Sterilization

Sterilization is the killing or removal of all microorganisms, including bacterial spores which are
highly resistant. Sterilization is an absolute term, i.e. the article must be sterile meaning the absence
of all microorganisms.

Disinfection is the killing of many, but not all microorganisms. It is a process of reduction of number
of contaminating organisms to a level that cannot cause infection, i.e. pathogens must be killed.
Some organisms and bacterial spores may survive.

Disinfectants are chemicals that are used for disinfection. Disinfectants should be used only on
inanimate objects.

Antiseptics are mild forms of disinfectants that are used externally on living tissues to kill
microorganisms, e.g. on the surface of skin and mucous membranes.

Uses of Sterilization
1. Sterilization for Surgical Procedures: Gloves, aprons, surgical instruments, syringes etc. are to be
sterilized.

2. Sterilization in Microbiological works like preparation of culture media, reagents and equipments
where a sterile condition is to be maintained.

CLASSIFICATION OF METHODS
Sterilization and disinfection are done by :
(A). Physical Agents
1. Heat
2. Radiation
3. Filtration

(B). Chemical Agents

In practice, certain methods are placed under sterilization which in fact do not fulfill the definition of
sterilization such as boiling for 1/2 hr and pasteurization which will not kill spores.

STERILIZATION BY HEAT
Heat is most effective and a rapid method of sterilization and disinfection. Excessive heat acts by
coagulation of cell proteins. Less heat interferes metabolic reactions. Sterilization occurs by heating
above 100C which ensure lolling of bacterial spores. Sterilization by hot air in hot air oven and
sterilization by autoclaving are the two most common method used in the laboratory.

Types of Heat :
A. Sterilization by moist heat
B. Sterilization by dry heat
A. Sterilization by Moist Heat
Moist heat acts by denaturation and coagulation of protein, breakage of DNA strands, and loss of
functional integrity of cell membrane.

(I). Sterilization at 100C

1. Boiling. Boiling at 100C for 30 minutes is done in a water bath. Syringes, rubber goods and
surgical instruments may be sterilized by this method. All bacteria and certain spores are killed. It
leads to disinfection.

2. Steaming. Steam (100C) is more effective than dry heat at the same temperature as: (a) Bacteria
are more susceptible to moist heat, (b) Steam has more penetrating power, and (c) Steam has more
sterilizing power as more heat is given up during condensation.

Steam Sterilizer. It works at 100C under normal atmospheric pressure i.e. without extra pressure. It
is ideally suitable for sterilizing media which may be damaged at a temperature higher than 100C.
It is a metallic vessel having 2 perforated diaphragms (Shelves), one above boiling water, and the
other about 4" above the floor. Water is boiled by electricity, gas or stove. Steam passes up. There is
a small opening on the roof of the instrument for the escape of steam. Sterilization is done by two
methods :

(a) Single Exposure for 11/2 hours. It leads to disinfection.

(b) Tyndallization (Fractional Sterilization). Heat labile media like those containing sugar, milk,
gelatin can be sterilized by this method. Steaming at 100C is done in steam sterilizer for 20 minutes
followed by incubation at 37C overnight. This procedure is repeated for another 2 successive days.
That is 'steaming' is done for 3 successive days. Spores, if any, germinate to vegetative bacteria
during incubation and are destroyed during steaming on second and third day. It leads to sterilization.

II. Sterilization above 100C: Autoclaving

Autoclaving is one of the most common methods of sterilization. Principle: In this method
sterilization is done by steam under pressure. Steaming at temperature higher than 100C 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 100C. At 15 ib per sq. inch pressure, 121C temperatures is obtained. This is kept for 15
minutes for sterilization to kill spores. It works like a pressure cooker.

'Sterilization holding time' is the time for which the entire load in the autoclave requires to be
exposed.

Autoclave is a metallic cylindrical vessel. On the lid, there are : (1) A gauge for indicating the
pressure, (2) A safety valve, which can be set to blow off at any desired pressure, and (3) A stopcock
to release the pressure. It is provided with a perforated diaphragm. Water is placed below the
diaphragm and heated from below by electricity, gas or stove. Working of Autoclave. (a) Place
materials inside, (b) Close the lid. Leave stopcock open, (c) Set the safety valve at the desired
pressure, (d) Heat the autoclave. Air is forced out and eventually steam ensures out through the tap,
(e) close the tap. The inside pressure now rises until it reaches the set level (i.e. 15 Win), when the
safety valve opens and the excess steam escapes, (f) Keep it for 15 minutes (holding time), (g) Stop
heating, (h) Cool the autoclave below 100C, (i) Open the stopcock slowly to allow air to enter the
autoclave.

