Culture Media

Culture medium: A nutrient material prepared for the growth of microorganisms in a laboratory is called a
culture medium.
Inoculum: When microbes are introduced into a culture medium to initiate growth, they are called an
inoculum.
Culture: The microbes that grow and multiply in or on a culture medium are referred to as a culture.
Cultivation is the process of growing microorganisms in culture by taking bacteria from the infection site
(i.e. in vivo environment) by some means of specimen collection and growing them in the artificial
environment of the laboratory (i.e. the in vitro environment). By appropriate procedures they have to be
grown separately (isolated) on culture media and obtained as pure cultures for study. Once grown in culture,
most bacterial populations are easily observed without microscopy and are present in sufficient quantities to
allow laboratory testing to be performed.
Main Purposes of Bacterial Cultivation
Bacterial cultivation has three main purposes:
i. To grow and isolate all bacteria present in an infection.
ii. Infection and contaminants or colonizers: To determine which of the bacteria that grow are most likely
causing infection and which are likely contaminants or colonizers.
iii. Identification and characterization: To obtain sufficient growth of clinically relevant bacteria to allow
identification and characterization.
Common Ingredients of Culture Media
Some of the components of culture media are as follows:
1. Water: Tap water is often suitable for culture media, particularly if it has a low mineral content, but if the
local supply is found unsuitable, glass-distilled or demineralized water must be used instead.
2. Agar: Agar (or agar-agar) is prepared from a variety of seaweeds and is now universally used for pre-
paring solid media. The chief component of agar is a long-chain polysaccharide. It also contains a variety
of impurities including inorganic salts, a small amount of protein like material and sometimes traces of
long-chain fatty acids, which are inhibitory to growth. The minerals present are mainly magnesium and
calcium.
Agar does not add to the nutritive properties of a medium and is not affected by the growth of bacteria. The
melting and solidifying points of agar solutions are not the same. At the concentrations normally used,
most bacteriological agars melt at about 95°C and solidify only when cooled to about 42°C. There are
considerable differences in the properties of the agars manufactured in different places, and even between
different batches from the same source. A concentration of 1-2 percent usually yields a suitable gel.
3. Peptone: Another almost universal ingredient of common media is peptone. It is a complex mixture of
partially digested proteins. The important constituents are peptones, proteoses, amino acids, a variety of
inorganic salts including phosphates, potassium and magnesium, and certain accessory growth factors such
as nicotinic acid and riboflavin.
4. Yeast extract: It contains a wide range of amino acids, growth factors and inorganic salts. Yeast extract is
used mainly as a comprehensive source of growth factors and may be substituted for meat extract in
culture media.
5. Malt extract: It consists mainly of maltose (about 50%), starch, dextrins and glucose, and contains about
5 percent of proteins and protein breakdown products, and a wide range of mineral salts and growth
factors.
6. Blood and serum: These are used for enriching culture media. Either human or animal blood can be used.
Usually 5-10 percent blood is used and the most usual concentration is 10 percent. Serum is used in certain
 media.
Classification of Media
Growth media are used in either of two phases: liquid (broth) or solid (agar). In some instances (e.g. certain
blood culture methods), a biphasic medium that contains both a liquid and a solid phase (Semi-solid) is used.
1. Liquid (broth) media: In broth media nutrients are dissolved in water, and bacterial growth is indicated
by a change in broth’s appearance from clear to turbid, (i.e. cloudy).
2. Solid (agar) media: Solid media are made by adding a solidifying agent to the nutrients and water.
Agarose is the most common solidifing agent. The petri dish containing the agar is referred to as agar.
 Colonies: While bacteria grow diffusely in liquids, they produce discrete visible growth on solid media.
If inoculated in suitable dilutions, bacteria form colonies, which are clones of cells originating from a
single bacterial cell. Bacterial cultures derived from a single colony or clone are considered “pure”. On
solid media, bacteria have distinct colony morphology and exhibit many other characteristic features
such as pigmentation or hemolysis, making identification easy.
3. Semisolid media: For special purposes where agar is added to media in concentrations that are too low to
solidify them. At 0.2 to 0.5% it yields a semisolid medium through which motile, but not non-motile,
bacteria may spread.

Table 6.1: Classification of media

A. Based on phases of C. Special media
growth media (i) Enriched media
1. Liquid (broth) media (ii) Enrichment media
2. Solid (agar) media (iii) Selective media
3. Semi-Solid (agar) (iv) Indicator or differential
media media
(v) Transport media
(vi) Sugar media

B. Based on nutritional D. Reducing media

factors
1. Simple media 1. Liquid (broth) media
(Basal media) 2. Solid (agar) media
2. Complex media 3. Semisolid media
3. Synthetic or defined
media
Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient
broth and nutrient agar considered basal medium

Nutrient media
Enriched media are used to grow nutritionally exacting (fastidious) bacteria Addition of extra nutrients in the
form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Blood agar, chocolate agar,
Loeffler’s serum slope etc are few of the enriched media.
 Blood agar is prepared by adding 5-10% (by volume) to a basal medium such as nutrient agar or other
blood agar bases. Since blood cannot be sterilized, it has to be collected aseptically from the animal.
 Animals have to be bled and the blood is collected in sterile containers with anticoagulant or glass beads.
While sheep blood is preferred, blood from rabbit, horse and ox can also be collected. Human blood must
be avoided since it may contain inhibitory substances including antibiotics. After the blood agar base is
autoclaved, blood is added to the medium at temperature just above the solidifying point of agar. The
mixture is then poured on to the plates and allowed to solidify. Blood agar is useful in demonstrating
hemolytic properties of certain bacteria.
 Chocolate agar is also known as heated blood agar or lysed blood agar. The procedure is similar to that
of blood agar preparation except that the blood is added while the molten blood agar base is still hot. This
lyses the blood cells and releases their contents into the medium. This process turns the medium brown,
hence the name. This medium is especially useful in growing Hemophilus sp and Neisseria sp. Serum for
medium can be obtained from animal blood but must be filtered through membrane or seitz filter before
use.

Blood agar Chocolate agar

Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria and
help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media
are liquid in consistency. Various approaches to make a medium selective include addition of antibiotics,
dyes, chemicals, alteration of pH or a combination of these.
 Thayer Martin Agar used to recover N. gonorrhoeae contains Vancomycin, Colistin and Nystatin.
 Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contain 10% NaCl. Potassium tellurite
medium used to recover C.diphtheriae contains 0.04% potassium tellurite.
 McConkey’s Agar used for Enterobacteriaceae members contains Bile salt that inhibits most gram
positive bacteria.
 Pseudosel Agar (Cetrimide Agar) used to recover P.aeruginosa contains cetrimide.
 Crystal Violet Blood Agar used to recover S.pyogenes contains 0.0002% crystal violet.
 Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating malachite
green.
 Wilson & Blair’s Agar for recovering S.typhi is rendered selective by the addition of dye Brilliant green.
 TCBS Agar and Monsur’s Tellurite Taurocholate Gelatin Agar used for isolating V. cholerae from fecal
specimens have elevated pH (8.5-5.6), which inhibits most other bacteria.
 Effect of Selective/Differential media
Enrichment media
When a substance is added to a liquid medium, which inhibits the growth of unwanted bacteria and favors the
growth of wanted bacteria is known as enrichment medium. This medium for an enrichment culture is
usually liquid and provides nutrients and environmental conditions that favor the growth of a particular
microbe but not others. The result is an absolute increase in the numbers of the wanted bacterium relative to
the other bacteria. This is often the case for soil or fecal samples. In mixed cultures or in materials containing
more than one bacterium, the bacterium to be isolated is often overgrown by the unwanted bacteria. Usually
the non- pathogenic or commensal bacteria tend to overgrow the pathogenic ones, for example, S.Typhi, being
overgrown by E. coli in cultures from feces. In such situations, substances, which have a stimulating effect on
the bacteria to be grown or an inhibitory effect on those to be suppressed, are incorporated in the medium.
Examples
 Tetrathionate broth: Tetrathionate inhibits coliforms while allowing typhoid-paratyphoid bacilli to
grow freely in fecal sample.
 Selenite F (F for Feces) broth: It is used for dysentery bacilli.
 Alkaline peptone water: It is used for V. cholera from feces.

