Microbiology and sterilization finals

Course outline

01.Sterlization and disinfection (only short and mcqs)

02.Growth in bacteria
01. Sterilization and disinfection (only short and mcqs)

Introduction
Sterilization is the killing or removal of all microorganisms, including bacterial spores, which are
highly resistant. Sterilization is usually carried out by autoclaving, which consists of exposure to steam at
121°C under a pressure of 15 lb./in2 for 15 minutes. Surgical instruments that can be damaged by moist
heat are usually sterilized by exposure to ethylene oxide gas, and most intravenous solutions are
sterilized by filtration.

Physical agents

 Sunlight

 Drying

 Heat

Sunlight
Direct sunlight has an active germicidal effect due to the combined effect of the ultraviolet radiation and
heat. This is a natural method of sterilization.

Drying
Moisture is essential for the growth of bacteria. Drying in air has therefore a deleterious effect on many
bacteria. But spores are unaffected by drying. Hence this is very unreliable method.

Heat
Heat has a killing effect on microorganisms and is one of the most popular reliable methods to destroy.
Microorganisms has a minimum, optimum and maximum growth temperatures.

Dry heat
Many objects are best sterilized in the absence of water by dry heat sterilization; killing by dry heat is
due to protein denaturation, oxidative damage and toxic effect of elevated levels of electrolytes.

Methods of Sterilization by Dry Heat;

1. Flaming
Inoculating loops and points of forceps may be heated in the Bunsen flame, till they are red-hot. Articles
such as mouth of the culture tubes, cotton wool plugs, glass slides etc. are passed over the flame
without allowing it to become red hot.

2. Incineration
This is an excellent method for rapidly destroying, animal carcasses, pathological material and
disposables.

3. Hot Air Oven

This is the most widely adopted method of sterilization by dry heat. The hot air oven utilizes radiating
dry heat for sterilization. This type of energy does not penetrate materials easily and thus, long periods
of exposure to high temperature are necessary.

Moist heat
Moist heat kills microorganisms by coagulating their proteins and is much more rapid and effective than
dry heat because water molecules conduct heat better than air. Lower temperature and less time of
exposure are therefore required than for dry heat. Moist heat readily kills viruses, bacteria, fungi etc.

Temperature below 100 °C

Pasteurization of milk for pasteurization of milk, there are two methods · Holding Method or Low
Temperature Holding Method (LTH) In this method, the milk is exposed to a temperature of 63°C (145°F)
for 30 minutes in an appropriately designed equipment. This is followed by sudden cooling to 13°C or
below. · Flash Process or High Temperature Short Time (HTST) In this method, the milk is exposed to a
temperature of 72°C for 15 seconds in the equipment. This is followed by sudden cooling to 13°C or
below. The finished product should be stored at a low temperature to retard growth of microorganisms
and pasteurization removes the pathogenic bacteria in milk.

Temperature at 100°C

I. Boiling
Most of the vegetative forms of bacteria, fungi etc. are killed almost immediately at 90-100°C, but
sporing bacteria required considerable periods of boiling. Boiling water is not considered as a sterilizing
agent because destruction of bacterial spores and inactivation of viruses cannot always be assured.
Under ordinary circumstances, most species of microbes can be killed within 10 minutes. However,
spores of bacteria and fungi, protozoa cysts and large concentrations of Hepatitis A viruses requires up to
30 minutes exposure or more because inadequate information exists on the heat tolerance of many
microorganisms, boiling water is not reliable for sterilization purpose especially the sterilization of
instruments and for surgical procedures. In cases where boiling is considered adequate, the material
should be immersed in water and boiled for a period of 10-30 minutes.

Steam under atmospheric pressure 100°C

Steam under atmospheric pressure is used to sterilize culture media which may decompose if subjected
to higher temperature. A Koch or Arnold sterilizer is an instrument that generates free floating steam.
The container and the medium are simultaneously sterilized and evaporation from the medium is
prevented one exposure of 90 minutes usually ensures complete sterilization of the medium. This is an
inexpensive method.