Checking of Autoclave for Efficiency. Methods :

(i) Spores of Bacillus stearothermophilus are used. Spores withstand 121C heat for up to 12 min.
Strips containing this bacteria are included with the material being autoclaved. Strips are cultured
between 50C and 60C for surviving spores. If the spores are killed the autoclave is functioning
properly.
(ii)Automatic Monitoring System.

III. Sterilization below 100C

1. Pasteurization. Pasteurization is heating of milk to such temperature and for such a period of time
so as to kill pathogenic bacteria that may be present in milk without changing colour, flavour and
nutritive value of the milk. Mycobacterium bovis, Salmonella species, Escherichia coli and Brucella
species may be present in milk. It does not sterilize the milk as many living organisms including
spores are not destroyed..

Methods of Pasteurization
(i) Flash Method. It is "high temperature- short time method". Heating is done at 72C for 15
seconds.
(ii) Holding Method. Heating is done between 63C and 66C for 30 minutes.

2. Inspissation. Inspissation is done between 75C to 80C. Inspissation means stiffening of protein
without coagulation as the temperature is below coagulation temperature. Media containing serum or
egg is sterilized by heating for 3 successive days. It is done in 'Serum Inspissator'.

B. Sterilization by Dry Heat

Mechanisms. (1) Protein denaturation, (2) Oxidative damage, (3) Toxic effect of elevated electrolyte
(in absence of water).

Dry heat at 160C (holding temperature for one hour is required to kill the most resistant spores).
The articles remain dry. It is unsuitable for clothing which may be spoiled.

1. Red Heat. Wire loops used in microbiology laboratory are sterilized by heating to 'red' in bunsen
burner or spirit lamp flame. Temperature is above 100C. It leads to sterilization.

2. Flaming. The article is passed through flame without allowing it to become red hot, e.g. scalpel.
Temperature is not high to cause sterilization.

3. Sterilization by Hot Air

Hot Air Oven (Sterilizer). It Is one of the most common method used for sterilization. Glass wares,
swab sticks, all-glass syringes, powder and oily substances are sterilized in hot air oven. For
sterilization, a temperature of 160C is maintained (holding) for one hour. Spores are killed at this
temperature. It leads to sterilization.

Hot Air Oven 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 on Me top of the apparatus. A thermostat is fitted to maintain a
constant temperature of 160C.
BACTERIAL GROWTH
Growth of Bacteria is the orderly increase of all the chemical constituents of the bacteria.
Multiplication is the consequence of growth. Death of bacteria is the irreversible loss of ability to
reproduce.

Bacteria are composed of proteins, carbohydrates, lipids, water and trace elements.

Factors Required for Bacterial Growth

The requirements for bacterial growth are:
(A)) Environmental factors affecting growth, and
(B) Sources of metabolic energy.

A. Environmental Factors affecting Growth

1. Nutrients. Nutrients in growth media must contain all the elements necessary for the synthesis of
new organisms. In general the following must be provided : (a) Hydrogen donors and acceptors, (b)
Carbon source, (c) Nitrogen source, (d) Minerals : sulphur and phosphorus, (e) Growth factors:
amino acids, purines, pyrimidines; vitamins, (f) Trace elements: Mg, Fe, Mn.

Growth Factors: A growth factor is an organic compound which a cell must contain in order to
grow but which it is unable to synthesize. These substances are essential for the organism and are to
be supplied as nutrients. Thiamine, nicotinic acid, folic acid and para-aminobenzoic acid are
examples of growth factors.

Essential Metabolites: These metabolites are essential for growth of bacterium. These must be
synthesized by the bacterium, or be provided in the medium. Mg, Fe and Mn are essential trace
elements.

Autotrophs live only on inorganic substances, i.e. do not require organic nutrients for growth. They
are not of medical importance.

