Unit IV
Methods of Microbiology:
Principles of Microbial nutrition – Carbon, nitrogen, sulphur, growth factors, nutritional
requirements of Bacteria, Nutritional classification of Bacteria, Culture media preparation, Types
of media Selective media, Enrichment media and Differential media, Sterilization methods
(physical, Chemical and mechanical)
EXPLAIN PRINCIPLES OF MICROBIAL NUTRITION – CARBON, NITROGEN,
SULPHUR, GROWTH FACTORS, NUTRITIONAL REQUIREMENTS OF
BACTERIA&NUTRITIONAL CLASSIFICATION OF BACTERIA: (SECTION B & C)
MICROBIAL NUTRITION :
a. A list of vocabulary words is found toward the end of this document
b. Nutrients may be divided into three general, often overlapping categories: Those
that supply energy, those that supply carbon, and those that supply any and
everything else. In this lecture we will consider nutrition from the point of view of
microorganisms, though many of the principles apply to macroorganisms equally
well.
2. Nutrient
a. "Something that nourishes or promotes growth and repairs the natural wastage of
organic life." (Websters, 1986)
b. More specifically, nutrients are elements and specific combinations of elements
(chemicals) that specific organisms require for growth and repair.
3. Nutritional pattern
a. Organisms can be classified according to their nutritional pattern. This, minimally,
is where they obtain their energy and where they obtain their carbon.
4. Energy source
a. This is the source of high energy electrons used to make ATP.
5. Conversion of food to energy
a. Conversion to ATP:
i.
To be utilized as energy, an organic compound must first be converted to
ATP.
1
ii.
This is often done by first metabolically converting the nutrient molecule
to a common intermediate, very often glucose (i.e., different catabolic
biochemical pathways will terminate in a common product, e.g., glucose,
which, in the case of glucose, is always treated as glucose regardless of the
source).
b. Energy in bonds:
i.
Glucose as well as other foodstuff intermediates contain large amounts of
energy in their molecular bonds.
ii.
This energy may be converted to the high energy bonds found in ATP, by
a variety of processes, by different organisms.
c. Generally these processes by which ATP is generated are referred to as
fermentation and cellular respiration.
6. Other substrates for ATP yielding catabolism
a. Organic, energy containing substances other then glucose include:
i.
lipids
ii.
amino acids
iii.
other carbohydrates
b. Inorganic, energy containing substances include various containing or consisting
of:
i.
sulfur
ii.
ammonia
iii.
H2
7. Carbon source
a. This is the source of the carbon atoms used in the organic compounds found in
organisms.
8. Phototroph
a. An organism whose energy source is light.
9. Chemotroph
a. An organism whose energy sources are electron-donating compounds such as
glucose.
b. This compound(s) is not necessarily an organic compound (i.e., above).
10. Autotroph [lithotroph]
a. An organism whose principle carbon source is carbon dioxide.
b. Producers:
2
i.
The autotrophs are what make non-CO2 carbon compounds, using CO2 as
their starting compound.
ii.
Because the autotrophs are what make non-CO2 carbon compounds for
everything else (i.e., other organisms which eat the autotrophs), they are
know ecologically as the producers.
iii.
Indeed, autotrophs are "self-feeders."
11. Heterotroph [organotroph]
a. An organism which uses organic compounds as its principle source of carbon.
b. Consumers:
i.
Heterotrophs feed on others.
ii.
That is, Heterotrophs obtain their carbon compounds by consuming other
organisms.
iii.
Heterotrophs are thus consumers, decomposers, scavengers, predators,
herbivors, etc.
12. Photoautotroph
a. An organism whose energy source is light and whose principle carbon source is
carbon dioxide.
b. Photoautotrophs include plants, alage, cyanobacteria, as well as some
photosynthesizing bacteria.
13. Photoheterotroph
a. An organism whose energy source is light and which uses organic compounds as
its principle source of carbon.
b. These organisms are unable to convert CO2 to sugar nor produce O2.