Sterilization above 100°C

Heat in the form of saturated steam under pressure is the most practical and dependable agent for
sterilization. Steam under pressure provides temperature above those obtainable by boiling. Moreover,
it has advantages of rapid heating, penetration and moisture in abundance, which facilitates the
coagulation of the protein of microorganisms, resulting in complete destruction of all forms of microbial
life, including bacterial endospores. It is important to note that the sterilizing agent is the moist heat not
the pressure. The laboratory apparatus designed to use steam under regulated pressure is called an
autoclave.

Filtration and irradiation

 Irradiation;
 The process of exposing organisms to anyone of the radiation such as UV-rays, X-rays, gamma
rays etc. is known as irradiation. Irradiation is an effective method of sterilization. Two types of
radiations are used for sterilization.

a) Non ionizing radiation

 UV radiation

 Infrared radiation

b) Ionizing radiation
 X rays

 Gamma rays

 Filtration;
Filtration is the process of removal of microorganisms from liquid or gases using a mechanical device
known as filter. This is an excellent way to reduce the microbial population in solution of thermo
labile materials such as sera, antibiotic solutions, intravenous solutions, carbohydrates solutions
used in the preparation of culture media etc. As fluid passes through the filter, microorganisms are
trapped in the pores of the filtering material. The solution that drips through the filter is collected in
a previously sterilized container. Porosity, electric charges of the filter, electric charge carried by the
organisms, nature of the fluid being filtered etc. can influence efficiency of filtration. Types of filters:
Seltz Filter, Beresfield Filter, Membrane Filter, High Efficiency Particulate Air (HEPA) filter.

Control of Microbes by Chemical Agents

• A large number of chemical compounds have the ability to inhibit the growth and metabolism of
microorganisms or to kill them.

• Commercial products which incorporate these compounds are available for controlling microbial
populations in many different circumstances.

• For example,
• Solutions of some chemical compounds are used to reduce the microbial flora of the oral cavity;
other chemical compounds are recommended for reducing the microbial population in the dust
of hospital floors. No single chemical agent is best for any and all purposes.

Characteristics Of An Ideal Agents

1. Antimicrobial activity

2. Solubility

3. Stability

4. Non toxicity to humans and other animals

5. Homogeneity

6. Capacity to penetrate

7. Detergent capacities

8. Availability

1) Antimicrobial activity. The capacity of the substance to kill or inhibit microorganisms is the first
requirement. The chemical, at a low concentration, should have a broad spectrum of
antimicrobial activity.

2) Solubility. The substance must be soluble in water or other solvents to the extent necessary for
effective use.

3) Stability. Changes in the substance upon standing should be minimal and should not result in
significant loss of germicidal action.

4) Nontoxicity to humans and other animals. Ideally, the compound should be lethal to
microorganisms and no injurious to humans and other animals.

5) Homogeneity. The preparation must be uniform in composition so that active ingredients are
present in each application. Pure chemicals are uniform, but mixtures of materials may lack
homogeneity.

6) Capacity to penetrate. Unless the substance can penetrate through surfaces, its germicidal
action is limited solely to the site of application. Sometimes, of course, surface action is all that is
required.

7) Detergent capacities. A disinfectant which is also a detergent (cleaning agent) accomplishes the
cleansing action improves the effectiveness of the disinfectant.

8) Availability. The compound must be available in large quantities at a reasonable price.

Different Terms:
• The following terms are used to describe the processes and chemical, agents employed in
controlling microorganisms.

 Sterilization

 Disinfectant

 Antiseptic

 Sanitizer

 Germicide (Microbicide)

 Bactericide

 Bacteriostasis

 Antimicrobial agents

Sterilization

 The process of destroying all forms of microbial life. A sterile object, in the microbiological sense,
is free of living microorganisms.