Heterotrophs require organic materials for growth, e.g. proteins, carbohydrates, lipids as source of
energy. All bacteria of medical importance belong to heterotrophs. Parasites may depend on the host
for certain foods. Saprophytes grow, on dead organic matter.

2. pH of the medium. Most pathogenic bacteria grow best in pH 7.2-7.4. Vibno cholerae can grow
in pH 8.2-9.0.
3. Gaseous Requirement
(a) Role of Oxygen. Bacteria may be classified into four groups on oxygen requirement :
(i) Aerobes. They cannot grow without oxygen, e.g. Mycobacterium tuberculosis.
(ii) Facultative anaerobes. These grow under both aerobic and anaerobic conditions. Most bacteria
are facultative anaerobes, e.g. Enterobacteriaceae.
(iii)Anaerobes. They only grow in absence of free oxygen, e.g. Clostridium, Bacteroides.
(iv) Microaerophils grow best in oxygen less than that present in the air, e.g. Campylobacter.

Aerobes and facultative anaerobes have the metabolic systems: (1) cytochrome systems for the
metabolism of oxygen, (2) Superoxide dismutase, (3) catalase.

Anaerobic bacteria do not grow in the presence of oxygen. They do not use oxygen for growth and
metabolism but obtain their energy from fermentation reactions. Anaerobic bacteria are killed by
oxygen or toxic oxygen radicals. Multiple mechanisms play role for oxygen toxicity : (1) They do
not have cytochrome systems for oxygen metabolism, (2) They may have low levels of superoxide
dismutase, and (3) They may or may not have catalase.

(b) Carbon dioxide. All bacteria require CO2 for their growth. Most bacteria produce CO2. N.
gonorrhoeae and N. meningitides and Br abortus grow better in presence of 5 per cent CO2.

4. Temperature. Most bacteria are mesophilic. Mesophilic bacteria grow best at 30-37C.
Optimum temperature for growth of common pathogenic bacteria is 37C. Bacteria of a species will
not grow but may remain alive at a maximum and a minimum temperature.

5. Ionic strength and osmotic pressure.

6. Light. Optimum condition for growth is darkness.

B.Sources of Metabolic Energy

Mainly three mechanisms generate metabolic energy. These are fermentation, respiration and
photosynthesis. An organism to grow, at least one of these mechanisms must be used.

REPRODUCTION
Bacteria reproduce by binary fission. Multiplication takes place in geometric progression. The
nucleus (chromosome) undergoes duplication prior to cell division. When the cell grows about twice
its size, the nuclear material divides, and a transverse septum originates from plasma membrane and
cell wall and divides the cell into two parts. The two daughter cells receive an identical set of
chromosomes. The daughter cells separate and may be arranged singly, in pairs, clumps, or chains.

GROWTH CURVE
The growth curve indicates multiplication and death of bacteria. When a bacterium is inoculated in a
medium, it passes through four growth phases which will be evident in a growth curve drawn by
plotting the logarithm of the number of bacteria against time. Number of bacteria in the culture at
different periods may be : (1) Total count. It includes both living and dead bacteria, or (2) Viable
count. It includes only the living bacteria. Microbial concentration can be measured in terms of cell
concentration, i.e. the number of viable cells per unit volume of culture, or of biomass concentration,
i.e. dry weight of cells per unit volume of culture.

Bacterial count is of use in the examination of water and milk.

Growth Phases

1. Lag Phase. In this phase there is increase in cell size but not multiplication. Time is required for
adaptation (synthesis of new enzymes) to new environment. During this phase vigorous metabolic
activity occurs but cells do not divide. Enzymes and intermediates are formed and accumulate until
they are present in concentration that permits growth to start. Antibiotics have little effect at this
stage.

2. Exponential Phase or Logarithmic (Log) Phase. The cells multiply at the maximum rate in this
exponential phase, i.e. there is linear relationship between time and logarithm of the number of cells.
Mass increases in an exponential manner. This continues until one of two things happens: either one
or more nutrients in the medium become exhausted, or toxic metabolic products, accumulate and
inhibit growth. Nutrient oxygen becomes limited for aerobic organisms. In exponential phase, the
biomass increases exponentially with respect to time, i.e. the biomass doubles with each doubling
time. The average time required for the population, or the biomass, to double is known as the
generation time or doubling time. Linear plots of exponential growth can be produced by plotting the
logarithm of biomass concentration as a function of time. Importance : Antibiotics act better at this
phase.