Differential/Indicator media
Differential media or indicator media distinguish one microorganism type from another growing on the same
media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of
specific nutrients or indicators (such as neutral red, phenol red or methylene blue) added to the medium to
visibly indicate the defining characteristics of a microorganism.
When a particular substrate (carbohydrate) is incorporated into a medium and a mixture of bacteria inoculated
on it, only that bacterium that can ferment it produces acid. This change in pH is detected by using a pH
indicator incorporated in the medium and the bacterium that can ferment the sugar appears in a different
colour. This approach is used in MacConkey’s agar, CLED agar, TCBS agar, XLD agar etc.
MacConkey’s agar is the most commonly used media to culture and identify gram negative bacilli
(especially enterobacteriaceae members). It contains bile salts (selective agent), lactose (sugar), peptone and
neutral red (pH indicator), agar and water. Those bacteria that can ferment lactose produce pink coloured
colonies where non-lactose fermenting colonies produce colourless colonies.
Similarly, Vibrio cholerae produces yellow coloured colonies on sucrose containing TCBS medium.
Reduction of potassium tellurite to metallic tellurium by Corynebacterium diphtheriae results in production of
black coloured colonies on KT agar. Production of H2S by Salmonella typhi results in production of black
coloured colonies on Wilson & Blair’s medium.
Enterococcus fecalis produces black coloured colonies on bile esculin agar due to reduction of esculin to
esculetin.

Transport media
Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth
of contaminating organisms or commensals. This can be achieved by using transport media. Such media
prevent drying (desiccation) of specimen, maintain the viability of all organisms in the specimen without
altering their concentration. Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition
of charcoal serves to neutralize inhibitory factors. Cary Blair medium is used to transport feces from
suspected cholera patients. Sach’s buffered glycerol saline is used to transport feces from patients suspected
to be suffering from bacillary dysentery. Pike’s medium is used to transport streptococci from throat
specimens.

Anaerobic media
Anaerobic bacteria need reduced oxidation –reduction potential and extra nutrients. Such media may be
reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. Addition
of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a
medium reduced.
 Robertson cooked meat that is commonly used to grow Clostridium spps medium.
 Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein
hydrolysate.
 Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the
medium. Under reduced condition, methylene blue is colourless.

Type
1. Complex media
2. Chemically defined media
3. Special media
Characteristics
Composed of ingredients such as peptones and extracts, which may
vary in their chemical composition.
Composed of precise mixtures of pure chemicals such as ammonium sulfate

i. Enriched media They are used to grow bacteria which are more exacting in their
nutritional needs. Sub- stances such as blood, serum, or egg are
added to a basal medium
ii. Enrichment media Similar to selective media but designed to increase numbers of
desired microbes to detectable levels.
iii. Selective media Suppression of unwanted microbes and encouraging desired microbes
iv. Indicator media Contain an indicator which changes color when a bacterium grows in them
v. Differential media Medium that contains an ingredient that can be changed by certain
bacteria in a recognizable way. Differentiation of colonies of
desired microbes from others
vi. Sugar media Sugar fermentation reactions are carried out for the identification
of most of the organ- isms
vii. Transport media Transport medium is a holding medium designed to preserve the
viability of microorganisms in the specimen but not allow
multiplication

4. Reducing media Growth of obligate anaerobes

 Sterilization and Disinfection

Sterilization is defined as the process where all the living microorganisms, including bacterial spores are
killed. Sterilization can be achieved by physical, chemical and physiochemical means. Chemicals used as
sterilizing agents are called chemisterilants.

Disinfection is the process of elimination of most pathogenic microorganisms (excluding bacterial spores)
on inanimate objects. Disinfection can be achieved by physical or chemical methods. Chemicals used in
disinfection are called disinfectants. Different disinfectants have different target ranges, not all
disinfectants can kill all microorganisms. Some methods of disinfection such as filtration do not kill
bacteria, they separate them out. Sterilization is an absolute condition while disinfection is not. The two
are not synonymous.

Decontamination is the process of removal of contaminating pathogenic microorganisms from the articles
by a process of sterilization or disinfection. It is the use of physical or chemical means to remove,
inactivate, or destroy living organisms on a surface so that the organisms are no longer infectious.

Sanitization is the process of chemical or mechanical cleansing, applicable in public health systems.
Usually used by the food industry. It reduces microbes on eating utensils to safe, acceptable levels for
public health.
Asepsis is the employment of techniques (such as usage of gloves, air filters, uv rays etc) to achieve
microbe-free environment.

Antisepsis is the use of chemicals (antiseptics) to make skin or mucus membranes devoid of pathogenic
microorganisms.

Bacteriostasis is a condition where the multiplication of the bacteria is inhibited without killing them.

Bactericidal is that chemical that can kill or inactivate bacteria. Such chemicals may be called variously
depending on the spectrum of activity, such as bactericidal, virucidal, fungicidal, microbicidal, sporicidal,
tuberculocidal or germicidal.

Antibiotics are substances produced by one microbe that inhibits or kills another microbe. Often the term
is used more generally to include synthetic and semi-synthetic antimicrobial agents.
PHYSICAL METHODS OF STERILIZATION:

Sunlight: The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays in it. It is
responsible for spontaneous sterilization in natural conditions. In tropical countries, the sunlight is more
effective in killing germs due to combination of ultraviolet rays and heat. By killing bacteria suspended in
water, sunlight provides natural method of disinfection of water bodies such as tanks and lakes. Sunlight is
not sporicidal, hence it does not sterilize. Heat: Heat is considered to be most reliable method of
sterilization of articles that can withstand heat. Heat acts by oxidative effects as well as denaturation and
coagulation of proteins. Those articles that cannot withstand high temperatures can still be sterilized at
lower temperature by prolonging the duration of exposure.

Factors affecting sterilization by heat are:

o Nature of heat: Moist heat is more effective than dry heat
o Temperature and time: temperature and time are inversely proportional. As temperature increases
the time taken decreases.
o Number of microorganisms: More the number of microorganisms, higher the temperature or longer
the duration required.
o Nature of microorganism: Depends on species and strain of microorganism, sensitivity to heat may
vary. Spores are highly resistant to heat.
o Type of material: Articles that are heavily contaminated require higher temperature or prolonged
exposure. Certain heat sensitive articles must be sterilized at lower temperature.
o Presence of organic material: Organic materials such as protein, sugars, oils and fats increase the
time required.

