Coal City University, Emene Enugu, Enugu State, Nigeria.

Faculty of Natural and Applied Sciences.

Department of Microbiology

Name of Lecturer: Dr. JaneSylvia Uzoeto.

Course code: MCB 201: General Microbiology 1

Course Description:

(Pre-requisites BIO 151 and BIO 152)

General review of the nature and biology of protozoa, fungi, algae, bacteria, archae and

viruses.

General characteristics.

Growth and reproduction of microorganisms.

Microbiology methods and instrumentation.

Sterilization and disinfection.

Microorganisms in various environments- soil, water, food, etc

Industrial use of microorganisms

Pathogenic role of microorganisms

Growth and Reproduction of Microorganisms.

Introduction: This topic explains growth and reproduction which is a characteristic of living

organisms as it concerns microorganisms.

Objectives: At the end of this topic students should be able to describe the following:

Define growth.

Define closed and open system.

Describe different growth phases.

Describe different measuring instruments involve in the growth of microorganisms.

Definition of growth: It is defined as an increase in cellular constituents which leads to a rise in

cell when microorganisms reproduce by processes like budding or binary fission. It is also

defined as a co-ordinated and orderly increase in all the chemical components in a biological

system.

Terminology:

Specific growth rate (N): This is the rate of growth per unit organism; that is µs

µs= dm/dt x I/ms

Where dm = is the increase in mass over a time dt.

Growth rate (µg): Is the rate of total mass increase or total number increase of a whole culture.

It increases as the number of dividing cells in the culture increases.

µg = dm/dt

Generation Time (tg): Is the time taken by a particular cell from its formation by cell division to

its own fission.

Mean generation time (tgm): Is the arithmetic mean of a number of observations of individual

generation times of cells growing in the same culture during the same period.

Doubling time: This is the time taken for the number of cells in a population to double.

In binary fission, a new cell increases until it doubles its size and subsequently divides into two

approximately equal halves; 20, 21, 22, 23, 24......

Students’ assignment 1: Diagrammatic representation of binary fission.

An example of Exponential growth

Time Division number 2n Population (No X 2n) Log10 Nt

0 0 20 = 1 1 0.00020 1 21 = 2 2 0.30140 2 22 = 4 4 0.60260 3 23 = 8 8 0.90380 4 24 = 16 16 1.204100 5 25 = 32 32 1.505120 6 26 = 64 64 1.806

The Growth Curve: The nature of growth of microorganism (bacteria) has not been fully

elucidated or understood. The maintenance of microorganisms in an artificial medium with static

environmental conditions leads to a batch culture or a closed system of maintaining growth. The

growth of microorganisms reproducing by binary fission can be plotted as the logarithm of the

number of viable cells versus the incubation time.

Students’ assignment 2: Draw the diagram of growth curve.

GROWTH PHASES IN A SYSTEM:

Lag Phase: This is the period required by the cells to adjust to a new environment. There is no

appreciable multiplication in this phase although the cells may increase considerable in size

which indicates marked metabolic activity. The duration of this phase varies according to the

condition and number of cells on the inoculums. The cells in the inoculums may be depleted in

enzymes, metabolic intermediates and other factors. If the composition of the new medium

differs significantly in composition from that in which the inoculums was growing previously,

entirely new enzymes may be require to be synthesized.

The Exponential phase or Log phase: After the lag phase, cells now enter a phase which for a

given organism, the growth rate is both constant and maximal for a particular medium and

growth condition. During this period, the doubling of biomass is usually accompanied by the

doubling of all measurable properties such as the DNA, RNA, and others. Exponential growth is

called balanced growth.

Stationary phase: In this phase, population growth ceases and the growth curve becomes

horizontal. This stationary phase usually is attained by bacteria at a population level of around

109 cells per ml; other microorganisms normally do not reach such high population densities.

Protozoan and algae culture often have maximum concentration of about 106 cells ml.

Microbial population enter the stationary phase for the following reasons:

Nutrient limitation: If an essential nutrient is severally depleted, population growth will

slow down.

Availability of oxygen: Aerobic organisms often are limited by low oxygen.

Accumulation of toxic waste products slows down on population growth.

Death Phase: Death phase is linear decrease in the number of viable cells with time. The death

rate of bacteria varies from one bacterium to the other and also with the environmental

conditions. The main cause of this loss in viability depends mainly on the cause of the onset of

stationary phase.

Continuous Culture: This is an ‘open’ system in which there is a continuous removal of grown

bacteria by means of a constant level device. It is used for research and industrial purposes. In a

continuous culture, the growth chamber is connected to a reservoir of sterile medium. Once

growth has been initiated, fresh medium is continuously supplied from the reservoir to the

growth chamber. The volume of the liquid in the growth chamber is maintained constant,

allowing the excess volume to be removed continuously through the siphon overflow.