3. Maximal Stationary Phase. Due to exhaustion of nutrients or accumulation of toxic products

death of bacteria starts and the growth cease completely. The count remains stationary due to balance
between multiplication and death rate. Importance: Production of exotoxins, antibiotics,
metachromatic granules, and spore formation takes place in this phase.

4. Decline phase or death phase. In this phase there is progressive death of cells. However, some
living bacteria use the breakdown products of dead bacteria as nutrient and remain as persister.

Recently, some authors are dividing the growth curve into six phases by the letters A to F as follows
(a) Lag phase- Growth rate is zero.
(b) Acceleration phase- Increasing growth rate. C Exponential phase Constant growth rate.
(c) Retardation phase- Growth rate is decreasing.
(e) Maximum stationary phase- Growth rate is zero. F Decline phase- Growth rate is negative
(death).
GENETIC ELEMENTS

Nucleoid- Bacterial Chromosome

Bacterial nucleoid consists of a single circular molecule. It is a single, haploid chromosome of about
1 mm long in unfolded state. Bacterial chromosome (DNA) is attached to a septa) mesosome.
Nucleoids consist of DNA, smaller amounts of RNA, RNA polymerase and probably other proteins.
The nucleoid replicates by fission. It has no nucleolus, nuclear membrane, spindle or non-identical
chromosomes. Genes of the nucleus determines the amino acid sequences and hence the structure of
a protein and thereby all the properties of the organism. These account for heredity. Recently it has
been found that some prokaryotes have a linear chromosome. Staining of Nucleus. Nuclei are stained
with Feulgan stain which is specific for DNA.

Plasmid
Certain bacteria contain plasmid. These are extra-chromosomal genetic material (DNA), ie. genetic
determinant, and are mostly circular. Certain properties are determined by plasmid:

1.Virulence factors, eg. In enteropathogenic E. coli strains, both enterotoxins and colonization
antigens (pill). Other exotoxin productions are frequently controlled by plasmids.

2.Drug resistance by R factors (Resistance plasmids).

3.Metabolic activities may be determined. Examples: Sucrose uptake and metabolism, and citrate
uptake by Escherichia coli.

4.Production of colicins (Bacteriocins) by many Gram-negative bacteria is controlled by plasmids.

5.Transfer of genetic material by sex factors (See Chap. 40).

Transposon
Transposons are genetic elements with DNA sequences

CYTOPLASMIC STRUCTURES
Mitochondria are absent in prokaryotic cells.

1. Ribosomes. They are present as minute granules in large number. RNA is the principal component
of ribosomes.

2. Nutrient granules. These are not essential or permanent structures: These may be:

(a)Metachromatic, Volutin or Babes-Ernst granules. These may be present in Corynebacterium

diphtheriae and may be used for identification. These act as reserve of energy. These have affinity for
basic dyes. Albert staining is done to demonstrate metachromatic granules.
(b)Lipid granules. These are P- hyd roxybu rate.
(c)Polysaccharide granules, eg. glycogen, starch.
(d)Other granules, eg. sulphur.

3. Carotenoids- Photosynthetic pigments are present in photosynthetic bacteria.

FLAGELLA
A large number of bacteria possess flagella. Flagellum is the organ of locomotion. Flagellated
bacteria are motile. Nonflagellated bacteria are non-motile.

Examples:
Motile Bacteria: Escherichia, Salmonella, Proteus, Pseudomonas, Vibrio, Aeromonas, Plesiomonas,
Campylobacter, Helicobacter, Clostridium tetani.

Nonmotile Bacteria: All Cocci, Shigella, Klebsiella, Corynebacterium diphtheriae, Clostridium

perfringens, Bacillus anthraces, Haemophilus, Bordetella, Brucella, Yersinia, Pasteurella, Francisella.

Structure . Flagella are long, slender thread-like (12-30 nm in diameter) organs of locomotion. A
flagellum is attached to the bacterial cell body by a complex structure consisting of filament, hook
and basal body. The basal body has one pair of rings in Gram-positive bacteria and two pairs in
Gram-negative bacteria. A bacterial flagellum is composed entirely of protein. A flagellum is made
up of several thousand molecules of a protein subunit called flagellin. The flagellum is formed by the
aggregation of subunits to form a helical structure.

