Assignment

Instructor: Dr. Usama Masood.

Student name: Amina Hassan.

Student ID: Bc210205940.

Practical No. 4: Sterilization techniques (Physical/Heat method of sterilization)

Solution:

Sterilization is defined as total destruction of all microorganisms (whether or not pathogenic) and
their spores, usually through the use of drastic methods such as concentrated toxic chemicals like
alcohols, chlorine, formaldehyde, glutar-aldehydes etc. Very high temperature, or intense
radiation.
Methods involved in sterilization:
1. Heat: a. dry heat
b. Moist heat
2. Chemical: Ethylen oxide
3. Filtration: a. Membrane filter
b. Seitz filter
c. Candle filter
Heat:
This is the most common method of sterilization. The heat used kills the microbes in the
substance. The temperature of the heat and duration of heating are the factors that affect the
extent of sterilization. In heat sterilization process, the longer the exposure to heat the better is
the sterilization at a given temperature. As the temperature of heat raises the time span required
for sterilization decreases. Further, the sterilization time increases with a decrease in temperature
and vice-versa. But one needs to maintain minimum sterilization time or minimum contact time
for the heat to be in touch with microbes or bacteria and thereby kill them. The heat method of
sterilization is again of two types based on the type of heat used.
A) Moist heat methods. B) Dry heat methods.
A. Moist heat method of sterilization:
Here heat is applied in the form of steam or just boiling. This method includes techniques like
1. Boiling.
2. Pasteurization.
3. By use of steam (Autoclave).
1. Boiling:
Boiling is preferred for metallic devices like surgical scissors, scalpels, needles, etc.

2. Pasteurization:
Pasteurizatio is the process of heating the milk at a temperature of 6o degrees
or 72 degrees three to four times. Here alternative heating and cooling kills all the microbes and
molds without boiling the milk.
3. Using Steam (autoclaving):
It is the most common method used for drugs as it is powerful
enough even to kill bacterial spores. Bacterial spores are the forms of bacteria which are inert.
They form a rigid cover over the cell wall during harsh climate. This cover prevents any damage
to cell and drying of the cell. By steam sterilization, these forms of bacteria are also killed as
steam destroys the cell wall.
Principle:
In this method sterilization is done by steam under pressure. Steaming at temperature
higher than 100°C is used in autoclaving. The temperature of boiling depends on the surrounding
atmospheric pressure. A higher temperature of steaming is obtained by employing a higher
pressure. When the autoclave is closed and made air-tight, and water starts boiling, the inside
pressures increases and now the water boils above 100°C. At 15 ib per sq. inch pressure, 121°C
temperatures is obtained. This is kept for 15 minutes for sterilization to kill spores. It works like
a pressure cooker.
Advantages:
The penetrating nature of steam makes it a great solution to destroy proteins in any
microorganism after a certain amount of time. It is environmentally safe having no toxic
byproducts.
B. Dry heat methods
Here the substances are subjected to dry heat like
1. Flaming
2. Incineration
3. Hot air oven.
4. Radiation sterilization
1. Flaming:
It is the process of exposing metallic device like the needle, scalpels, and scissors to
flame for few minutes. The fire burns the microbes and other dust on the instrument directly.
2. Incineration:
It is done especially for inoculating loops used in microbe cultures. The metallic
end of the loop is heated to red hot on the flame. This exposure kills all the germs.
3. Hot Air Oven:
It is one of the most common methods used for sterilization. Glass wares, swab
sticks, all glass ware, syringes, powder and oily substances are sterilized in hot air oven. For
sterilization, a temperature of 160°C is maintained (holding) for one hour. Spores are killed at
this temperature.
It leads to sterilization. Hot air oven is suitable for dry material like powders, metal devices,
glassware, etc.
Working and Principle:
It is an apparatus with double metallic walls and a door. There is an air
space between these walls. The apparatus is heated by electricity or gas at the bottom. On
heating, the air at the bottom becomes hot and passes between the two walls from below
upwards, and then passes in the inner chamber through the holes of the apparatus. A thermostat
is fitted to maintain a constant temperature of 160°C.

Advantages: Dry heat sterilization is preferred for heat stable products that are sensitive to
moisture.
4. Radiation:
This method involves exposing the packed materials to radiation for sterilization.
There are two types of radiations available for sterilization i.e. non-ionic and ionic radiation.
Non-ionic radiations are safe to the operator of sterilization, and they are like Ultra Violet
radiations, they can be used even at the door entrances to prevent entry of live microbes through
the air.
Ionizing radiation sterilization They are powerful radiation and very useful for sterilization.
The operator needs to protect himself from exposure from these radiations by use of special
clothing.