Two major types of Continuous culture system commonly use are:

Turbidostat

Chemostat

CHEMOSTAT: In a Chemostat, the flow rate is set at a particular value and the rate of growth

of the culture adjusts to the rate of fresh medium. A Chemostat is constructed so that sterile

medium is fed into the culture vessel at the same rate as the media containing microorganisms is

removed.

TURBIDOSTAT: Turbidostat is an optical sensing device which measures the absorbency of

the culture. It has photocell that measures the absorbance or turbidity of the culture in the growth

vessel. The flow rate of media through the vessel is automatically regulated to maintain a

predetermined turbidity or cell density. Turbidostat operates best at high dilution rates while

chemostat is most stable and effective at lower dilution.

Measurement of Microbial Growth:

Measurement of cell numbers: To determine microbial numbers direct counting chambers is

easy, inexpensive and relatively quick; it also gives information about the size and morphology

of microorganism. Petroff–Hausser counting chambers can be used for counting prokaryotes.

Haemocytometers can be used for both prokaryotes and eukaryotes.

Coulter counter: This is used for larger microorganisms such as protozoa, algae. Non

filamentous yeasts can be directly counted with electronic counters.

Measurement of cell mass (Total): The only direct way of measuring cell mass is to determine

the dry weight of the cell material in a fixed volume of the suspension. This is done by filtering

out cells from the culture, drying them and weighing them. But such determination is time

consuming and relatively insensitive since it is difficult to weigh less than 1mg resulting from

more than 5 billion bacteria with accuracy.

Spectrophotometer: Is an optical instrument used in measuring cell mass, it is preferable and

more reliable. Nephelometry is a type of spectrophotometer, it is use to determine the

concentration of a cell suspension (often in cell mass by measuring the amount of light scattering

that occurs if a beam of light is passed through the suspension. The technique is based on the fact

that small particles scatter light proportionally (within certain limits) to their concentration.

Membrane Filtration: Lipid samples containing low concentrations of cells can be filtered

using membrane filter; the cells are stained for clarity and counted on the membrane under the

microscope.

Dilution Method (Multiple Tube Method): Is a method for estimating the concentration of

viable cells in a suspension. It gives the most probable number (MPN) of viable cells.

Colony Count (viable): Measures the number of viable cells in the suspension. Bacteria

colonies commonly consist of a compact mass of individual cell which have usually resulted

from the multiplication of a single cell at that location. The size of the colony depends on the

species of bacteria, type of medium, growth condition and on the time during which growth

(multiplication) has occurred. These methods discussed are usually applicable to most bacteria

and spores. Some of the methods are also good for algae but the algal colony is a more or less

stable form which, in some species, their rate of movement is lowered by using viscous

suspending media like 2-5% methyl cellulose. Viruses are usually counted by plaque forming

unit (PFU), Electron Microscopy and Phase contrast illumination while larger ones can be

stained and counted under the light microscope.

Plaque Assay: This is a discrete, macroscopic or microscopic, usually circular region in a cell

monolayer or in a film or layer of bacterial growth in which some or all the cells have been lysed

as a result of viral activity. PFU, a virion has the ability to infect and lyse particular cells and to

give rise to a plaque under suitable conditions. Plaque assay is a technique used for counting the

number of infective viral particles in a given suspension.

Phase Contrast Illumination: It involves spreading an appropriate diluted sample of the cell

suspension in the surface of a sterile glass slide. The slide is incubated briefly just to permit a

few cell divisions and the slide is then viewed under a microscope with phase contrast

illumination. Individual viable cells develop into micro-colonies and are therefore, seen and

counted under phase contrast illumination. In the same process, the non-viable cells are also

identified relative to the viable cells.

Growth Yield or Efficiency of Growth: The difference in initial cell number (cell mass) and

the final cell number or cell mass in a microbial growth monitoring exercise is the growth yield.

Where the final cell number is the number just before the population enters the stationary phase.

Where a particular nutrient constitutes part of the growth limiting factor, a fixed linear

relationship exists between the concentration of that nutrient and the net growth that result. The

cell mass produced per unit of limiting nutrient is accordingly a constant.

Factors affecting the growth of microorganisms:

Age of the culture.

Temperature of the environment.

The oxidation – reduction potential of the environment.

The level of acidity PH.

Availability of nutrients.

