled thermostat controlling the temperature. Their double walled insulation keeps the heat in . There is also an air filled space in between to aid insulation. An air circulating fan helps in uniform distribution of the heat.

They are fitted with the adjustable wire mesh plated trays or aluminum trays. Temperature sensitive tapes or other devices like those utilizing bacterial spores can be used to work as controls, to test for the efficacy of the device in every cycle.

Advantages disadvantages They do not require water and there is not much pressure build up within the oven, unlike an autoclave, making them safer to work with. disadvantages As they use dry heat instead of moist heat, some organisms may not be killed by them every time.

Uses: These are widely used to sterilize articles that can withstand high temperatures and not get burnt, like glassware. The standard settings for a hot air oven are: 1.5 to 2 hours at 160 °C ....plus the time required to preheat the chamber before beginning the sterilization cycle.

C. Incineration: complete burning of material in an incinerator. This is used for the safe disposal of items such as contaminated dressings and lab cultures.

II. Moist heat: Mechanism: Moist heat, as the name indicates, utilizes hot air that is heavily loaded with water vapour and where this moisture plays the most important role in the process of sterilization. It is more rapid and efficient than dry heat. Mechanism: Moist heat coagulates the proteins in any organism and this is aided by the water vapour that has a very high penetrating property, leading to their death.

I. Autoclave Autoclaves commonly use steam heated to 121 °C or 134 °C. To achieve sterility, a holding time of at least 15 minutes at 121 °C or 3 minutes at 134 °C is required. The pressure reached is 15 pounds /square inch. Additional sterilizing time is usually required for liquids and instruments packed in layers of cloth, as they may take longer to reach the required temperature.

Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant.

To ensure the autoclaving process was able to cause sterilization, most autoclaves have meters and charts that record or display information such as temperature and pressure as a function of time. Biological indicators can also be used to confirm autoclave performance. Most contain spores of the heat resistant microbe Bacillus stearothermophilus, among the toughest organisms for an autoclave to destroy.

There also chemical indicator strips that contains a compound that changes color upon an efficient sterilization cycle. For effective sterilization, steam needs to penetrate the autoclave load uniformly, so an autoclave must not be overcrowded, and the lids of bottles and containers must be covered. During the initial heating of the chamber, residual air must be removed.

II. Boiling water: III. Pasteurization: For 10 to 30 min It is still used in some clinical situations but they don’t destroy spores. III. Pasteurization: Used to destroy vegetative cells in milk (Mycobacteria,Salmonella, Brucella), not the spores. The main principal involves heating followed by sudden cooling.

It is done by one of three methods: Holder method - peaks at 63°C for 30 minutes . Flash method - peaks at 72°C for 20 seconds. Ultra high temperature (UHT) method - peaks at 125°C for a few seconds

III. Filtration: Clear liquids that would be damaged by heat, irradiation or chemical sterilization can be sterilized by mechanical filtration. This method is commonly used for sensitive pharmaceuticals and protein solutions in biological research. A filter with pore size 0.2 µm will effectively remove bacteria.

If viruses must also be removed, a much smaller pore size is needed. Vacuum pump is usually used since solutions filter slowly through membranes with smaller pore diameters. To ensure the best results, pharmaceutical sterile filtration is performed in a room with highly filtered air or in a laminar flow cabinet.

IV. Radiation sterilization Many bacteria are readily killed by radiation. Gamma rays - They are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets. -Gamma radiation requires storage of a radioisotope (usually Cobalt-60), which continuously emits gamma rays.

- This process does not result in rise in temp; but the time required for sterilization is long (48hrs). - Only used within hospitals due to its hazardous effects. b. X-rays They are less penetrating than gamma rays and tend to require longer exposure times, but require less shielding.

C. Ultraviolet light irradiation - It is useful only for sterilization of surfaces and some transparent objects. - Many objects that are transparent to visible light absorb UV. - UV irradiation is routinely used to sterilize the interiors of biological safety cabinets

B. Chemical methods Advantages: Chemicals are also used for sterilization of heat-sensitive materials such as biological materials, fiber optics, electronics, and many plastics where heat sterilization can’t be used. The chemicals used as sterilants are designed to destroy a wide range of pathogens.

Disadvantages: We must be sure that article to be sterilized is chemically compatible with the sterilant being used.

I. Gases a. Ethylene Oxide (EO) Ethylene oxide gas is commonly used to sterilize objects sensitive to temperatures greater than 60 °C such as plastics, optics and electrics. Advantages: penetrates well, moving through paper, cloth, and some plastic films highly effective Ethylene oxide sterilizers are used to process sensitive instruments which cannot be adequately sterilized by other methods It can kill all known viruses, bacteria and fungi, including bacterial spores and is satisfactory for most medical materials, even with repeated use.

Disadvantages: highly flammable requires a longer time to sterilize than any heat treatment The process also requires a period of post-sterilization aeration to remove toxic residues.

Spore testing Bacillus atrophaeus a very resistant organism, is used as a rapid biological indicator for EO sterilizers. If sterilization fails, incubation at 37 °C causes a fluorescent change within four hours. Fluorescence is emitted if a particular (EO resistant) enzyme is present, which means that spores are still active.

b. Ozone Ozone is used in industrial settings to sterilize water and air, as well as a disinfectant for surfaces. Advantages: ozone is a very efficient sterilant because of its strong oxidizing properties and it is capable of destroying a wide range of pathogens. Able to oxidize most organic matter.