 The terms sterile, sterilize, and sterilization therefore refer to the complete absence or
destruction of all microorganisms and should not be used in a relative sense.

 An object or substance is sterile or no sterile; it can never be semi sterile or almost sterile.

Disinfectant

 An agent, usually a chemical, that kills the growing forms but not necessarily the resistant spore
forms of disease-producing microorganisms.

 The term is commonly applied to substances used on inanimate objects. Disinfection is the
process of destroying infectious agents.

Antiseptic

 A substance that opposes sepsis, i.e., prevents the growth or action of microorganisms either by
destroying microorganisms or by inhibiting their growth and metabolism. Usually associated
with substances applied to the body.

Sanitizer

 An agent that reduces the microbial population to safe levels as judged by public health
requirements.

 Usually, it is a chemical agent that kills 99.9 percent of the growing bacteria. Sanitizers are
commonly applied to inanimate objects and are generally employed in the daily care of
equipment and utensils in dairies and food plants and for glasses, dishes, and utensils in
restaurants
 Germicide (Microbicide)

 An agent that kills the growing forms but not necessarily the resistant spore forms of germs; in
practice a germicide is almost the same thing as a disinfectant, but germicides are commonly
used for all kinds of germs (microbes) for any application.

Bactericide and Bacteriostasis

• Bactericide. An agent that kills bacteria (adjective, bactericidal). Similarly, the terms
fungicide, virucide, and sporicide refer to agents that kill fungi, viruses, and spores, respectively.

• Bacteriostasis. A condition in which the growth of bacteria is prevented (adjective,

bacteriostatic). Similarly, fungistatic describes an agent that stops the growth of fungi.

• Agents that have in common the ability to inhibit growth of microorganisms are collectively
designated microbistatic agents.

Antimicrobial Agent

 One that interferes with the growth and metabolism of microbes. In common usage the term
denotes inhibition of growth, and with reference to specific groups of organisms such terms as
antibacterial or antifungal are frequently employed.

 Some antimicrobial agents are used to treat infections, and they are called chemotherapeutic
agents.

Selection of a Chemical Agent

• The major factors that need to be assessed in the process of selecting the most appropriate
chemical agent for a specific practical application are:

1. Nature of the material to be treated

2. Types of microorganisms

3. Environmental conditions

Major Groups of Antimicrobial Agents

1) Phenol and phenolic compounds

2) Alcohols

3) Halogens

4) Aldehydes

Phenol and Phenolic Compounds

Phenol and phenolic compounds are very effective disinfectants. A 5% aqueous solution of phenol
rapidly kills the vegetative cells of microorganisms; spores are much more resistant. Many
derivatives of phenol have been prepare and evaluated for their antimicrobial activity.
• Practical Application
• Phenolic substances may be either bactericidal or bacteriostatic, depending upon the
concentration used. Bacterial spores and viruses are more resistant than are vegetative cells.
Some phenolics are highly fungicidal.

• The antimicrobial activity of phenolics is reduced at an alkaline pH and by organic material. Low
temperatures and the presence of soap also reduce antimicrobial activity.

• Mode of Action
• Exposure of microbial cells to phenolic compounds produces a variety of effects. Depending
upon the concentration of the phenolic compound to which microbial cells were exposed,
researchers have described results such as disruption of cells, precipitation of cell protein,
inactivation of enzymes, and leakage of amino acids from the cells.

• Although the specific mode of action is not clear, there is a consensus that the lethal effect is
associated with physical damage to the membrane structures in the cell surface, which initiates
further deterioration.

Alcohol
• Ethyl alcohol, in concentrations between 50 and 90%, is effective against vegetative or
nonsporeforming cells, for practical application a 70% concentration of alcohol is generally used.

• Methyl alcohol is less bactericidal than ethyl alcohol: furthermore, it is highly poisonous. Even
the fumes of this compound may produce permanent injury to the eyes, and is not generally
employed for the destruction of microorganisms.