Practical No.5: Sterilization techniques (Chemical method of sterilization)

Solution:

Here the articles are subjected to sterilization by using toxic gasses. The gas penetrates
quickly into the material like steam so, the sterilization is effective. But the chances of explosion
and cost factors are to be considered. The gasses used for sterilization are very poisonous. The
commonly used gas is ethylene oxide with a combination of carbon-dioxide. Carbon dioxide is
added to minimize the chances of an explosion.
Modes of action of chemicals:
 Protein coagulation
 Disruption of cell membrane resulting in exposure, damage or loss of the contents.
 Removal of free sulfhydryl groups essential for the functioning of the enzymes.
 Substrate competition – a compound resembling the essential substrate of the enzyme diverts
or misleads the enzymes necessary for the metabolism of the cell and causes cell death.
Alcohols:
The types of alcohol used in disinfection are ethanol (80%), propanol (60%), and
isopropanol (70%). Alcohols are quite effective against bacteria and fungi, less so against
viruses. They do not kill bacterial spores. Due to their rapid action and good skin penetration, the
main areas of application of alcohols are surgical and hygienic disinfection of the skin and hands.
One disadvantage is that their effect is not long-lasting (no depot effect). Alcohols denature
proteins.
Aldehydes:
Formaldehyde (HCHO) is the most important aldehyde. It can be used in a special
apparatus for gas sterilization. Its main use, however, is in disinfection. Formaldehyde is a water-
soluble gas. Formalin is a 35% solution of this gas in water. Formaldehyde irritates mucosa; skin
contact may result in inflammations or allergic eczemas. Formaldehyde is a broad-spectrum
germicide for bacteria, fungi, and viruses. At higher concentrations, spores are killed as well.
The mechanism of action of formaldehyde is based on protein denaturation.
Ethylene oxide:
This highly reactive gas (C2H4O) is flammable, toxic, and a strong mucosal
irritant. Ethylene oxide can be used for sterilization at low temperatures (20–60 8C). The gas has
a high penetration capacity and can even get through some plastic foils. One drawback is that
this gas cannot kill dried microorganisms and requires a relative humidity level of 40–90% in the
sterilizing chamber. Ethylene oxide goes into solution in plastics, rubber, and similar materials,
therefore sterilized items must be allowed to stand for a longer period to ensure complete
desorption.
Chlorine compounds:
Chlorine denatures proteins by binding to free amino groups. Generally,
chlorine is used in the form of sodium hypochlorite which is commonly known as household
bleach. It is very useful for sterilization purpose as it is very inexpensive and also effective
against bacteria and viruses.
But it has some disadvantages as well; First of all high concentration of this agent corrodes
metals and also damages cloths. Secondly the strength of this solution decreases with time so
whenever you want to use it always prepare its fresh solution.
Oxidants:
This group includes ozone, hydrogen peroxide, and peracetic acid. Their relevant
chemical activity is based on the splitting off of oxygen. Most are used as mild antiseptics to
disinfect mucosa, skin, or wounds.
Equipment:

 A spray bottle with 70% ethanol

 A contaminated surface (Bench top).
 Cotton

Procedure:

1. Spray the 70% ethanol evenly over the contaminated surface, making sure to cover all
areas. This helps in effectively killing or reducing microorganisms on the surface.
2. Dry the surface with Cotton Budd.

Practical No.6: Sterilization techniques (Mechanical/Filtration methods)

Solution:
This method is used for sterilizing thermolabile solutions, which will otherwise be
degraded by other conventional heating methods. The drug solutions are passed through the
sterile bacteria proof filter unit and subsequently transferring the product aseptically into the
sterile containers which are then sealed
Procedure:
The solutions to be sterilized is passed through the filter and collected in the sterile receiver by
the application of positive pressure to the non-sterile compartment or negative pressure to the
sterile side.
Membrane filters
 Membrane filters are made of cellulose-derivative (acetate or nitrate). They are very fine.
They are fixed in some suitable holders.
 Nominal pore size is 0. 22 ± 0. 02 µm or less is required.
 The membranes are brittle when dry. In this condition they can be stored for years together.
They become very tough when dipped in water.
 They are sterilized by autoclaving or by ethylene oxide gas. They cannot be sterilized by
dry heat as they decompose above 1200C.
 They are suitable for sterilizing aqueous and oily solutions but not for organic solvents
such as alcohol, chloroform etc.
 Membrane filters are generally blocked by dirt particles and organisms. Pre-filtration
(through glass-fibre paper prefilter) reduces the risks of blockage of the final filter.
Examples of membrane filters:
MF-Millipore – it is a mixture of cellulose esters

Sintered (or fritted) glass filters

Borosilicate glass is finely powdered in a ball-mill and the particles of required size are
separated. This is packed into disc mounted and heated till the particles get fused. The discs thus
made are used for filtration. As these are made of glass and hence do not absorb liquids during
filtration. The disadvantage is that they are very brittle and break easily
They are cleaned with the help of sulfuric acid.
Seitz filter:
These are made of asbestos or other material. They are pad like and thicker than membrane
filters. They do not rupture during filtration. But the solution might get absorbed by the filter pad
itself

Equipment:

 Sample to be filtered
 Sterile Syringe 5cc
 Syringe Filters 0.22µ
 Falcon tubes 15ml and 50ml

Procedure:
 1. Syringe filters are available in a variety of pore sizes, with the most common pore
sizes being 0.22 microns that will exclude most microbial contaminants
2. Take sample in a sterile syringe.
3. Open the disposable syringe filter.
4. Attach syringe to the filter carefully.
5. Collect the filtrate in a sterile falcon tube.