Disadvantages: toxic unstable gas that must be produced on-site.

a.Glutaraldehyde and Formaldehyde II.Liquids a.Glutaraldehyde and Formaldehyde They are accepted liquid sterilizing agents, provided that the immersion time is sufficiently long. To kill all spores in a clear liquid can take up to 12 hours with glutaraldehyde and even longer with formaldehyde.

The presence of solid particles may lengthen the required period or render the treatment ineffective. Glutaraldehyde and formaldehyde are volatile, and toxic by both skin contact and inhalation. Many vaccines, such as the original Salk polio vaccine, are sterilized with formaldehyde.

b. Hydrogen Peroxide Hydrogen peroxide is another chemical sterilizing agent. It is relatively non-toxic when diluted to low concentrations (3 % ). Advantages: Hydrogen peroxide is strong oxidant and these oxidizing properties allow it to destroy a wide range of pathogens. It is used to sterilize heat or temperature sensitive articles such as rigid endoscopes. Short cycle time compared to ethylene oxide but its penetrating ability is not as good as ethylene oxide.

Disadvantages: Hydrogen peroxide is primary irritant the contact of the liquid solution with skin will cause bleaching or ulceration depending on the concentration and contact time. The vapor is also hazardous with the target organs being the eyes and respiratory system.

Disinfection: It is usually by chemical agents. Disinfectants are generally not intended to achieve sterilization. Most reduce the microbial populations to safe levels or remove pathogens from objects. An ideal disinfectant or antiseptic kills microorganisms in the shortest possible time without damaging the material treated. Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms while antiseptics destroy microorganisms on living tissue.

Ethanol: Examples of commonly used disinfectants: The choice of disinfectant for different hospital applications is made on the basis of their antimicrobial activity, inactivation by organic materials and their toxicity. Examples of commonly used disinfectants: Ethanol: used at concentrations between 70 – 95% Denature proteins; disrupt membranes

2. phenols: Kills vegetative cells of bacteria & fungi but not spores. Used in disinfecting surfaces; thermometers; “ethanol-flaming” technique used to sterilize glass plate spreaders or dissecting instruments at the lab bench. 2. phenols: Aromatic organic compounds with attached -OH Denature protein & disrupt membranes Commonly used as disinfectant and is effective in presence of organic matter. It has disagreeable odor & is skin irritant.

Phenol coefficient test (Rideal Walker method) Evaluation methods of disinfectants: To evaluate an antiseptic or disinfectant, the phenol coefficient test is used. Phenol coefficient test (Rideal Walker method) It is a measure of the bactericidal activity of a chemical compound in relation to phenol. Phenol Coefficient Test is done by measuring the concentration at which a chemical is equal in effectiveness to phenol.

Phenol Coefficient = Conc. of chemical Conc. of phenol a. If a chemical is equal in effectiveness to phenol at the same concentration, its phenol coefficient is 1. b. If the concentration of the chemical to be tested must be twice that of phenol, its phenol coefficient is 1/2. c. If the solution is less concentrated than the phenol standard, its phenol coefficient is greater than 1. Phenol Coefficient = Conc. of chemical Conc. of phenol

Phenol Coefficient Test A series of dilutions of phenol and the experimental disinfectant are inoculated with Salmonella typhi Samples are removed at 2.5 min intervals and inoculated into fresh broth. The cultures are incubated at 37°C for 2 days The highest dilution that kills the bacteria after a 7.5min exposure, but not after 5 min, is used to calculate the phenol coefficient

The reciprocal of the maximum effective dilution for the test disinfectant is divided by the reciprocal of the maximum effective dilution for phenol to get the phenol coefficient For example: Suppose that, on the test with Salmonella typhi The maximum effective dilution for phenol is 1/90 The maximum effective dilution for “Disinfectant X” is 1/450 The phenol coefficient for “Disinfectant X” with S. typhi = 450/90 = 5

Evaluation of the efficacy of an antiseptic by Rideal Walker test Materials: Salmonella typhi suspension 5ml of phenol (1:95) 5 ml of different dilutions of the antiseptic under test.(1:50,1:100,1:150………………. 4 sterile broth tubes for each dilution. Loop 1sterile pipette 1 ml

Methods: Add 0.2 ml of S.typhi suspension to each of the dilutions of the phenol and antiseptic leaving 30 seconds intervals between each tube. At 2.5 minutes intervals inoculate one loopful of each of the previous dilution tubes into a sterile broth tubes. Shake all the tubes and incubate at 37oC for 2 days. Record the results as – if there is no growth and + if growth occurs. Calculate the phenol coefficient of the given antiseptic.

Results: Antiseptic 1:50 1:100 1:150 1:200 phenol 1:95 Time of 1:50 1:100 1:150 1:200 phenol 1:95 Time of Sampling(min) + _ 2.5 5 7.5 10

Disadvantages: Absence of organic matter. (Chick Martin Test: yeast extract) Test time is so short: 10 min (Chick Martin: 30min) It is only applicable for phenolic compounds It uses Salmonella typhi as the test organism only.

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