• Propyl and isopropyl alcohols in concentrations ranging from 40 to 80% are bactericidal for
vegetative cells.

• Practical Application
• Alcohol is effective in reducing the microbial flora of skin and for the disinfection of clinical oral
thermometers. Alcohol concentrations above 60% are effective against viruses; however, the
effectiveness is influenced considerably by the amount of extraneous protein material in the
mixture, The extraneous protein reacts with the alcohol and thus protects the virus.

• Mode of Action
• Alcohols are protein denaturants, and this property may, to a large extent, account for their
antimicrobial activity. Alcohols are also solvents for lipids, and hence they may damage lipid
complexes in the cell membrane.

• They are also dehydrating agents. This may account for the relative ineffectiveness of absolute
alcohol on "dry" cells; it is possible that very high concentrations remove so much water from
the cell that the alcohol is unable to penetrate.
Halogens
• Iodine is one 'of the oldest and most effective germicidal agents. A bluish-black crystalline
element having a metallic Luster. It is only slightly soluble in water but readily soluble in alcohol
and aqueous solutions of potassium or sodium iodide.

• The element is traditionally used as a germicidal agent in a form referred to as tincture of iodine.

• Iodine is also used in the form of substances known as iodophors, lodophors are mixtures of
iodine with surface- active agents which act as carriers and solubilizers for the iodine.

• Practical Application
• Iodine is a highly effective bactericidal agent and is unique in that it is effective against all kinds
of bacteria. Iodine also possesses sporicidal activity; however, the rate at which the spores are
killed is markedly influenced by the conditions under which they are exposed, e.g., amount of
organic material and extent of dehydration. In addition, it is highly fungicidal and is to some
extent virucidal.

• Iodine solutions are chiefly used for the disinfection of skin, and for this purpose they rank
among the best disinfectants.

 Mode of Action
• The mechanism by which iodine exerts its antimicrobial activity is not specifically understood.
Iodine is an oxidizing agent, and this fact may account for its antimicrobial action.

• Oxidizing agents can irreversibly oxidize and thus inactivate essential metabolic compounds such
as proteins with sulfhydryl groups. It has also been suggested that the action may involve the
halogenation of tyrosine units of enzymes and other cellular proteins requiring tyrosine for
activity.

Aldehydes
• Among the class of chemicals with the general formula RCHO (aldehydes), several of the low-
molecular-weight compounds are antimicrobial.

• Two of the most effective are

1. Formaldehyde

2. Glutaraldehyde
• Both are highly microbicidal, and both have the ability to kill spores (sporicidal).

• Practical Applications
Formaldehyde in solution is useful for sterilization of certain instruments. Formaldehyde in gaseous
form can be used for disinfection and sterilization of enclosed areas. Formalin and paraformaldehyde
are two principal sources of formaldehyde when it is used for gaseous disinfection.
3. Vaporization of formaldehyde from either of these sources into an enclosed area for art time will
cause sterilization, vegetative cells being killed more quickly than spores.

4. Humidity and temperature have a pronounced effect on the microbicidal action of

formaldehyde; in order to sterilize an enclosure, the temperature must be about room
temperature (22°C) and the relative humidity between 60 to 80 percent. One of the
disadvantages of this process is the limited ability of the formaldehyde vapors to penetrate
covered surfaces

• Mode of Action
• Formaldehyde is an extremely reactive chemical. It combines readily with vital organic nitrogen
compounds such as proteins and nucleic acids. It is likely that interaction of formaldehyde with
these cellular substances’ accounts for its antimicrobial action.
 02. REPRODUCTION IN BACTERIA:

Modes of Cell Division

Asexual means of reproduction

1. Binary fission
2. Budding
3. Fragmentation
4. Formation of Conidiospores or Sporangiospores
Binary Fission

 The most common, and no doubt the most important, mode of cell division in the usual growth
cycle of bacterial populations is transverse binary fission, in modes of Cell Division which a single
cell divides after developing a transverse septum (crosswall).