A flagellated bacterium may become non-flagellated but not the reverse. The arrangement of flagella
for a particular bacterium is constant and is one of the four types.

1. Peritrichous. Flagella are distributed over the entire cell, e.g. Escherichia coli, Salmonella genus.

2. Amphitrichous. There is a single flagellum at each pole.

3. Lophotrichous. There is a bunch of flagella at one or both poles, e.g. Spirillum minus.

4. Monotrichous. There is a single polar flagellum at one pole, e.g. Vibrio cholerae.

They have no function in the virulence of the bacteria. Antigenic structure of the flagella (H
antigens) may be used for identification and classification of bacteria.

Sensory transduction is the behavior of the flagellated bacteria in response to the environment. This
may be: (1) Chemotaxis is the movement of the bacterium towards a chemical attractant, e.g. sugar,
amino-acid, (2) Aerotaxismovement towards the optimal oxygen concentration, (3) Electron acceptor
taxis- movement towards alternative electron receptors, (4) Phototaxis- movement towards light of
photosynthetic bacteria.

Motility Test. Hanging-drop preparation is done by placing a drop of bacterial suspension between a
slide and cover slip separated by a circular layer of vaseline on the surface of the slide.

Flagella Staining. They are demonstrated by special staining. Flagella are not visible under light
microscope. Bacteria are first treated with tannic acid salt which precipitates on flagella and cell
walls and increases the diameter of the flagella. They are then visible under light microscope.
PILI (FIMBRIAE)
Pill or fimbriae are very thin, hair-like short rigid surface appendages. Many Gram-negative bacteria
possess pili or fimbriae. There may be 100-500 pili in one cell. They are shorter and finer than
flagella and composed of protein subunits termed pilins. Minor proteins, located at the tips of pili, are
responsible for the attachment properties. They are seen by electron microscope. Pellicle formation
in the fluid media by bacteria is due to fimbriae. Pill of different bacteria are antigenically distinct.
Some bacteria, e.g. N. gonorrhoeae are able to make pili of different antigenic types (antigenic
variation) and this can still adhere to cells by a type in presence of antibodies to other types. Fimbriae
of group A streptococci are the site of the surface antigen, M protein. Lipoteichoic acid, associated
with these fimbriae is responsible for adherence of these streptococci to epithelial cells of the hosts.
There are two types of pili on function:

1. Ordinary pill. These play roles in adherence of bacteria to host cell surfaces. 'Colonization
antigens' are ordinary pili that provide the cells with adherent properties, genetically determined by
plasmids.

2. Sex pill. These give the 'maleness'. They are few in number and very long, e.g. in enterobacteria.
During bacterial conjugation the male (donor) cell adhere with its sex pili and transfer DNA to non-
male (recipient) cell. The pili act as conjugation tubes. In this way the genetic material which
determines antibiotic resistance may pass from one bacterium to another. Sex pili possess receptors
to which bacteriophage can become attached.

BACTERIAL SPORES (ENDOSPORES)

Bacterial endospores are formed when conditions for vegetative growth are not favourable.

Spore-Forming Bacteria
1.Bacillus and Clostridium genera form spores.
2. Sporosarcina (Gram-positive coccus) and possibly Coxiella burnetii can produce spores.

Demonstration of Spore:
1.In Gram stain, the spore appears as unstained colourless areas in cells.
2.Spores are stained with malachite green, or carbol fuchsin (by modified Ziehl-Neelsen stain).

Nature. The spore is a resting cell. Spores are in a subdued metabolic state and non-reproducing.
Each cell forms a single internal spore that is liberated when the mother cell undergoes autolysis. The
spore is highly resistant to heat, chemical agents and desiccation. In sporulation, each vegetative cell
forms only one spore, and in subsequent germination each spore gives rise to a single vegetative cell.

Genetic.
Genetic is the science that deals with heredity. Thus, it is the study of the mechanisms by which
different characteristics are passed on from parents to offsprings (progeny).