14. Chemoautotroph
a. Chemical plus CO2:
i.
An organism whose energy sources are electron donating compounds and
whose principle carbon source is carbon dioxide.
ii.
These organisms tend to use inorganic electron donors (i.e., they eat
rocks!).
iii.
That is, chemoautotrophs obtain their energy from something other than
light or carbon compounds.
b. Deep-sea vent producers:
3
i.
Chemoautotrophs constitute the producers at the deep sea vents, i.e., the
extract energy from inorganic compounds dissolved in sea water which are
vented at these locations.
ii.
As the producers the chemoautotrophic bacteria at deep sea vents basically
constitute the first link of the deep sea vent food chain.
15. Chemoheterotroph
a. An organism whose energy source is electron donating compounds and which
uses organic compounds as its principle source of carbon.
b. For these organisms energy and carbon sources tend to be the same organic
compounds.
c. Humans are an example of chemoheterotrophs.
16. Nutritional patterns of pathogens
a. Chemoheterotrophs:
i.
Medically relevant microorganisms are almost always chemoheterotrophs.
ii.
This is because pathogens tend to derive both their energy and their carbon
from organic compounds obtained from their hosts, e.g., human bodies.
b. All of the organisms on your binimials list are chemoheterotrophs.
17. Microbial nutrient requirements
a. Common microbial nutritional requirements include:
i.
water
ii.
a carbon source
iii.
an energy source
iv.
nitrogen
v.
sulfur
vi.
phosphorus
vii.
potassium
viii.
magnesium
ix.
calcium
x.
oxygen*
xi.
various trace elements
xii.
various organic growth factors**
xiii.
*
Not molecular oxygen but oxygen atoms incorporated into compounds
other than O2.
4
xiv.
**
Of all common nutrient requirements, the need for specific organic
growth factors is least shared among microorganisms.
b. Source utilization variation:
i.
Note that not all microorganisms do or are even able to assimilate all of
these nutrients from the same source(s).
ii.
"There are many types of laboratory prepared media available for the
isolation and the cultivation of bacteria. It is important to understand that
whatever growth medium is used, it must provide the necessary nutritional
requirements for the organism you wish to grow." (Krueger & Kolodziej,
1986)
18. Nitrogen
a. Amino acids:
i.
Used in amino acids and nucleic acids.
ii.
Possible organic source = amino acids.
b. Inorganic sources:
i.
Possible inorganic sources include:
1. NH4+ (ammonium---nitrogen in its lowest oxidation state)
2. NO3- (nitrate---nitrogen in its highest oxidation state)
3. atmospheric nitrogen (nitrogen fixing)
19. Nitrogen fixation
a. Nitrogen from air:
i.
The conversion of gaseous, elementary nitrogen (N2) into nitrogen
available to cellular metabolism.
ii.
Ultimately this is where all of the nitrogen found in all organisms comes
from.
b. Uncommon metabolic pathway:
i.
Only a minority of bacteria are capable of nitrogen fixation.
ii.
See particularly Rhizobium spp..
20. Sulfur
a. Amino acids:
i.
Sulfur is found in some amino acids and in various vitamins.
ii.
Possible organic source is sulfur-containing amino acids.
b. Possible inorganic sources included SO42- (sulfate ion).
21. Phosphorus
5
a. Phosphorus is found in nucleic acids and phospholipids.
b. The dominant inorganic source of phosphorus is phosphate ion (PO43-).
22. Trace elements
a. Usually present:
i.
Trace elements are often assumed to be present unless highly pure
synthetic components are utilized.
ii.
Even distilled water often contains adequate amounts of these element for
growth.
b. Enzyme cofactors are basically used as enzyme cofactors.
c. Examples of cofactors include:
i.
copper
ii.
iron
iii.
molybdenum
iv.
zinc
v.
cobalt
vi.
manganese
d. "Many microorganisms require a variety of trace elements, tiny amounts of
minerals such as copper, iron, zinc, and cobalt, usually in the form of ions. Trace
elements often serve as cofactors in enzymatic reactions. All organisms require
some sodium and chloride, and halophiles require large amounts of these ions.