 Transverse binary fission is an asexual reproductive process.

Budding:
  Some bacteria, such as Rhodopseudomonas acidophila, reproduce by budding process in which a
small protuberance (bud) develops at one end of the cell; this enlarges and eventually develops
into a new cell which separates from the Parent.

 In Hyphomicrobium species, the bud may develop at the end of a prostheca (semi-rigid
extensions of the cell wall and cytoplasmic membrane)

Budding:

Fragmentation:
 Fragmentation in multicellular or colonial organisms is a form of asexual reproduction or
cloning, where an organism is split into fragments. Each of these fragments develop into
mature, fully grown individuals that are clones of the original organism.

 Example Bacteria that produce extensive filamentous growth, such as Nocardia species,
reproduce by fragmentation of the filaments into small bacillary or coccoid cells, each of which
gives rise to new growth.

Formation of Conidiospores or Sporangiospores:

Species of the genus Streptomyces and related bacteria produce many spores per organism by
developing crosswalls (septation) at the hyphal tips; each spore gives rise to a new organism.

Bacterial Genetics:

In conjugation, DNA is transferred between bacteria through a tube between cells.

In transformation, a bacterium takes up a piece of DNA floating in its environment.

In transduction, DNA is accidentally moved from one bacterium to another by a virus.

Conjugation:

 In conjugation, DNA is transferred from one bacterium to another. After the donor cell pulls
itself close to the recipient using a structure called a pilus, DNA is transferred between cells. In
most cases, this DNA is in the form of a plasmid.

 Donor cells typically act as donors because they have a chunk of DNA called the fertility
factor (or F factor). This chunk of DNA codes for the proteins that make up the sex pilus. It also
contains a special site where DNA transfer during conjugation begins .

 If the F factor is transferred during conjugation, the receiving cell turns into an F donor that
can make its own pilus and transfer DNA to other cells. Here's one analogy: this process is sort
of like how a vampire can turn other people into vampires by biting them.

Transformation:
 In transformation, a bacterium takes in DNA from its environment, often DNA that's been shed
by other bacteria.If the DNA is in the form of a circular DNA called a plasmid, it can be copied
in the receiving cell and passed on to its descendants.

Transduction:
  In transduction, viruses that infect bacteria move short pieces of chromosomal DNA from one
bacterium to another "by accident."

 Sometimes, chunks of host cell DNA get caught inside the new bacteriophage as they are
made. When one of these "defective" bacteriophages infects a cell, it transfers the DNA. Some
bacteriophages chop the DNA of their host cell into pieces, making this transfer process more
likely . can get a virus! The viruses that infect bacteria are called bacteriophages.

Normal Growth Cycle of Bacteria/Growth Curve

Bacterial colonies progress through four phases of growth:

1. The lag phase

2. The log phase

3. The stationary phase, and

4. The death phase

Lag Phase:
 This initial phase is characterized by cellular activity but not growth. A small group of cells are
placed in a nutrient rich medium that allows them to synthesize proteins and other molecules
necessary for replication. These cells increase in size, but no cell division occurs in the phase.

Exponential (Log) Phase:

 After the lag phase, bacterial cells enter the exponential or log phase. This is the time when
the cells are dividing by binary fission and doubling in numbers after each generation time.
Metabolic activity is high as DNA, RNA, cell wall components, and other substances necessary
for growth are generated for division.
 Generation time is the average interval between the birth of an individual and the birth of its
offspring.

Stationary Phase:
 Eventually, the population growth experienced in the log phase begins to decline as the
available nutrients become depleted and waste products start to accumulate. Bacterial cell
growth reaches a plateau, or stationary phase, where the number of dividing cells equal the
number of dying cells. This results in no overall population growth.

 Under the less favorable conditions, competition for nutrients increases and the cells become
less metabolically active.