Summary: Students were given assignments and spot test assessment to test their ability to

understand growth in microorganisms and the difference between closed and open system in the

growth of organisms. Their ability to recognise instruments used in measuring growth were

enhanced at the end of the lecture.

References

Lansing, M.P., John, P. H. and Donald A. K. (2005) Microbiology.6th edition Published by Mc Graw Hill.

Onyeagba, R.A and Isu, N.R. (2004). General Microbiology. Research and Academic publishers Owerri.

Ogbulie, J.N., Onyeagba, R. and Isu, N.R.(1997). Introductory Microbiology. CRC publications Nig.Ltd. Owerri.

TOPIC: STERILIZATION AND DISINFECTION

Objectives:

Define sterilization and disinfection.

Discuss different methods of sterilization.

Discuss different methods of disinfection.

Introduction: Sterilization and disinfection are two procedures that are aimed at eliminating or

reducing the number of viable microorganisms in the treated object. Both processes employ the

use of physical or chemical agents to inactivate, destroy or remove microorganisms from

substances or enclosures.

Definition: Sterilization is derived from the Latin word ‘sterile’ which means incapable of

producing offspring. Sterilization is the process by which all living cells, viable cells (bacteria,

virus, fungi, viroid and their spores or endospore) are either destroyed or removed from an object

or habitat. A sterile object is totally free of viable microorganisms, spore and others infectious

agents. Sterilization is also define as the process by which all forms of living organisms are

eliminated; virus, bacteria and fungi their spores or endospore. It is an irreversible process.

Disinfection: Is the process that eliminated many or all pathogenic micro organisms except

bacterial spores on inanimate objects. Also it is define as the destruction, inactivation or removal

of those microorganisms likely to cause infection or to give rise to other undesirable effects.

Terminology:

Sterilants: These are chemical agents used to achieve sterilization.

Disinfectants: are agents usually chemical, used to carry out disinfection. They are normally

used only on inanimate or non-living objects. Disinfection does not necessarily sterilise an object

because viable spores and a few microorganisms may remain.

Antisepsis: This is derived from the Greek word (‘anti’ meaning against and ‘sepsis’ meaning

putrefaction) is the prevention of infection or sepsis. Antisepsis achieved by the use of

antiseptics.

Antiseptics: These are chemical agents applied to tissues to achieve antisepsis or prevent

infection by killing or inhibiting pathogen growth, it also reduces microbial load or population.

Sanitization: The word sanitization is closely related to disinfection. In sanitization, the

microbial population is reduced to levels that are considered safe by public health standards.

Decontamination: This is the process of removing pathogenic microorganisms from objects so

that they are safe for handling, use or discard.

Methods of Sterilization:

Physical method

Chemical method

Gaseous method

Physical method

a. Thermal (heat)

b. Radiation method

c. Filtration method

Thermal or Heat: Heat sterilization is the most widely used and reliable method of Sterilization.

It involves destruction of enzymes and other essential cell constituents. Heating under moisture

or humidity is more effective because hydrolysis and denaturalization occurs faster and lower

heat input is required, while under dry state, oxidation changes takes place and higher heat input

is required.

Mode of action: The lethal action is largely due to the denaturation of microbial proteins and

nucleic acids which are deaminated or degraded. Dry heat: This involves sterilization by heat in

the absence of moisture.

Incineration: Is the most effective method of sterilization. It is used to discard

contaminated swabs, paper material, cloth, etc.

Red heat: This is used for sterilizing inoculating wireloops, and metal instrument which

are not damaged by direct exposure to heat.

Flaming: This method is frequently used for scapels, spatula, glass rods, and hockey

sticks. Dipping this instrument in ethanol before flaming is more effective.

Hot air oven: This method involves heating either by electricity or gas. It is normally

thermostatically controlled. Sterilization in hot air oven is used for dry glassware, such as

test tubes, glass petri-dishes, flasks, pipettes, canisters.

Dry heat is used for moisture sensitive materials at a temperature as high as (160 – 180oC).

Benefits of Dry heat

Good penetration

Non-corrosive nature

Moist Heat: This involves sterilization in the presence of moisture. This process is more rapidly lethal than dry heat. It requires lower working temperature and shorter periods of exposure. Moist heat denature proteins with relative ease due to facilitated rupture of proteins, stabilizing hydrogen bonds for example C=O......HN bonds. These bonds are more easily broken down when water molecules are available for hydrogen bonding. Temperature/ time combination used in the autoclave:

1210 C for 15-20 minutes at 151b/in2 (1.5kg/cm2 )

1150C for 35 minutes at 101bs/in2.(1.0KG/cm2)

Sterilization by moist heat is done at three different temperatures for different purposes:

At temperature below 1000C as used in Pasteurisation originally to sterilise wine but now

used primarily to prevent milk borne infections and delay milk decomposition. It involves

heating 620 for thirty minutes. Milk borne pathogens like the tuberculin bacilli,

Streptococcus species and Brucella species are all killed by this treatment.