Action of heat:
Dry heat acts by protein denaturation, oxidative damage and toxic effects of elevated levels of electrolytes.
The moist heat acts by coagulation and denaturation of proteins. Moist heat is superior to dry heat in
action. Temperature required to kill microbe by dry heat is more than the moist heat. Thermal death time
is the minimum time required to kill a suspension of organisms at a predetermined temperature in a
specified environment.

DRY HEAT:
Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing spatulas are
sterilized by holding them in Bunsen flame till they become red hot. This is a simple method for effective
sterilization of such articles, but is limited to those articles that can be heated to redness in flame.

Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to redness.
Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are passed through the
flame a few times. Even though most vegetative cells are killed, there is no guarantee that spores too
would die on such short exposure. This method too is limited to those articles that can be exposed to
flame. Cracking of the glassware may occur.

Incineration: This is a method of destroying contaminated material by burning them in incinerator.

Articles such as soiled dressings; animal carcasses, pathological material and bedding etc should be
subjected to incineration. This technique results in the loss of the article, hence is suitable only for those
articles that have to be disposed. Burning of polystyrene materials emits dense smoke, and hence they
should not be incinerated.

Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are exposed to high
temperature (160o C) for duration of one hour in an electrically heated oven. Since air is poor conductor of
heat, even distribution of heat throughout the chamber is achieved by a fan. The heat is transferred to the
article by radiation, conduction and convection. The oven should be fitted with a thermostat control,
temperature indicator, meshed shelves and must have adequate insulation.

Articles sterilized: Metallic instruments (like forceps, scalpels, scissors), glasswares (such as petri-dishes,
pipettes, flasks, all-glass syringes), swabs, oils, grease, petroleum jelly and some pharmaceutical products.

Sterilization process: Articles to be sterilized must be perfectly dry before placing them inside to avoid
breakage. Articles must be placed at sufficient distance so as to allow free circulation of air in between.
Mouths of flasks, test tubes and both ends of pipettes must be plugged with cotton wool. Articles such as
petri dishes and pipettes may be arranged inside metal canisters and then placed. Individual glass articles
must be wrapped in kraft paper or aluminum foils.

Sterilization cycle: This takes into consideration the time taken for the articles to reach the sterilizing
temperature, maintenance of the sterilizing temperature for a defined period (holding time) and the time
taken for the articles to cool down. Different temperature-time relations for holding time are 60 minutes at
160oC, 40 minutes at 170oC and 20 minutes at 180oC. Increasing temperature by 10 degrees shortens the
sterilizing time by 50 percent. The hot air oven must not be opened until the temperature inside has fallen
below 60oC to prevent breakage of glasswares.

Sterilization control: Three methods exist to check the efficacy of sterilization process, namely physical,
chemical and biological.
 Physical: Temperature chart recorder and thermocouple.
 Chemical: Browne’s tube No.3 (green spot, color changes from red to green)
 Biological: 106 spores of Bacillus subtilis var niger or Clostridium tetani on paper strips are placed
inside envelopes and then placed inside the hot air oven. Upon completion of sterilization cycle,
the strips are removed and inoculated into thioglycollate broth or cooked meat medium and
incubated at 37oC for 3-5 days. Proper sterilization should kill the spores and there should not be
any growth.

Advantages: It is an effective method of sterilization of heat stable articles. The articles remain dry after
sterilization. This is the only method of sterilizing oils and powders.

Disadvantages:
 Since air is poor conductor of heat, hot air has poor penetration.
 Cotton wool and paper may get slightly charred.
 Glasses may become smoky.
 Takes longer time compared to autoclave.

Infra red rays: Infrared rays bring about sterilization by generation of heat. Articles to be sterilized are
placed in a moving conveyer belt and passed through a tunnel that is heated by infrared radiators to a
temperature of 180oC. The articles are exposed to that temperature for a period of 7.5 minutes. Articles
sterilized included metallic instruments and glassware. It is mainly used in central sterile supply
department. It requires special equipments, hence is not applicable in diagnostic laboratory.

MOIST HEAT:
Moist heat acts by coagulation and denaturation of proteins.
At temperature below 100°C:
 Pasteurization: This process was originally employed by Louis Pasteur. Currently this procedure is
employed in food and dairy industry. There are two methods of pasteurization, the holder method
 (heated at 63oC for 30 minutes) and flash method (heated at 72oC for 15 seconds) followed by quickly
cooling to 13oC. Other pasteurization methods include Ultra-High Temperature (UHT), 140oC for 15
sec and 149oC for 0.5 sec. This method is suitable to destroy most milk borne pathogens like
Salmonella, Mycobacteria, Streptococci, Staphylococci and Brucella, however Coxiella may survive
pasteurization. Efficacy is tested by phosphatase test and methylene blue test.

 Vaccine bath: The contaminating bacteria in a vaccine preparation can be inactivated by heating in a
water bath at 60oC for one hour. Only vegetative bacteria are killed and spores survive.

 Serum bath: The contaminating bacteria in a serum preparation can be inactivated by heating in a
water bath at 56oC for one hour on several successive days. Proteins in the serum will coagulate at
higher temperature. Only vegetative bacteria are killed and spores survive.

 Inspissation: This is a technique to solidify as well as disinfect egg and serum containing media. The
medium containing serum or egg are placed in the slopes of an inspissator and heated at 80-85oC for
30 minutes on three successive days. On the first day, the vegetative bacteria would die and those
spores that germinate by next day are then killed the following day. The process depends on
germination of spores in between inspissation. If the spores fail to germinate then this technique
cannot be considered sterilization.

At temperature 100oC:
 Boiling: Boiling water (100oC) kills most vegetative bacteria and viruses immediately. Certain
bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Some bacterial spores
are resistant to boiling and survive; hence this is not a substitute for sterilization. The killing
activity can be enhanced by addition of 2% sodium bicarbonate. When absolute sterility is not
required, certain metal articles and glasswares can be disinfected by placing them in boiling water
for 10-20 minutes. The lid of the boiler must not be opened during the period.

 Steam at100oC: Instead of keeping the articles in boiling water, they are subjected to free steam at
100oC. Traditionally Arnold’s and Koch’s steamers were used. An autoclave (with discharge tap
open) can also serve the same purpose. A steamer is a metal cabinet with perforated trays to hold
the articles and a conical lid. The bottom of steamer is filled with water and heated. The steam that
is generated sterilizes the articles when exposed for a period of 90 minutes. Media such as TCBS,
DCA and selenite broth are sterilized by steaming. Sugar and gelatin in medium may get
decomposed on autoclaving, hence they are exposed to free steaming for 20 minutes for three
successive days. This process is known as tyndallisation (after John Tyndall) or fractional
sterilization or intermittent sterilization. The vegetative bacteria are killed in the first exposure and
the spores that germinate by next day are killed in subsequent days. The success of process
depends on the germination of spores.