Death Phase:
 As nutrients become less available and waste products increase, the number of dying cells
continues to rise. In the death phase, the number of living cells decreases exponentially and
population growth experiences a sharp decline.
O3. ISOLATION OF BACTERIA:

METHODS OF ISOLATING PURE CULTURE

PLATING TECHNIQUES:

 Pour plate method

 Spread plate method

• Streak plate method

 Pour Plate Method

• In pour plate method, successive dilutions of the inoculum (serially diluting the original
specimen) are added into sterile petri plate to which is poured melted and cooled (42ºC - 45ºC)
agar medium and thoroughly mixed by rotating the plates which is then allowed to solidify.

• After incubation, the plates are examined for the presence of individual colonies.

• The pure colonies may be isolated and transferred into test tube culture media for making pure
cultures. This technique is employed to estimate the viable bacterial count in a suspension.

• Prior to performing the pour plate technique, the sample must be serially diluted to make the
microbial load in the sample between 20 – 300 CFU/mL (suitable colony counting range is 20 –
200, some consider it to 30 – 300, and in average it is taken as 25 – 250).

• If the sample is liquid, then it can be serially diluted with sterile distilled water or sterile broth. If
the sample is solid or semisolid, it must be first emulsified and then serially diluted to reduce
microbial load up to the permitted range.

Interpretation
• For optimum count, the number of colonies must be between 20 – 300 CFU/mL. Beyond this
limit, the whole procedure must be repeated. If the number of the colony is less than 20, it is
suggested to use the sample of lower dilution, whereas, if the total number of colonies exceeds
300, it is suggested to use the sample of higher dilution on successive repeats.

• If the colonies are fused or the whole plate is covered with a single colony, then report as “too
numerous to count” (TNTC) and repeat the process of taking the sample at a higher dilution.

• Less than 20 (TFTC) “too few to count”

• Following the incubation, the viable microorganisms in the sample will grow into visible colonies
on the surface of and within the medium. The visible colonies can be counted and CFU/mL can
be calculated using the following formula;

 Spread Plate Method

• The spread plate technique is used for the separation of a dilute, mixed population of the
microorganisms so that individual colonies can be isolated.

• In this technique, a small volume of dilute microbial mixture is transferred to the center of an
agar plate and spread evenly over the surface with a sterile L-shaped bent glass rod, while the
Petri dish is spun, at some stage, single cells will be deposited with the bent glass rod on the agar
surface. Incubate the agar plate at 37ºC for 24 hours, in the inverted position.

• The dispersed cells will

develop into isolated
colonies. Because the
number of colonies will
be equal to the
 number of viable organisms in the sample spread plates can be used to count the microbial
population.

• The sample in the spread plate method must be liquid or in suspension. Before plating, the
samples are serially diluted. If the objective is to count the CFU/mL then the sample must be
diluted to make the microbial load in the sample between 20 – 300 CFU/mL (suitable colony
counting range is 20 – 200, some consider it to be 30 – 300, and in average it is taken as 25 –
250).

• The visible colonies can be counted and CFU/mL can be calculated using the following formula;

 Streak Plate Method

• Streak literally means “a long, thin line”: and the streak plate method is a microbiological culture
technique where a sample is spread in a petri dish in the form of a long, thin line over the
surface of solid media.

• This method was developed by two bacteriologists, Leoffler and Gaffkey in the laboratory of
Robert Koch.

• This method is routinely employed for the isolation of bacteria in pure culture.

• In this method a sterilized inoculating loop or transfer needle is dipped into a suitable diluted
suspension of microorganisms which is then streaked on the surface of an already solidified agar
plate to make a series of parallel, non-overlapping streaks. The process is known as streaking and
the plate so prepared is called a streak plate.

• The main objective of the streak plate method is to produce well separated colonies of bacteria
from concentrated suspensions of cells.

• A sterilized inoculating needle with a loop made up of either platinum or nichrome wire is used
for streaking.