Potassium, zinc, magnesium, and manganese are used to activate certain enzymes.
Cobalt is required by organisms that can synthesize vitamin B12. Iron is required
for the synthesis of heme-containing compounds (such as cytochromes of the
electron transport system) and for certain enzymes. Although little iron is
required, a shortage severly retards growth. Calcium is required by gram-positive
bacteria for synthesis of cells walls and by spore-forming organisms for synthesis
of spores." (p. 149, Black, 1996)
23. Organic growth factors
a. Strict environmental requirement:
i.
Organic growth factors are organic compounds that are products of the
metabolism (particularly anabolism) of some organisms, but cannot be
synthesized by all microorganisms (or all organisms, for that matter), but
nevertheless are required for growth by the latter organisms.
ii.
Typical organic growth factors are vitamins and amino acids.
6
b. Note, under no circumstances would common carbon sources be considered
organic growth factors and, in fact, it is debatable whether carbon source (no
matter how obscure) and organic growth factor could ever be considered
synonymous.
24. Essential amino acids
a. Synthesis capability:
i.
Some but not all organisms are able to synthesize all 20 amino acids
directly from intermediates of carbohydrate metabolism.
ii.
Escherichia coli is an example of an organism which can synthesize all 20
amino acids.
b. Those organisms which are unable to synthesize all 20 amino acids must obtain
these amino acids (or, in some cases, their anabolic intermediates) from their
environment.
c. Strict environmental requirement:
i.
These must-be-obtained amino acids are termed essential.
ii.
Essential amino acids are examples of organic growth factors.
25. Fastidious (adj.)
a. To be fastidious means to have complex nutrient requirements.
b. Examples of fastidious genera include:
i.
Neisseria
ii.
Mycoplasma
iii.
Mycobacterium
iv.
etc.
c. Note that fastidiousness has nothing to do with oxygen requirements.
26. Auxotroph [auxotrophic mutation]
a. Mutational deficiency:
i.
An auxotroph is a microorganism possessing a mutation in a gene that
affects its ability to synthesize a crucial organic compound.
ii.
In addition, the auxotroph must be recently descended (e.g., since being
domesticated) from a microorganism of the same species that both lacks
the mutation and is able to synthesize the organic compound in question.
b. Auxotrophs thus a have novel requirement that a given nutrient be present in their
extracellular environment for growth to occur.
7
STERILIZATION
(Section-C)
Write short notes on physical agents of sterilization?
Introduction:
Microorganisms are ubiquitous. Since they cause contamination, infection & decay, it
becomes necessary to remove or destroy them from materials or from areas. This is the object
of sterilization.
Sterilization is defined as the process by which an article, surface or medium is freed of
all microorganisms either in the vegetative state or spore state. Sterilis (Latin) – Unable to
produce offspring.
Sanitization – Closely related to disinfection – the microbial population is reduced to
levels that are considered safe by public health standards.
Disinfection means the destruction of all pathogenic organisms or organisms capable of
giving rise to infection / killing, inhibition or removal of microorganisms that may cause
disease.
Disinfectants are defined as the agents usually chemical used only on inanimate objects.
Antiseptics are used to indicate the prevention of infection, usually by inhibiting the growth
of bacteria. Chemical disinfectants are used to prevent infection by inhibiting the growth of
bacteria can be safely applied to skin or mucous membrane surfaces are called antiseptics /
Chemical agents applied to tissues to prevent infection by killing / inhibiting the pathogen
growth.

Antisepsis – Greek anti – against, Sepsis – Putrefaction. Is the prevention of infection.

Germicide – Latin cida – to kill, to kill the pathogens.

Bactericidal – able to kill bacteria.

Bacteriostatic – agents which prevents the bacterial multiplication.