Sterilization at 1000C involves heating at 1000C for few minutes, breaking and

subsequently heating at 1000C for the same time and repeating for three days. It is

employed for heat-labile substance which could be damaged by heating at 1000C for

longer periods. It is called Tyndalization.

Sterilization at temperatures above 1000 C is possible only due to the fact that at pressures

above atmospheric pressures, water can be brought to boil above 1000C.It is usually

carried out in an autoclave. Autoclaving is the most efficient method of sterilizing

microbiological culture media, especially if they are capable of withstanding the high

temperature without decomposition.

Uses of Autoclave

Decontamination laboratory and hospital waste

Sterilization of glasswares, etc

Media and reagents.

General uses of dry and moist heat sterilization: Sterilization is required in the preparation of

culture media, suspension fluid, reagents, containers and equipments used in microbiology. It is

important for medical and surgical instruments and other materials used in procedures that

required or involve sterility.

FILTRATION STERILIZATION: Filtration is the process or practical method of removing

microorganism. Filtration is usually employed in the sterilization of liquids and gases that are

liable to be spoiled by heat or heat-sensitive liquid. Filtration is capable of preventing the

passage of both viable and non-viable sensitive liquid.

Mechanism of action: Filtration is sieving absorption and trapping within the matrix of the filter

material. Examples of material that use filtration sterilization; heat-sensitive injections,

ophthalmic solutions, biological products and air and other gases for supply to aseptic areas.

Filtration is used in industry as part of the venting systems on fermentors, centrifuges,

autoclaves, freezer, driers etc.The effectiveness of the bacterial filters depends upon the size of

the pores, the electric charge on the filter materials, etc.

TYPES OF FILTER: Earthenware candles: for example; Berkfeld and the Chamberland.

Asbestos filters: usually available in a number of graded porosities.

Membrane filters: usually made of highly porous cellulose acetate and are available in a wide

range of different porosities.

CHEMICAL METHOD STERILIZATION: Chemical method sterilization can be referred to

as disinfection. This method is carried out using appropriate antimicrobial agents called sterilants

at concentrations that are lethal enough to ensure sterility.

QUALITIES OF A GOOD DISINFECTANT OR ANTISEPTICS

Broad spectrum of activity

Ability to destroy microbe within practical period of time

Active in the presence of organic matter

Active in any pH

It has long shelf life

It should have long penetrating power

It should not have toxic, non-allergic, non-irritative or non-corrosive

It should not have bad ordure

It should be volatile enough to be able to escape soon after application.

CLASSIFICATION OF DISINFECTANT: Disinfectants and antiseptics are actually

differentiated by the levels of toxicity:

LEVEL OF TOXICITY: Strong disinfectants: These are potent microbiocidal chemicals that

rapidly kill vegetative cells of pathogens but often ineffective against spores. They are relatively

toxic substances for example, phenol (carbolic acid), Lysol, etc.

Mild disinfectant (antiseptics): These may kill microorganisms (microbiocidal) or merely

prevent their growth,(microbiostatic).Their non-toxicity permits their being used as antiseptics

which can be applied on living tissues like skin, example dettol.

There are chemical agents considered to be reliable sterilants: ethylene oxide, glutaraldehyde,

toluene and chloroform

Based on mechanism of action

a. Action on membrane (e.g. Alcohol, detergent)

b. Denaturation of cellules proteins (Alcohol, phenol)

c. Oxidation of essential sulphyryl groups of enzymes (e.g. H202 (hydrogen peroxide),

halogens

d. Alkylation of amino, carboxyl and hydroxyl group (e.g. formaldehyde)

e. Damage to nucleic acid (formaldehyde)

Example when alcohol is used as disinfectant, it dehydrate cells of microorganisms, disrupt

membrane and cause coagulation of protein. Example of alcohol: Ethyl alcohol (spirit), isopropyl

(ethanol), and methyl alcohol. 70% aqueous solution of alcohol is more effective than absolute

alcohols, methyl alcohol kills fungal spores.

Disadvantages: It causes skin irritation, volatile (evaporates rapidly) and it is inflammable.

Aldehydes: Mode of action: it acts by alkylation of amino carboxyl or hydroxyl group and

probably damages nucleic acids. It kills all microorganisms including spores.

Application: 40% formaldehyde (formalin) is used for surface disinfection and fumigation of

rooms, chambers, operation theatres, biological safety laboratory, wards sick rooms.