• One loopful of specimen is transferred onto the surface of the agar plate in a sterile Petri dish
and streaked across the surface in the form of a zig-zag line. This process is repeated thrice to
streak out the bacteria on the agar plate so that some individual bacteria are separated from
each other.
• The first streak will contain more organisms than the second and the second more than the third
and so on.

• The last streaks should thin so on. The last streaks should thin out the culture sufficiently to give
isolate colonies.

 Quadrant Streaking

• It is the most commonly used and the most preferred method where four equal-sized sections of
the agar plate are streaked. It is also referred to as the “four-quadrant streak” or “four sectors”
or “four-way streak” method.

• In this method, each plate is divided into four equal sectors and each adjacent sector is streaked
sequentially. The sector which is streaked first is called the first sector or the first quadrant, and
it has the highest concentration of inoculum. Gradually the second, third, and fourth quadrants
will have diluted inoculum. By the time the fourth quadrant is streaked, the inoculum is highly
diluted giving rise to isolated colonies following the incubation.

• Mostly, a discontinuous fashion of streaking is followed where the loop is sterilized at the end of
each quadrant prior to streaking over the next quadrant. However, if the bacterial load is too
small (or highly diluted), continuous fashion can also be used. In the latter, the loop needs not be
sterilized at the end of every quadrant.

Although being the most popular method, it limits us to use only one specimen per plate. If we try
two or more specimens in a single 10 cm plate, this method is not suitable.

Quadrant Streaking Procedure

• Lift the Petri plate in your left hand and hold it at an angle of 60°. (if you are left-handed, hold
the plate in your right hand)

• The sample is spread over about 1/4th of the media in the Petri plate from the rim to the center
of the plate using a rapid, gentle, back and forth motion. (For ease, a beginner can draw two
diameters intersecting each other diagonally at the back of the petri dish to divide the media
into 4 equal sections)

• Re-flame the loop and allow it to cool. Turn the Petri plate by 90° anticlockwise, and place the
loop to the last streaks of the first quadrant. Move the loop back and forth to spread the
inoculum over the last half of the streaks in the first quadrant into the empty second quadrant.
(Be sure not to move the loop to the streaks in the first half of the first quadrant.)

• Repeat the process (iii) for streaking the third

quadrant and the fourth quadrant.

• For the fourth quadrant similar step can be

followed. However, many people prefer to draw a
few (6 to 7 streaks) well-separated streaks by
touching the second half of streaks in the third
quadrant. Also, some prefer to make the final streak
in a zigzag fashion making a tail.
04.Bacterial Structure
• Examination of a bacterial cell reveals various component structures.

• Some of these structures are external to the cell wall others are internal to the cell wall.

• Some structures are present in only certain species; some are more characteristic of certain
species than of others; and still other cellular parts, such as the cell wall, are naturally common
to almost all bacteria. The following are brief descriptions of the readily evident structures of
bacteria.

Structure of bacteria

 External structures
 Internal structures

EXTERNAL STRUCTURE TO THE CELL WALL

• Flagella

• Pili

• Capsules

• Sheaths

• Prosthecae and
stalks

 FLAGELLA AND
MOTILITY

• Bacterial
flagella (singular.
flagellum) are hairlike,
helical appendages that pro-trude through the cell wall and are responsible for swimming
motility.

• Size: They are much thinner than the flagella or cilia of eukaryotes, being 0.01 to 0.02 µm in
diameter, and they are also much simpler in structure.

• Location: Their location on the cell varies depending on the bacterial species and may be
polar. (at one or both ends of the bacterium) or lateral (along the sides of the bacterium).
 A flagellum is composed of three parts
1. A basal body associated with the cytoplasmic membrane and cell wall,

2. A short hook,

3. And a helical filament which is usually several times as long as the cell.

 Some Gram-negative bacteria have a sheath surrounding the flagellum: this sheath is continuous
with the outer membrane of the Gram-negative cell wall. The chemical composition of the basal
body is unknown, but the hook and filament are composed of protein subunits (monomers)
arranged in a helical fashion.