At temperature above 100oC:

 Autoclave: Sterilization can be effectively achieved at a temperature above 100oC using an autoclave.
Water boils at 100oC at atmospheric pressure, but if pressure is raised, the temperature at which the
water boils also increases. In an autoclave the water is boiled in a closed chamber. As the pressure
rises, the boiling point of water also raises. At a pressure of 15 lbs inside the autoclave, the
temperature is said to be 121oC. Exposure of articles to this temperature for 15 minutes sterilizes them.
To destroy the infective agents associated with spongiform encephalopathies (prions), higher
temperatures or longer times are used; 135oC or 121oC for at least one hour are recommended.
  Advantages of steam: It has more penetrative power than dry air, it moistens the spores (moisture is
essential for coagulation of proteins), condensation of steam on cooler surface releases latent heat,
condensation of steam draws in fresh steam.

 Different types of autoclave:

Simple “pressure-cooker type” laboratory autoclave, Steam jacketed downward displacement
laboratory autoclave and high pressure pre-vacuum autoclave

Construction And Operation Of Autoclave:

A simple autoclave has vertical or horizontal cylindrical

body with a heating element, a perforated try to keep the
articles, a lid that can be fastened by screw clamps, a
pressure gauge, a safety valve and a discharge tap. The
articles to be sterilized must not be tightly packed. The
screw caps and cotton plugs must be loosely fitted. The lid
is closed but the discharge tap is kept open and the water is
heated. As the water starts boiling, the steam drives air out
of the discharge tap. When all the air is displaced and
steam start appearing through the discharge tap, the tap is
closed. The pressure inside is allowed to rise upto 15 lbs
per square inch. At this pressure the articles are held for 15
minutes, after which the heating is stopped and the
autoclave is allowed to cool. Once the pressure gauge
shows the pressure equal to atmospheric pressure, the
discharge tap is opened to let the air in. The lid is then
opened and articles removed.

Articles sterilized: Culture media, dressings, certain equipment, linen etc.

Precautions:
Articles should not be tightly packed, the autoclave must not be overloaded, air discharge must be complete
and there should not be any residual air trapped inside, caps of bottles and flasks should not be tight,
autoclave must not be opened until the pressure has fallen or else the contents will boil over, articles must
be wrapped in paper to prevent drenching, bottles must not be overfilled.

Advantage: Very effective way of sterilization, quicker than hot air oven.

Disadvantages: Drenching and wetting or articles may occur, trapped air may reduce the efficacy, takes
long time to cool

Sterilization control: Physical method includes automatic process control, thermocouple and temperature
chart recorder. Chemical method includes Browne’s tube No.1 (black spot) and succinic acid (whose
melting point is 121oC) and Bowie Dick tape. Bowie Dick tape is applied to articles being autoclaved. If the
process has been satisfactory, dark brown stripes will appear across the tape. Biological method includes a
paper strip containing 106 spores of Geobacillus stearothermophilus.

RADIATION:
Two types of radiation are used, ionizing and non-ionizing. Non-ionizing rays are low energy rays with
poor penetrative power while ionizing rays are high-energy rays with good penetrative power. Since
radiation does not generate heat, it is termed "cold sterilization". In some parts of Europe, fruits and
vegetables are irradiated to increase their shelf life up to 500 percent.

 Non-ionizing rays: Rays of wavelength longer than the visible light are non-ionizing.
Microbicidal wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being most
effective. UV rays are generated using a high-pressure mercury vapor lamp. It is at this wavelength
that the absorption by the microorganisms is at its maximum, which results in the germicidal
effect. UV rays induce formation of thymine-thymine dimers, which ultimately inhibits DNA
replication. UV readily induces mutations in cells irradiated with a non-lethal dose.
Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to the effective UV radiation
are inactivated within seconds. Since UV rays don’t kill spores, they are considered to be of use in
surface disinfection. UV rays are employed to disinfect hospital wards, operation theatres, virus
laboratories, corridors, etc. Disadvantages of using uv rays include low penetrative power, limited
life of the uv bulb, some bacteria have DNA repair enzymes that can overcome damage caused by
uv rays, organic matter and dust prevents its reach, rays are harmful to skin and eyes. It doesn't
penetrate glass, paper or plastic.

 Ionizing rays: Ionizing rays are of two types, particulate and electromagnetic rays.
o Electron beams are particulate in nature while gamma rays are electromagnetic in nature.
High- speed electrons are produced by a linear accelerator from a heated cathode. Electron
beams are employed to sterilize articles like syringes, gloves, dressing packs, foods and
pharmaceuticals. Sterilization is accomplished in few seconds. Unlike electromagnetic rays,
the instruments can be switched off. Disadvantage includes poor penetrative power and
requirement of sophisticated equipment.

FILTRATION:
Filtration does not kill microbes, it separates them out. Membrane filters with pore sizes between 0.2-0.45
µm are commonly used to remove particles from solutions that can't be autoclaved. It is used to remove
microbes from heat labile liquids such as serum, antibiotic solutions, sugar solutions, urea solution.
Various applications of filtration include removing bacteria from ingredients of culture media, preparing
suspensions of viruses and phages free of bacteria, measuring sizes of viruses, separating toxins from
culture filtrates, counting bacteria, clarifying fluids and purifying hydatid fluid. Filtration is aided by using
either positive or negative pressure using vacuum pumps. The older filters made of earthenware or
asbestos are called depth filters.

Different types of filters are:

1. Earthenware filters: These filters are made up of diatomaceous earth or porcelain. They are
usually baked into the shape of candle. Different types of earthenware filters are:

a. Pasteur-Chamberland filter: These candle filters are from France and are made up of
porcelain (sand and kaolin). Similar filter from Britain is Doulton. Chamberland filters are
made with various porosities.

b. Berkefeld filter: These are made of Kieselguhr, a fossilized diatomaceous earth found in
Germany. They are available in three grades depending on their porosity (pore size); they
are V (veil), N (normal) and W (wenig).

c. Mandler filter: This filter from America is made of kieselguhr, asbestos and plaster of Paris.
 2. Asbestos filters: These filters are made from chrysotile type of asbestos, chemically composed of
magnesium silicate. They are pressed to form disc, which are to be used only once. The disc is held
inside a metal mount, which is sterilized by autoclaving.

3. Sintered glass filters: These are made from finely ground glass that are fused sufficiently to make
small particles adhere to each other. They are usually available in the form of disc fused into a
glass funnel. Filters of Grade 5 have average pore diameter of 1-1.5 µm. They are washed in
running water in reverse direction and cleaned with warm concentrated H2SO4 and sterilized by
autoclaving.
4. Membrane filters: These filters are made from a variety of polymeric materials such as cellulose
nitrate, cellulose diacetate, polycarbonate and polyester. The older type of membrane, called
gradocol (graded colloidion) membrane was composed of cellulose nitrate. Gradocol membranes
have average pore diameter of 3-10 µm. The newer ones are composed of cellulose diacetate.
These membranes have a pore diameter ranging from 0.015 µm to 12 µm. These filters are
sterilized by autoclaving. Membrane filters are made in two ways, the capillary pore membranes
have pores produced by radiation while the labyrinthine pore membranes are produced by forced
evaporation of solvents from cellulose esters.

The disadvantages of depth filters are migration of filter material into the filtrate, absorption or retention
of certain volume of liquid by the filters, pore sizes are not definite and viruses and mycoplasma could
pass through. The advantages of membrane filters are known porosity, no retention of fluids, and reusable
after autoclaving and compatible with many chemicals. However, membrane filters have little loading
capacity and are fragile.