The various agents used in sterilization can be classified as follows :
1. Physical Agents :
Sunlight, Drying, Dry heat – flaming, incineration, hot air, Moist heat –
pasteurization, Boiling, Steam under pressure, Filtration – Candles, Asbestos pads,
membranes, Radiation.
2. Chemical agents :
8
Alcohols – Ethyl, Isopropyl, Trichlorobutanol, Aldehydes – formaldehyde,
Glutaraldehyde, Dyes, Halogens, Phenols, Surface active agents, Metallic salts, Gases –
Ethylene oxide, Formaldehyde,  - propiolactone.
Physical Agents :
Sunlight:
It possesses bactericidal activity & plays an important role in the spontaneous
sterilization under natural conditions. The action is due to the UV rays. Direct sunlight has an
germicidal effect due to the combined effect of the UV and heat rays where it is not filtered by
the impurities in the atmosphere.
Semple & Grieg showed in India, that Typhoid bacilli exposed to the sun on pieces of
white drill cloth were killed in 2 hours, whereas controls kept in the dark were still alive after 6
days. Bacteria suspended in water are readily destroyed by exposure to sunlight.
Drying :
Moisture is essential for the growth of bacteria.4/5 by weight of the bacterial cell
consists of water. Drying in air has a deleterious effect on many bacteria. Spores are unaffected
by drying.
Heat :
It is the most reliable method of sterilization. Materials damageable by heat can be
sterilized at low temperatures, for longer periods or by repeated cycles. The factors influencing
sterilization are :
1. Nature of heat – dry or moist heat,
2. Temperature & time,
3. The number of microorganisms present,
4. The characteristics of the organisms such as species, strain, spores &
5. The type of material from which the organisms have to be destroyed / removed.
The killing effect of dry heat is due to protein denaturation, oxidative damage & the
elevated levels of electrolytes. The lethal effect of moist heat is due to the denaturation &
coagulation of protein.
The time required for sterilization is inversely proportional to the temperature of
exposure. TDT – Thermal death time, is the time required to kill a suspension of organism at a
definite temperature.
The material in which organisms heated affects the rate of killing – proteins, nucleic
acid, starch, gelatin, sugar, fats & oils – increases the time.
(Section – B)
Dry Heat:
9
1. Flaming :
Inoculation loops or wires, forceps, spatulas are held in a Bunsen flame till they become
red hot, inorder to be sterilized. If the loops contain infective pertinacious material they should be
first dipped in chemical disinfectants before flaming to spattering.
Scalpels, needles, culture tube mouths, glass slides, cover slips could be passed a few
minutes through the Bunsen flame without allowing them to become red hot. The bacteria gets
destroyed.
Needles & scalpels are immersed in methylated spirit & the spirit burnt off them is
however unsatisfactory.
(Section – A)
2. Incineration :
This is an excellent method for rapidly destroying materials such as soiled dressings,
animal carcasses, bedding & pathological materials.
(Section – B)
3. Hot air oven :
It is the most widely used method of sterilization by dry heat. A holding period of 160 0
C for 1 hour is used. It is used to sterilize glasswares, forceps, scissors, scalpels, glass syringes,
swabs, pharmaceutical products – liquid paraffin, sulphonamides, dusting powder, fats, greases,
etc.
The oven is heated by electricity, with heating elements in the wall of the chamber & it
must be fitted with a fan to ensure even distribution of air.
The material should be arranged for free air circulation in-between the objects.
Glasswares should be dried before placing in the oven. Test tubes, flasks should be wrapped in
kraft paper. At 180 degree C cotton wool plugs may get charred.
Cutting instruments of ophthalmic surgery needs a sterilization time of 2 hrs at 150
degree C. The British pharmacopoeia recommends a holding time of 2 hrs – 150 degree C for
oils, glycerol & dusting powder. The oven must be allowed to cool slowly for about 2 hrs before
the door is opened, Since the glassware may get cracked by sudden or uneven cooling.