• The protein of the filament is known as flagellin.

• Unlike a hair. a flagellum grows at its tip rather than at the base.

• Flagellin monomers synthesized within the cell are believed to pass along the hollow center of
the flagellum and are added to the distal end of the filament.

BACTERIAL CHEMOTAXIS
• Many, perhaps most, motile bacteria are capable of directed swimming toward or away from
various chemical compounds—a phenomenon called bacterial chemotaxis.

• Swimming toward a chemical is termed positive chemotaxis

• Swimming away is negative chemotaxis

• Although chemicals may act as attractants or repellents, the stimulus is in fact not the chemical
itself but rather a change in the concentration of the chemical with time.

Pili(fimbriae)
• Pili (singular, - pilus) are hollow, nonhelical, filamentous appendages that are thinner, shorter
and more numerous than flagella.

• They do not function in motility, since they are

found on nonmotile as well as motile species.

• There are, however, several functions associated

with different types of pili.

• One type, known as the F pilus (or sex pilus), serves

as the port of entry of genetic material during bacterial mating.

• Some pili play a major role in human infection by allowing pathogenic bacteria to attach to
epithelial cells lining the respiratory, intestinal, or genitourinary tracts.

• This attachment prevents the bacteria from being washed away by the flow of mucous or body
fluids and permits the infection to be established.
Capsule
• Some bacterial cells are surrounded by a viscous substance forming a covering Capsules layer or
envelope around the cell wall.

• If this layer can be visualized by light microscopy using special staining methods, it is termed a
capsule

• If the layer is too thin to be seen by light microscopy it is termed a micocapsule if it is so

abundant that many cells are embedded in a common matrix, the material is Called slime.

• By Iight microscopy, capsules appear to be amorphous gelatinous areas surrounding a cell .

• In many instances capsular material is not highly water-soluble and therefore does not readily
diffuse away from the cells that produce it.

• In other instances, the material is highly water-soluble and dissolves in the medium, sometimes
dramatically increasing the viscosity of the broth in which the organisms are cultured.

Functions Of Capsule:
• Capsules can serve a number of functions, depending on the bacterial species.

• They may provide protection against temporary drying by binding water molecules.

• They may block attachment of bacteriophages.

• They may be antiphagocytic; i.e., they inhibit the engulfment of pathogenic bacteria by white
blood cells and thus contribute to invasive or infective ability (virulence).

• They may promote attachment of bacteria to surfaces; for example, Streptococcus mutans, a
bacterium associated with producing dental caries, firmly adheres to the smooth surfaces of
teeth because of its secretion of a water-insoluble capsular glucan.

• Most bacterial capsules are composed of polysaccharides.

 Homopolysaccharides
 Capsules composed of a single kind of sugar are termed homopolysaccharides; are usually
synthesized outside the cell from disaccharides by exocellular enzymes- The synthesis of glucan
(a polymer of glucose) from sucrose by S. mutans is an example.

 Heteropolysaccharides
 Other capsules are composed of several kinds of sugars and are termed heteropolysaccharides;
these are usually synthesized from sugar precursors that are activated (energized) within the
cell, attached to a lipid carrier molecule, transported across the cytoplasmic membrane, and
polymerized outside the cell.

 The capsule of klebsiella pneumoniae is an example.

Prosthecae and Stalk
• Prosthecae (singular, prostheca) are semirigid extensions of the cell wall and cytoplasmic
membrane and have a diameter that is always less than that of the cell. They are characteristic
of a number of aerobic bacteria from
freshwater and marine environments.

• Some bacterial genera such as Caulobacter

have a single prostheca; others such as
Stella and Ancalomicrobium have several.

• Prosthecae increase the surface area of the

cells for nutrient absorption, which is
advantageous in dilute environments. Some
prosthecate bacteria may form a new cell
(bud) at the end of a prostheca; others have
an adhesive substance at the end of a
prostheca that aids in attachment to

surfaces.