Air Filters: Air can be filtered using HEPA (High Efficiency Particle Air) filters. They are usually used in
biological safety cabinets. HEPA filters are at least 99.97% efficient for removing particles >0.3 µm in
diameter. Examples of areas where HEPA filters are used include rooms housing severely neutropenic
patients and those operating rooms designated for orthopedic implant procedures. HEPA filter efficiency
is monitored with the dioctylphthalate (DOP) particle test using particles that are 0.3 µm in diameter.

CHEMICAL METHODS OF DISINFECTION:

Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. Some
chemical have very narrow spectrum of activity and some have very wide. Those chemicals that can
sterilize are called chemisterilants. Those chemicals that can be safely applied over skin and mucus
membranes are called antiseptics. An ideal antiseptic or disinfectant should have following properties:

 Should have wide spectrum of activity

 Should be able to destroy microbes within practical period of time
 Should be active in the presence of organic matter
 Should make effective contact and be wettable
 Should be active in any pH
 Should be stable
 Should have long shelf life
 Should be speedy
 Should have high penetrating power
 Should be non-toxic, non-allergenic, non-irritative or non-corrosive
 Should not have bad odour
  Should not leave non-volatile residue or stain
 Efficacy should not be lost on reasonable dilution
 Should not be expensive and must be available easily
Such an ideal disinfectant is not yet available. The level of disinfection achieved depends on contact time,
temperature, type and concentration of the active ingredient, the presence of organic matter, the type and
quantum of microbial load. The chemical disinfectants at working concentrations rapidly lose their
strength on standing.

Classification of disinfectants:
1. Based on consistency
a. Liquid (E.g., Alcohols, Phenols)
b. Gaseous (Formaldehyde vapor, Ethylene oxide)
2. Based on spectrum of activity
a. High level
b. Intermediate level
c. Low level
3. Based on mechanism of action
a. Action on membrane (E.g., Alcohol, detergent)
b. Denaturation of cellular proteins (E.g., Alcohol, Phenol)
c. Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2, Halogens)
d. Alkylation of amino-, carboxyl- and hydroxyl group (E.g., Ethylene Oxide, Formaldehyde)
e. Damage to nucleic acids (Ethylene Oxide, Formaldehyde)

ALCOHOLS:
Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.

Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol

Application: A 70% aqueous solution is more effective at killing microbes than absolute alcohols. 70%
ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred to ethanol. It can also be
used to disinfect surfaces. It is used to disinfect clinical thermometers. Methyl alcohol kills fungal spores,
hence is useful in disinfecting inoculation hoods.

Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

ALDEHYDES:
Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably damages
nucleic acids. It kills all microorganisms, including spores.

Examples: Formaldehyde, Gluteraldehyde

Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms,
chambers, operation theatres, biological safety cabinets, wards, sick rooms etc. Fumigation is achieved by
boiling formalin, heating paraformaldehyde or treating formalin with potassium permanganate. It also
sterilizes bedding, furniture and books. 10% formalin with 0.5% tetraborate sterilizes clean metal
instruments. 2% gluteraldehyde is used to sterilize thermometers, cystoscopes, bronchoscopes,
centrifuges, anasethetic equipments etc. An exposure of at least 3 hours at alkaline pH is required for
action by gluteraldehyde. 2% formaldehyde at 40oC for 20 minutes is used to disinfect wool and 0.25% at
60oC for six hours to disinfect animal hair and bristles.
Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor penetration, leaves non-
volatile residue, activity is reduced in the presence of protein. Gluteraldehyde requires alkaline pH and
only those articles that are wettable can be sterilized.

PHENOL:
Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of enzymes.

Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene, chlorhexidine,

chloroxylenol (Dettol)

Applications: Joseph Lister used it to prevent infection of surgical wounds. Phenols are coal-tar
derivatives. They act as disinfectants at high concentration and as antiseptics at low concentrations. They
are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses. They are
not readily inactivated by organic matter. The corrosive phenolics are used for disinfection of ward floors,
in discarding jars in laboratories and disinfection of bedpans. Chlorhexidine can be used in an isopropanol
solution for skin disinfection, or as an aqueous solution for wound irrigation. It is often used as an
antiseptic hand wash. 20% Chlorhexidine gluconate solution is used for pre-operative hand and skin
preparation and for general skin disinfection. Chlorhexidine gluconate is also mixed with quaternary
ammonium compounds such as cetrimide to get stronger and broader antimicrobial effects (eg. Savlon).
Chloroxylenols are less irritant and can be used for topical purposes and are more effective against gram
positive bacteria than gram negative bacteria. Hexachlorophene is chlorinated diphenyl and is much less
irritant. It has marked effect over gram positive bacteria but poor effect over gram negative bacteria,
mycobacteria, fungi and viruses. Triclosan is an organic phenyl ether with good activity against gram
positive bacteria and effective to some extent against many gram negative bacteria including
Pseudomonas. It also has fair activity on fungi and viruses.
Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is inactivated by anionic soaps.
Chloroxylenol is inactivated by hard water.

HALOGENS:
Mode of action: They are oxidizing agents and cause damage by oxidation of essential sulfydryl groups
of enzymes. Chlorine reacts with water to form hypochlorous acid, which is microbicidal.

Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture iodine,
iodophores)

Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Iodine can be combined with
neutral carrier polymers such as polyvinylpyrrolidone to prepare iodophores such as povidone-iodine.
Iodophores permit slow release and reduce the irritation of the antiseptic. For hand washing iodophores
are diluted in 50% alcohol. 10% Povidone Iodine is used undiluted in pre and postoperative skin
disinfection. Chlorine gas is used to bleach water. Household bleach can be used to disinfect floors.
Household bleach used in a stock dilution of 1:10. In higher concentrations chlorine is used to disinfect
swimming pools. 0.5% sodium hypochlorite is used in serology and virology. Used at a dilution of 1:10 in
decontamination of spillage of infectious material. Mercuric chloride is used as a disinfectant.

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.

HYDROGEN PEROXIDE:
Mode of action: It acts on the microorganisms through its release of nascent oxygen. Hydrogen peroxide
produces hydroxyl-free radical that damages proteins and DNA.
 Application: It is used at 6% concentration to decontaminate the instruments, equipments such as
ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising wounds and
ulcers. Strong solutions are sporicidal.
Disadvantages: Decomposes in light, broken down by catalase, proteinaceous organic matter drastically
reduces its activity.

PHYSIO-CHEMICAL METHOD:
Mode of action: A physio-chemical method adopts both physical and chemical method. Use of steam-
formaldehyde is a physio-chemical method of sterilization, which takes into account action of steam as
well as that of formaldehyde. Saturated steam at a pressure of 263 mm has a temperature of 70 oC. The air
is removed from the autoclave chamber and saturated steam at sub-atmospheric pressure is flushed in.
Formaldehyde is then injected with steam in a series of pulses, each of 5-10 minutes. The articles are held
at this holding temperature for one hour. Formaldehyde is then flushed by inflow of steam.

Disadvantages: Condensation of formaldehyde occurs and induction of large volume of formaldehyde

wets the steam resulting in loss of latent heat.