Sterilization control for Dry heat (Hot air oven):
(Section – B)
The spores of a non-toxigenic strain of Clostridium tetani are used as a microbiological
test of dry heat efficiency. Paper strips impregnated with 106 spores are placed in envelopes &
inserted into suitable packs.
After sterilization is over, the strips are removed & inoculated into thioglycollate or
Robertson cooked meat media & incubated for sterility test under strict anaerobic conditions for
5 days at 37 degree C.
10
A browne’s tube is available & is convenient for routine use. After proper sterilization,
a green color is produced. Thermocouples may also be used.
Moist Heat:
1. Temperatures below 1000C (Pasteurization) :
(Section – B)
Milk are treated with controlled heating at temperatures below boiling is known as
pasteurization. The temperature employed is 63oC – 30 mins (Holder method) or 720C – 15 – 20
secs (Flash method) – HTST – high temperature short time or Ultrahigh temperature (UHT) –
140 – 1500C – 1 – 3 secs. By this method all nonsporing pathogens such as Mycobacteria,
Brucella & Salmonella are destroyed. Examples are :
a) Nonsporing bacterial vaccine – Inactivated – 600C – 1 hr.
b) Lowenstein-Jensen’s & Loeffler’s serum media – 80 – 850C – ½ hr – 3 days.
c) Staphylococcus aureus & Streptococcus faecalis – 600C – 1 hr.
d) Clostridium botulinum – 1200C – 4 mins.
e) Polio myelitis – 600C – 30 mins.
f) Hepatitis Virus – 600C – 10 hrs.
Clothing, bed clothes, eating utensils, equipment may be disinfected by washing in
water at 70 – 800C for several mins.
2. Temperature at 100oC (Boiling) :
Vegetative bacteria are killed at 90 – 100oC and not sporing bacteria. It is not
recommended for the surgical instruments. Sterilization is prompt by the addition of 2 % sodium
bicarbonate to the water. If boiling is necessary, the material should be immersed in water &
boiled for a period of 10 – 30 mins. The lid of the sterilizer should not be opened during boiling.
3. Steam at atmospheric pressure – 100oC (Tyndallisation) :
(Section – A)
Heat-sensitive material can be heat sterilized by tyndallisation or fractional steam
sterilization or intermittent sterilization. The container with the material to be sterilized is heated
at 90 – 100oC for 30 mins for 3 consecutive days & incubated at 37oC in-between.
The first heating will destroy all organisms but bacterial endospores germinate during
the incubation & are killed by second incubation period. Any remaining spores are destroyed by
the second incubation & third heat treatment.
(Section – B)
4. Steam under pressure (Autoclave) :
Autoclave is like a fancy pressure cooker developed by Chamberland in 1884. Water
is boiled to produce steam, which is released through the jacket & into the autoclave’s chamber.
11
The air initially present in the chamber is forced out until the chamber is filled with saturated
steam & the outlets are closed.
Hot, saturated steam continues to enter until the desired temperature & pressure is
reached – 121oC – 15 pounds of pressure. At this temperature, saturated steam destroys all
vegetative cells & endospore in a small volume of liquid within 10 – 12 mins. Treatment is for 15
mins to provide a margin of safety. Moist heat kill by degrading nucleic acids, denaturing
enzymes & proteins & also disrupt cell membranes.
If all air has not been flushed out of the chamber, it will not reach 121oC even it reach
15 lb pressure. The chamber should not be packed too tightly because steam needs to circulate
freely & contact everything in the autoclave. When a large volume of liquid must be sterilized, an
extended time will be needed because it will take longer for the center of the liquid to reach
121oC – 5 litres – 70 mins.
Materials such as dressings, instruments, laboratory wares, media & pharmaceutical
products can be sterilized.
5. Sterilization control for Moist Heat (Autoclave) :
The biological indicator consists of a culture tube with a sterile ampoule of medium & a
Paper strip covered with spores of Bacillus stearothermophilus or Clostridium PA 3679. After
autoclaving, the ampoule is broken & the culture is incubated for several days. Sometimes, a
special tape that spells out the word sterile or a paper indicator strip that changes colour upon
sufficient heating is autoclaved with a load of material.