Advantages of sterilization by filtration:

1. Thermolabile solutions can be sterilized.
2. It removes all the living microorganisms.
Disadvantages of sterilization by filtration:
1. Filters may break down suddenly or gradually on use.
2. Sterility testing is obligatory on the filtered solution.
3. Filter media may be absorbed on the filter surface.
4. Viruses are not removed by filtration.
5. Suspensions and oils cannot be sterilized by this method due to their heavy load of
particulate matters and viscosity.
Practical No. Growth Kinetics
Solution:

Objectives: To establish growth curves for an unknown bacterial species and observe
the different phases of growth.
Introduction:
Bacteria normally reproduce by binary fission, forming two equal-sized progeny cells and thus
doubling their number with each division. This type of cell division is called exponential; i.e.,
one cell divides to form two, each of these cells divides so that four cells are obtained, and so
forth in a geometric progression. The unit of microbial growth is "generation time", which is the
time required to achieve a doubling of the population size and is designated as tgen. Generation
time can be estimated by determining cell numbers during the period of active cell division and
is expressed mathematically as:
tgen = t ln 2/ ln Xt − ln X0= t log 2 /log Xt − log X0
In the above equations, t is the elapsed time during which growth is measured, and Xo and Xt are
the number of bacteria at times zero and t, respectively. The presence of 2 in the numerator of the
equations is due to the fact that when doubling of the population number occurs, the quantity
Xt/Xo (called the doubling constant) will be equal to 2. The generation time of many bacteria is
usually several hours. However, under optimum conditions, E. coli has a generation time of
about 20 minutes. Thus it can be calculated that a single E. coli cell will produce about a
thousand progeny in 3.3 hours and over a million in 6.6 hours. If the generation time and the
elapsed time are known, then the number of generations can be estimated as follows:
Number of generations = t/ tgen

A bacterial population follows a characteristic growth curve which has four phases: the lag
phase, the log or exponential growth phase, the stationary phase, and the death phase.
1. Lag phase: During this phase, there is no increase in cell number; rather, bacteria are
preparing for reproduction and synthesizing DNA and various inducible enzymes needed
for cell division.

2. Log phase: This phase, also called the exponential phase, follows the lag phase and starts
with a rapid increase in bacterial number. At this stage, the logarithm of bacterial biomass
increases linearly with time so that numbers of bacterial cells in a given interval of time is
proportional to the biomass of bacteria present.
3. Stationary phase: The number of bacteria reaches a maximum in this phase and does not
increase further (the growth rate is exactly equal to the death rate). This phase is
sometimes called the plateau stage. A bacterial population may reach stationary growth
when a required nutrient is exhausted, when inhibitory end products accumulate, or when
physical conditions are inappropriate for growth. The duration of the stationary phase
varies, with some bacteria exhibiting very long stationary phases.
4. Death phase: Eventually, the number of viable bacterial cells begins to decline, signaling
the onset of the death phase. No further divisions occur in this phase. Death rate, in many
cases, follows the same kinetics as the exponential growth.
Principle:
Turbidity, which is a measure of the growth of microorganisms, is determined via an instrument
called a spectrophotometer. The basis for the use of this instrument is that less light passes
through a turbid solution than through a clear solution; i.e., the more turbid a solution (more
growth), the less the amount of light transmitted through the solution. Since the optical density of
the culture is proportional to the cell density, measuring the turbidity of the culture solution can
be used in estimating numbers of bacterial cells, if a growth curve for the conditions used has
already been established. This is the most common method used to rapidly estimate bacterial
numbers.
Laboratory Supplies: 10 ml culture of an unknown microorganism, 50 ml LB broth in 125-ml
flasks, Spectrophotometer, Un-inoculated LB broth in small test tube for blank.
Procedure:
1. Obtain a flask of LB and transfer 5 ml of broth to a small test tube. This is your blank. Adjust
the wavelength to 550 nm on the spectrophotometer and calibrate the machine to 0.0 O.D. or
100% transmittance with your blank. Take the blank out but do not discard it.
2. Inoculate your flask with 10 ml of the culture provided and swirl the flask well.
3. Work quickly to withdraw 5 ml of the sample into a clean small test tube, using a pipette.
4. Place the culture flask immediately at the assigned incubating temperature and aeration
condition and record the time.
5. Measure the O.D. of your sample next. Record the 0 time absorbance. Discard the contents of
your sample test tube. This test tube can be re-used for your next sample without washing.
6. At 15-minute intervals for up to 90 minutes, shake your flask vigorously and transfer 5 ml of
the culture to your sample test tube.
7. Record the optical density at each time period. Use your blank each time to make sure your
readings are correct and other students have not altered the machine’s calibration. Your
instructor will ask all groups to write their results in a table on the blackboard. Copy these results
to your Results Sheet. If more than one group is doing a growth condition, get their average data.
8. Plot the optical density (y-axis) versus time (x-axis) for each growth condition on the same
regular graph paper.