PRINCIPLES OF BACTERIAL GROWTH

Growth may be defined as the orderly increase of all of the chemical constituents of the cell. Bacterial
growth involves both an increase in the size of individuals and increase in the number of individuals.
Whatever the balance between these two processes, the net effect is an increase in the total mass
(biomass).

Bacterial Division
Bacteria divide by binary fission where individual cells enlarge and divide to yield two progeny of
approximately equal size. Nuclear division precedes cell division and, therefore, in a growing population,
many cells carrying two nuclear bodies can be seen. The cell division occurs by a constrictive or pinching
process, or by the ingrowth of a transverse septum across the cell. The daughter cells may remain partially
attached after division in some species.

Generation Time or Doubling Time

The size of a population of bacteria in a favorable growth medium increases exponentially. The interval
of time between two cell divisions, or the time required for a bacterium to give rise to two daughter cells
under optimum conditions, is known as the generation time or doubling time.
Examples: In coliform bacilli and many other medically important bacteria, it about 20 minutes, in
tubercle bacilli it is about 20 hours and in lepra bacilli it is about 20 days. It is often difficult to grasp fully
the scale of exponential microbial growth.

Colonies: Bacteria growing on solid media form colonies. Each colony represents a clone of cells derived
from a single parent cell. In contrast to growth in broth, far less is known about the state of the bacteria in
a mature macroscopic colony on an agar plate. It is likely that all phases of growth are represented in
colonies, depending on the location of a particular cell and age of the culture. In liquid media, growth is
diffuse.

Bacterial count: Growth of bacteria is diffuse in liquid media and they form colonies on solid media.
Each colony consists of a clone of cells derived from a single par-ent cell. Bacteria in a culture medium or
clinical specimen can be counted by two methods:
 1. Total count
This is total number of bacteria present in a specimen irrespective of whether they are living or
dead. Total count is done by counting the bacteria under micro- scope using counting chamber and by
comparing the growth with standard opacity tubes.
2. Viable count
This measures only viable (living) cells, which are capable of growing and producing a colony on a
suitable medium. The viable count measures the number of living cells, that is, cells capable of
multiplication.

Determination of Viable Counts - Dilution or Plating Methods

1. Dilution method
In dilution method, the suspension is diluted to a point beyond which unit quantities do not yield
growth when inoculated into suitable liquid media. With varying dilutions several tubes are
inoculated and the viable counts calculated statistically from the number of tubes showing growth.
The method does not give accurate values, but is used in the ‘presumptive coliform count’ in
drinking water.
2. Plating method
In the plating method, appropriate dilutions are inoculated on solid media by:
i. Pour-plate method
ii. Spread plate method in which serial dilutions are dropped on the surface of dried plates and colony
counts obtained.
Detection and Measurement of Bacterial Growth
1. Direct cell counts
2. Viable cell counts
3. Measuring biomass
4. Measuring cell products

Bacterial Growth Curve

If a suitable liquid medium is inoculated with bacterium and incubated, its growth follows a definitive
course. Small samples are taken at regular intervals after inoculation and plotted in relation to time. A
plotting of the data will yield a characteristic growth curve. The changes of slope on such a graph indicate
the transition from one phase of development to another.

Phases of Bacterial Growth Curve

The bacterial growth curve can be divided into four major phases: (i) lag phase (ii) exponential or log
(logarithmic) phase (iii) stationary phase, and (iv) decline phase. These phases reflect the physiologic state
of the organisms in the culture at that particular time.

1. Lag phase
When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell
number occurs, and therefore this period is called the lag phase. After inoculation, there is an increase in
cell size at a time when little or no cell division is occurring. During this time, however, the cells are not
dormant. This initial period is the time required for adaptation to the new environment, during which the
necessary enzymes and metabolic intermediates are built up in adequate quantities for multiplication to
proceed. (The situation is analogous to a factory being equipped to produce automobiles; there is
considerable tooling-up activity but no immediate increase in the automobile population).
The lag phase varies considerably in length with the species, nature of the medium, size of inoculum
and environmental factors such as temperature and nutrients present in the new medium.

2. Log (logarithmic) or exponential phase

Following the lag phase, the cells start dividing and their numbers increase exponentially or by
 geometric progression with time. If the logarithm of the viable count is plotted against time, a straight
line will be obtained. The population is uniform in terms of chemical and physiological properties
during this phase. There- fore, exponential phase cultures are usually used in biochemical and
physiological studies.
Exponential phase is of limited duration because of
(i) exhaustion of nutrients;
(ii) accumulation of toxic metabolic end products;
(iii) rise in cell density,
(iv) change in pH; and
(v) decrease in oxygen tension (in case of aerobic organisms).

The log phase is the time when cells are most active metabolically and is preferred for industrial
purposes. However, during their log phase of growth, microorganisms are particularly sensitive to
adverse conditions. Radiation and many antimicrobial drugs, e.g. the anti- biotic penicillin exert their
effect by interfering with some important step in the growth process and are, therefore, most harmful to
cells during this phase.

3. Stationary phase
After a varying period of exponential growth, cell division stops due to depletion of nutrients and
accumulation of toxic products. Eventually growth slows down, and the total bacterial cell number
reaches a maximum and stabilizes. The number of progeny cells formed is just enough to replace the
number of cells that die. The growth curve becomes horizontal. The viable count remains stationary, as
an equilibrium exists between the dying cells and the newly formed cells.

4. Decline or death phase

The death phase is the period when the population decreases due to cell death. Eventually the rate of
death exceeds the rate of reproduction, and the number of viable cells declines. Like bacterial growth,
death is exponential cell death may also be caused by autolysis besides nutrient deprivation and buildup
of toxic wastes. Finally, after a variable period, all the cells die and culture becomes sterile.
When the total count is plotted, it parallels the viable count up to the stationary phase, but it continues
steadily without any phase of decline. Even the total count shows a phase of decline with autolytic
enzymes.

Bacterial growth curve. The viable count shows lag, log, stationary and decline phases. In the total count, the
phase of decline is not evident
Categories of Requirements for Microbial Growth
The requirements for microbial growth can be divided into two main categories: (i) chemical and (ii)
physical.
1. Chemical Requirements
Chemical requirements include sources of carbon, nitro- gen, sulfur, phosphorus, trace elements, oxygen,
and organic growth factors.
A. Major elements (Macroelements or macronutrients)
Elements that make up cell constituents are called major elements (macroelements or
macronutrients) and over 95 percent of cell dry weight is made up of a few major elements. These
include carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium, and
iron.

B. Trace elements
Some elements, termed trace elements or micronutri- ents are required in very minute amounts by
all cells. They include cobalt, zinc, copper, molybdenum, and manganese. These elements form
parts of enzymes or may be required for enzyme function. They aid in the catalysis of reactions and
maintenance of protein structure. Very small amounts of these trace elements are found in most natural
environments, including water.

2. Growth Factors
Some bacteria require certain organic compounds in minute quantities known as growth factors or
bacterial vitamins. A growth factor is an organic compound which a cell must contain in order to
grow, but which it is unable to synthesize. These low molecular weight compounds that must be
provided to a particular bacterium are called growth factors or bacterial vitamins. Growth factors
are called ‘essential’ when growth does not occur in their absence or ‘accessory when they enhance
growth without being necessary for it. In many cases, bacterial vitamins are identical with the vitamins
necessary for mammalian nutrition, particularly those belonging to the B group, thiamine, riboflavin,
nicotinic acid, pyridoxine, folic acid and vitamin B12.

