HOW TO CONTROL BACTERIAL GROWTH ?

Lab number 6

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.

SterilizationA. Physical methods :• Dry heat.• 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.

A. Physical methodsI. Dry heat: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:

oThey are electrical devices used in sterilization.

o There is a digitally controlled thermostat

controlling the temperature.

o Their double walled insulation keeps the heat in .

o There is also an air filled space in between to aid

insulation.

o An air circulating fan helps in uniform distribution

of the heat.

oThey are fitted with the adjustable wire mesh

plated trays or aluminum trays.

oTemperature 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.

disadvantagesAs they use dry heat instead of moist heat, some

organisms may not be killed by them every time.

Advantages

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.

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: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: 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 sterilizationMany bacteria are readily killed by radiation.

a.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:

1. 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.

2. 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:

1. penetrates well, moving through paper, cloth, and some plastic films

2. highly effective

3. Ethylene oxide sterilizers are used to process sensitive instruments which cannot be

adequately sterilized by other methods

4. It can kill all known viruses, bacteria and fungi, including bacterial spores and is

satisfactory for most medical materials, even with repeated use.

5.

Disadvantages:

1. highly flammable

2. requires a longer time to sterilize than any heat treatment

3. 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. OzoneOzone is used in industrial settings to sterilize water and

air, as well as a disinfectant for surfaces.

Advantages:

1. ozone is a very efficient sterilant because of its strong

oxidizing properties and it is capable of destroying a

wide range of pathogens.

2. Able to oxidize most organic matter.

Disadvantages:

1. toxic

2. unstable gas that must be produced on-site.

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 PeroxideHydrogen peroxide is another chemical sterilizing agent. It is

relatively non-toxic when diluted to low concentrations

(3 % ).

Advantages:

1. Hydrogen peroxide is strong oxidant and these oxidizing

properties allow it to destroy a wide range of pathogens.

2.It is used to sterilize heat or temperature sensitive articles

such as rigid endoscopes.

3.Short cycle time compared to ethylene oxide but its

penetrating ability is not as good as ethylene oxide.

Disadvantages:

1. Hydrogen peroxide is primary irritant

2. the contact of the liquid solution with skin will cause

bleaching or ulceration depending on the concentration

and contact time.

3. 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.

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:

1.Ethanol:• used at concentrations between 70 – 95%

• Denature proteins; disrupt membranes

• 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.

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.

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:

Antiseptic1:50 1:100 1:150 1:200

phenol1:95

Time of Sampling(min)

+ + + _ + 2.5

+ + _ _ + 5

+ _ _ _ _ 7.5

_ _ _ _ _ 10

Disadvantages:1. Absence of organic matter. (Chick

Martin Test: yeast extract)2. Test time is so short: 10 min

(Chick Martin: 30min)3. It is only applicable for phenolic

compounds4. It uses Salmonella typhi as the

test organism only.

THANK YOU