Disadvantages: Vapours are irritating, gave poor penetration, leave non-volatile residue, etc

Phenol: Mode of action: It acts by disruption of membrane precipitation of proteins and

inactivation of enzymes, examples; 5% of phenol, 1-5% cresol, 5% Lysol, (a saponified cresol)

hexachlorophene, chlorohexidine, chloroxylenol (dettol).Disadvantages: it is toxic, corrosive

and skin irritant.

Halogens: Mode of action: They are oxidizing agents and causes damage by oxidation of

essential sulfydryl group of enzymes. Chlorine reacts with water to form hypochlorous acid

which is microbicidal, example: chlorine compounds (chlorine, bleach, and hypochlorite) and

iodine compounds (tincture iodine, iodophore). Tincture iodine used is combination of (2%

iodine in 70% alcohol).

Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine is

corrosive and staining. Bleach solution is corrosive and will corrode stainless steel surfaces.

Surface active agents: (soaps and detergents): Soaps and detergent: Washing with soap and

detergent greatly aid hygiene because they aid mechanical removal of microorganism. As

anionic detergents, ordinary soaps also have measure of antibacterial activity. Soaps of saturated

fatty acid are mildly active against some pathogenic intestinal bacteria. On the other hand, soaps

of long chain unsaturated fatty acid are active against some of the respiratory pathogens.

Generally surface active agents posses wetting and cleaning properties.

Mode of action: They have the property of concentrating at interface between lipid containing

membrane of bacteria cells and surrounding aqueous medium. These compounds have long chain

hydrocarbons that are fat soluble and charged ions that are water-soluble, examples soaps and

detergents. Detergent can be anionic or cationic.

Application: active against vegetative cells, Mycobacterium and enveloped viruses.

Gaseous sterilization: Ethylene oxide sterilizer: it is used widely to process heat-sensitive

device but the aeration time needed at the end of the cycle to eliminate the gas made this method

slow.

Gaseous sterilization (Disinfectant): Formaldehyde: the gas is librated by spraying or heating

formalin or by heating solid para-formaldehyde. For effectiveness, the atmosphere should have a

temperature around 18oC and a relative humidity between 60 – 90%.

Ethylene oxide: this gas is highly lethal to all kinds of microbes and spores and is much more

capable of penetration into porous materials. This gas must be used only in special chambers or

appropriate because it is toxic and forms an explosive mixture when more than 3% is in air.

Factors affecting activity of disinfectant

Contact: moisture is essential in the contact between the chemical and the micro

organisms for effectiveness.

Concentration: The rate of killing of micro organism by the concentration.

Time: In a microbial population, the cells usually inhibit varying grades of resistance to

adverse condition including the presence of disinfectant. Susceptible cells die first, while

those of greater resistance die in successive internals.

Nature of medium: most disinfectants kill micro organism by virtue of their ability to

react with organic cell constituents.

Temperature: Efficiency of disinfectant is enhanced by mild increase in temperature.

Nature of microorganism: Sporulate and encapsulated bacteria are much more difficult to

kill that vegetative cell.

pH: micro organisms are usually more resistant when suspended in media of a reaction

satisfaction for growth. Micro organisms are killed when the pH departs from their

optimum pH

GAS VAPOUR STERILIZATION:

Ethylene oxide – 450 to 1200mg/l at 29oC to 65oC for 2 – 5hrs

Formaldehyde vapour – 2% to 5% at 60oC to 80oC

Hydrogen peroxide vapour – 30% at 55oC to 60oC

Plasma gas – highly ionized hydrogen peroxide

Chlorine dioxide gas – variable.

Questions:1. Define sterilization and disinfection.2. Differentiate between sterilization and antiseptics.3. Describe the characteristics of an ideal antiseptic.4. What are the major methods of sterilization?5. What technique is used to sterilize heat-sensitive materials?

ReferencesLansing, M.P., John, P. H. and Donald A. K. (2005) Microbiology.6th edition Published by Mc Graw Hill.

Onyeagba, R.A and Isu, N.R. (2004). General Microbiology. Research and Academic publishers Owerri.

Ogbulie, J.N., Onyeagba, R. and Isu, N.R.(1997). Introductory Microbiology. CRC publications Nig.Ltd. Owerri.

Murray,P., Rosenthal, K. and Michael,P (2005).Medical Microbiology.Chapter ten (10) 5th edition.

Samuel Baron (1996). Medical Microbiology. Chapter eleven (10) 4th edition.

Recommended books and articles:

Sridhar Rao,P.N(2008).Available online www.microrao.com