Gene. Genes are genetic determinants and thus control heredity and determine properties of the
organism. The genes are functional units of the chromosomes. Synthesis of protein components and
enzymes of a cell is regulated by genes. DNA is responsible for both gene function and replication.
By replication heredity or stability of a type is maintained. An organism containing a normal gene is
known as 'wild type'. Genes may rarely mutate (change) resulting in heritable variations called
mutations (See chapter 8). The changed organism is called a mutant.

Genotype. It is the genetic determinant of a cell.

Phenotype. This is the structural and physiological manifestations of the organism due to a particular
genotype.

Genome. Genome is the entire set of genes and thus is the totality of genetic information in an
organism.

BACTERIAL GENOME (PROKARYOTIC GENOME)

Genes are carried on :
1. Bacterial chromosome. Chromosomes carry most prokaryotic genes. Genes essential for bacterial
growth are carried on chromosome. It is present in all bacteria. Only one chromo-some is present in a
bacterial nucleoid. Bacterial chromosome is a single circle containing about 4000 kbp (kilobase
pairs) of DNA. DNA is a double stranded helical structure. A molecule of DNA consists of many
mononucleotides. A mononucleotide consists of a molecule of sugar, a molecule of phosphate and a
molecule of base. In each strand of DNA, molecules of phosphate and sugar alternate, and one of the
four bases present in DNA is attached with the sugar. The four bases are adenine, thymine, guanine
and cytosine. A base of a strand joins with its complementary base of the other strand. Adenine is
always complementary to thymine, and guanine to cytosine. During cell division, duplication of
chromosome occurs so that each daughter cell receives an identical set.

2.Plasmid. Certain bacteria contain plasmids. Plasmids carry genes associated with specialized
function. These are extra-chromosomal genetic elements. Plasmid is a circular double stranded DNA
having several to 100 kbp. Plasmids replicate autonomously and they code for functions which are
normally not indispensable. In some cases, plasmids may be transferred from one cell to another and
thus may carry sets of specialized genetic information through a population, e.g. Drug resistance
plasmids (R factors) may render diverse bacteria resistant to antimicrobial drugs.

3.Transposon. Transposons are genetic elements with DNA sequences of several kbp. They can
migrate from one genetic

locus to another. Transfer of transposons can occur between one plasmid to another, or between
plasmid and chromosome within a bacterial cell. The process is called transposition. Transposons
carry genes for specialized functions, e.g. antibiotic resistance. Transposons do not contain genetic
information for their replication.
NOTE: Bacteria infected with bacterial viruses (bacteriophages) contain the genes of the phage.
Lysogenic bacteria are the bacteria which contain nonlytic prophage state of temperate phages.

Phenotypic changes that can occur in bacteria due to Mutation:

1. Loss of capsule formation by capsulated bacteria.
2. Change in colony characters.
3. Change in fermentation activity.
4. Loss of sensitivity to antibiotics.
5. Loss of sensitivity to bacteriophage (Bacterial virus).

TRANSFER OF GENE
Transfer of DNA is widespread among bacterial cells. In intercellular transfer, the genetic material
passes from the donor cell to the recipient cell. It leads to genetic diversity of bacteria. Intercellular
transfer of DNA between bacterial strains can occur by conjugation, transduction and transformation.

1. Conjugation
In conjugation, a suitable donor cell (male) comes near a recipient cell (female), establishes direct
cell-to-cell contact and transfers genetic material. Plasmids are most frequently transferred by
conjugation. Plasmids can also mobilize portions of chromosome for transfer. This is mediated by a
fertility or F factor which is carried on a plasmid. Sex pilus is responsible for the attachment of donor
(F*) cell and recipient cell (F) in bacterial conjugation (mating) process. Cell with sex pilus is the
male and without sex pilus is the female bacteria.

Example: Transfer of antibiotic resistance can occur by conjugation. Transfer of drug resistant
plasmid is called R-factor (Resistance plasmid). This occurs via sex pilus from the male (donor) cell
to the female (recipient) cell.

2.Transduction
Transduction is phage-mediated. In transduction, genetic material of donor bacterial cell is enclosed
in a bacterial virus (phage) and transferred to the recipient cell.

3.Transformation
In transformation, the recipient cell directly takes up naked DNA released from the donor cell
altering its genotype. It can occur in the medium. Natural transformation also can occur.