Physical Factors Influencing Microbial Growth

1. Temperature
2. Oxygen
3. Carbon dioxide
4. Moisture and drying
5. pH
6. Light
7. Osmotic effect
8. Mechanical and sonic stresses

1. Temperature
Optimum Temperature
Each bacterial species has an optimal temperature for growth and a temperature range above and
below which growth is blocked. The temperature at which growth occur best is known as the
‘optimum temperature’. Thus, bacteria pathogenic for humans usually grow at 37ºC (our body
temperature). Bacteria are divided into three groups on the basis of temperature ranges through which
they grow:
i. Mesophilic: Bacteria which grow between 10ºC and 45ºC, with optimal growth between 20-
40ºC. Examples: All parasites of warm blooded animals are mesophilic and it is the largest
group of bacteria including all of the pathogenic forms.
ii. Psychrophilic: Psychrophilic bacteria (cold-loving) are organisms that grow between –5 to
30ºC, optimum at 10 to 20ºC.
 Examples: They are soil and water saprophytes and though not of direct medical importance,
may cause spoilage of refrigerated food. These organisms may be capable of growth in food and
pharmaceuticals stored at normal refrigeration temperatures (0-8ºC).
iii. Thermophilic: Thermophiles (heat-loving) have growth range 25-80ºC, optimum at 50-60ºC.
They may cause spoilage of underprocessed canned food and can be a source of proteins with
remarkable thermotolerant properties such as taq polymerase, the key enzyme used in the
polymerase chain reaction.
Examples: Some thermophiles (like Bacillus stearthermophilus) form spores that are
exceptionally thermotolerant.

2. Oxygen
Based on their O2 requirements, prokaryotes can be separated into aerobes and anaerobes.
A. Aerobic bacteria
Require oxygen for growth and may be:
i. Obligate aerobes: They have an absolute or obligate requirement for oxygen (O2), like the
cholera vibrio.
ii. Facultative anaerobes: They are ordinarily aerobic but can also grow in the absence of
oxygen, though less abundantly, e.g. Staphylococcus spp.; Escherichia coli, etc. Most bacteria
of medical importance are facultative anaerobes.
iii. Microaerophilic organisms: They grow best at low oxygen tension (~5%) e.g. Campylobacter
spp., Helicobacter spp.
B. Anaerobic bacteria
Grows in absence of oxygen.
Obligate anaerobes: They may even die on exposure to oxygen, e.g. Clostridium tetani,
Bacteroides fragilis.

3. Carbon Dioxide
All bacteria require small amount of carbon dioxide for growth. Thus, this requirement is
usually met by the carbon dioxide present in the atmosphere, or produced endogenously by cellular
metabolism. Some organisms such as Brucella abortus, require much higher levels of carbon
dioxide (5-10%) for growth, especially on fresh isolation (capnophilic). Pneumococci and
gonococci are other capnophilic which grow better in air supplemented with 5 to 10 percent CO2.
4. Moisture and Drying
Moisture is very essential for the growth of the bacteria because water is essential ingredient
of bacterial protoplasm and hence drying is lethal to cells. However, the effect of drying varies in
different species Examples:
i. Treponema pallidum are highly sensitive, while others like staphylococci withstand
resistant to desiccation for months.
ii. Bacterial spores: Bacterial spores are particularly resistant to desiccation and survive in
the dry state for several decades.
iii. Freeze drying or lyophilization: Drying in vacu- um in the cold (freeze drying or
lyophilization) is a method of preservation of bacteria, viruses and many labile biological
materials. On a larger scale, it is used for preserving therapeutic antisera, human plasma,
antibiotics and vaccines.
5. pH
Most bacteria can live and multiply within the range of pH 5 (acidic) to pH 8 (basic) and
have a pH optimum near neutral (pH 7). Most pathogenic bacteria grow best at a neutral or slightly
alkaline pH (7.2 to 7.6). Some acidophilic bacteria such as lactobacilli grow under acidic
conditions while cholerae vibrio, grow at high degrees of alkalinity (well above pH 8), whereas
most other bacteria grow in a range of 6 to 7.5. Numerous fungi grow well at pH 4 or 5.

6. Light
Darkness provides a favorable condition for growth and viability of bacteria. Bacteria are
sensitive to ultraviolet light and other radiations as ultraviolet rays from direct sunlight or a
mercury lamp are bactericidal. Bacteria are also killed by ionizing radiations.
Exposure to light may influence pigment production. Photochromogenic mycobacteria form
pigment only on exposure to light.

7. Osmotic Effect
Tolerance to osmotic variation: Bacteria are more tolerant to osmotic variation because of
the mechanical strength of the cell wall. Except for the mycoplasma and other cell wall defective
organisms, the majority of the bacteria are osmotically tolerant.
 Plasmolysis: When microorganisms with rigid cell walls are placed in a hypertonic
environment, water leaves and the plasma membrane shrinks away from the wall, a process
known as plasmolysis. This occurs more readily in gram-negative bacteria than in gram-positive
bacteria.

Plasmoptysis: Sudden transfer of bacteria from concentrated solution to distilled water may also
cause plasmoptysis due to excessive osmotic imbibition of water leading to swelling and rupture
of the cell.

8. Mechanical and Sonic Stresses

In spite of tough walls of bacteria, they may be ruptured by mechanical stress such as grinding or
vigorous shaking with glass beads. Exposure to ultrasonic vibration may also disintegrate
bacteria.

Nutritional Types of Microorganisms

Microbiologists use the term growth to indicate an increase in a population of microbes rather than an
increase in size. Microbial growth depends on the metabolism of nutrients, and results in the formation
of a discrete colony, an aggregation of cells arising from a single parent cell. A nutrient is any chemical
required for growth of microbial populations. The most important of these are compounds containing
carbon, oxygen, nitrogen, and/or hydrogen. All microorganisms require source of energy and hydrogen
atoms or electrons for their growth and development. There are two sources of energy available to
microorganisms, and based on this they are of two types:

Phototrophs – Energy for growth is derived from sunlight.

Chemotrophs – Energy for growth is derived from the oxidation of either organic or inorganic
chemical compounds.

Microorganisms also have two sources for hydrogen atoms and electrons and based on that they can be
grouped as:
Lithotrophs : Source of electrons is reduced inorganic compounds

Organotrophs : Source of electrons and hydrogen is organic compounds.

Some can be Chemoorganotrophs and Photoorganotrophs.
Thus, bacteria may be either:
1. Photo-lithotrops: These bacteria gain energy from light and use reduced inorganic compounds
such as H2S as a source of electrons. eg: Chromatium okeinii.
2. Photo-organotrophs: These bacteria gain energy from light and use organic compounds such as
Succinate as a source of electrons.eg; Rhodospirillum.
3. Chemo-lithotrophs: These bacteria gain energy from reduced inorganic compounds such as
NH3 as a source of electron eg; Nitrosomonas.
4. Chemo-organotrophs: These bacteria gain energy from organic compounds such as glucose
and ammino acids as a source of electrons.eg; Pseudomonas pseudoflora.
Some bacteria can live ether chemo-lithotrophs or chemoorganotrophs like Pseudomonas pseudoflora as
they can use either glucose or H2S as electron source.

On the basis of carbon source bacteria may be:

 All organisms require carbon in some form for use in synthesizing cell components.
 All organisms require at least a small amount of CO2.
 However, some can use CO2 as their major or even sole source of carbon; such organisms are
termed as Autotrophs (Autotrophic bacteria).
  Others require organic compounds as their carbon source and are known as Heterotrophs
(Heterotrophic bacteria).

Autotrophic Bacteria
These bacteria synthesize all their food from inorganic substances (H2O, CO2, H2S salts).
The autotrophic bacteria are of two types:
(i) Photoautotrophs
 These bacteria capture the energy of sunlight and transform it into the chemical energy.
 In this process, CO2 is reduced to carbohydrates.
 The hydrogen donor is water and the process produce free oxygen.
 Photoautotroph has Chlorophyll pigment in the cell and its main function is to capture sunlight
e.g., Cyanobacteria.
 Some photoautotrophic bacteria are anaerobes and have bacteriochlorophyll
and bacteriovirdin pigments respectively.
Purple Sulphur Bacteria:
These bacteria have the pigment bacteriochlorophyll located on the intracytoplasmic membrane i.e.,
thylakoids. These bacteria obtain energy from sulfur compounds e.g., Chromatiiun. Theopedia rosea,
Thiospirilium.

Green Sulphur Bacteria:

These bacteria use hydrogen sulfide (H2S) as hydrogen donor. The reaction takes place in the presence
of light and pigment termed as bacteriovirdin or bacteriopheophytin or chlorobium chlorophyll
e.g., Chlorobium limicola, Chlorobacterium etc.
These bacteria take hydrogen from inorganic sources like sulphides and thiosulphates. Therefore, these
bacteria are also known as photolithographs.

(ii) Chemoautotrophs
 These bacteria do not require light (lack the light phase but have the dark phase of photosynthesis)
and pigment for their nutrition.
 These bacteria oxidize certain inorganic substances with the help of atmospheric oxygen.
 This reaction releases the energy (exothermic) which is used to drive the synthetic processes of the
cell.
Sulphomonas (Sulphur bacteria):
These bacteria obtain energy by oxidation of elemental sulphur or H2S, e.g., Thiobacillus, Beggiatoa.
 Elemental Sulphur Oxidising Bacteria: Denitrifying sulphur bacteria oxidize elemental sulphur to
sulphuric acid e.g., Thiobacillus denitrificans
2S + 2H2O + 3O2 → 2H2SO4 + 126 kcal.
 Sulphide Oxidizing Bacteria: These bacteria oxidizes H2S and release the sulphur e.g., Beggiatoa.
2H2S +4O2 → 2H2O + 2S + 141.8 cal

Hydromonas (Hydrogen bacteria)
 These convert hydrogen into water, e.g., Bacillus pantotrophus, Hydrogenomonas.
2H2 + O2 → 2H2O + 55 kcal.
4H2 + CO2 → 2H2O + CH4 + Energy

Ferromonas (Iron bacteria)

 These bacteria inhabit water and obtain energy by oxidation of ferrous compounds into ferric forms.
e.g., Thiobacillus ferroxidans, Ferro bacillus, Leptothrix.
4FeCo3 + 6H2O + O2 → 4Fe (OH)3 + 4CO2 + 81 kcal.
 Methanomonas (Methane bacteria):
 These bacteria get their energy by oxidation of methane into water and carbon dioxide.

Nitrosomonas (Nitrifying bacteria):

 These bacteria get their energy by oxidation of ammonia and nitrogen compounds into nitrates.
 Nitrosomonas oxidises NH3 to nitrites. NH3 + ½O2 ® H2O + HNO2 + Energy
 Nitrobacter converts nitrites to nitrates. NO2 + ½O2 ® NO2 + Energy

Carbon Bacteria:
These bacteria oxidizes CO into CO2 e.g., Bacillus oligocarbophillous, Oligotropha
carboxydovorans
2CO + O2 → 2CO2 + Energy

Heterotrophic Bacteria
 The heterotrophic bacteria obtain their-ready made food from organic substances, living or dead.
 Most of pathogenic bacteria of human beings, other plants and animals are heterotrophs.
 Some heterotrophs have simple nutritional requirement while some of them require large amount of
vitamin and other growth promoting substance. Such organisms are called fastidious heterotrophs.
 Heterotrophic bacteria are of two types:

a) Photoheterotrophs
 These bacteria can utilize light energy but cannot use CO2 as their sole source of carbon.
 They obtain energy from organic compounds to satisfy their carbon and electron requirements.
Bacteriochlorophyll pigment is found in these bacteria. eg., Purple non-sulphur bacteria
(Rhodospirillum, Rhodomicrobium, Rhodopseudomonas palustris).

b) Chemoheterotrophic heterotrophs
These are also called as chemoheterotrophs. They use organic compounds like sugars and amino
acids as source of energy, hydrogen and carbon. The vast majority of pathogenic microorganisms
are chemoheterotrophs.

Glucose or Monosaccharide [(CH2O)n] + O2 → CO2 + H2O + Energy

There are three main categories that differ in how chemohetrotrophs obtain their organic nutrients.
(i) Saprophytic bacteria.
(ii) Parasitic bacteria.
(iii) Symbiotic bacteria.

(i) Saprophytic bacteria:

Saprophytic bacteria obtain their food from the dead and organic decaying matter such as leaves,
fruits, vegetables, meat, animal feces, leather, humus etc. These bacteria secrete enzymes to digest
the food and absorb it. The enzymes secreted break down the complex compounds such as
carbohydrate and protein, into simpler soluble compounds, which are easily absorbed. Examples are
Bacillus mycoides, B. ramosus, Acetobacter etc.

(ii) Parasitic bacteria:

These bacteria obtain their nutrition from the tissues of the hosts on which they grow. They may be
harmless or may cause serious diseases. Parasitic bacteria which cause various diseases in plants
and animals are known as pathogens, e.g., Bacillus typhosus, B. anthracis, B.tetani. B.diplheriae,
B.tuberculosis, B. pneumoniae, Vibrio cholerae, Pseudomonas citri etc.
 (iii) Symbiotic bacteria:
Symbiotic bacteria live in close association with other organisms as symbionts. They are beneficial
to the organisms. The common examples are the nitrogen-fixing bacteria, e.g., Bacillus radicicola,
B. azotobacter, Rhizobium, Ctostridium etc. Rhizobium spp., B. radicicola and B. azotobacter.
These bacteria live inside the roots of leguminous plants. These bacteria fix free atmospheric
nitrogen into nitrogenous compounds, which are utilized by the plants. In return, the plant provides
nutrients and protection to the bacteria.

4. Chemolithotrophic autotrophs
They oxidise inorganic compounds like iron, sulphur or nitrogen to derive both energy and
electrons for biosynthesis. Carbon dioxide is the carbon source for them. Few chemolihotrophs
have been recognized to derive their carbon from organic sources and are called chemolithotrophic
heterotrophs. Bacteria that obtain energy by utilizing inorganic electron donors but obtain most of
their carbon from organic compounds are called mixotrophs.