If the word appears on the tape or if the colour changes after autoclaving, the material is
sterile. It is safe & convenient but not reliable for spores.
Filteration :
It reduces the microbial population in solutions of heat-sensitive material. Rather than
destroying the microorganisms, filtration removes them. This is useful for antibiotic solutions,
sera & carbohydrate solutions, culture media, oil, etc. Two types of filters are:
1. Depth filters – fibrous or granular material of thick layer. The solution with
microorganisms is sucked through this layer under vacuum & microbial cells are removed
by physical screening. Eg. Berkefield filters, Chamberlain filters, Asbestos, etc.
2. Membrane filters – Circular, porous filters of 0.1 mm thick made of cellulose acetate,
cellulose nitrate, polycarbonate.
Different pore sizes are available but 0.2 µm diameter pore size is used to remove
vegetative cells from solutions. The solution is pulled or forced through the filter with a vacuum
or pressure from a syringe, pump & collected in pre-sterilized containers.
12
Membrane filters remove microorganisms by screening them out as a sieve separates
large sand particles from small ones. Air can be sterilized by filtration. Eg. Surgical masks &
cotton plugs on culture vessels that let air in but keep microorganisms out. Laminar air flow with
HEPA – high efficiency particulate air filters (Section A), which remove 99.97% of 0.3 µm
particles, is of the most important air filtration system.
a) Candle type filters – Berkefield filters are made of diatomaceous earth of various
porosity. V(viel) – Coarsest, W(wenig) – finest, N(normal) – intermediate.
b) Porcelain filters – The most commonest one is the chamberland filter – made of sand.
These are made in various porosities.
c) Asbestos filters (Seitz) – This is made from chrysotile asbestos – magnesium silicate. It
is available in different grades are : HP/PYR – removal of pyrogens, HP/EKS – absolute
sterility, HP/EK – clarifying.
d) Sintered glass filters – These are made of finely ground glass which is fused to make the
particles adhere. These are available in different pore sizes.
Radiation :
Two types of radiation are used for sterilizing purposes – nonionising & ionizing.
1. Nonionising radiation :
Infrared (IR) rays & ultraviolet(UV) rays are nonionising low energy type. IR rays is a
hot air sterilization & used for sterilization of syringes. UV rays is lethal but does not penetrate
glass, water, etc.
So, it is used for disinfecting enclosed areas such as entryways, hospital; wards,
operation rooms & virus inoculation rooms, laboratories, to sterilize the air or any exposed
surfaces.
UV radiation burns the skin & damages eyes, people working in such areas must take
care that the UV lamps are off when the areas are in use. Pathogens & other microorganisms are
destroyed when a thin layer of water is passed under the lamps.
2. Ionising radiation :
Excellent sterilizing agent & penetrate deep into the objects. X – rays, gamma rays &
cosmic rays are highly to DNA. There is no increase in temperature, so this method is cold
sterilization.
Gamma rays are used to sterilize antibiotics, hormones, disposable syringes, meat &
other foods. FDA (Food & Drug Administration) & WHO (World Health Organisation) have
approved food irradiation & declared it safe. It is used to destroy pathogens in poultry & extend
the shelf life of seafood’s & fruits.
13
(Section – C)
Write a short notes on chemical agents of sterilization?
Chemical agents
 A variety of chemical agents has been used as antiseptics & disinfectants. An ideal
antiseptics or disinfectant should be
1) Be effective against all microorganisms – bacteria including spores, protozoa &
fungi.
2) Be active in the presence of organic matter.
3) Be effective in acid & alkaline media
4) Have speedy action
5) Have high penetrating power
6) Be stable
7) Be compatible with other antiseptics & disinfectants
8) Not corrode metals
9) Not cause local irritation or sensitization
10) Not interfere with healing
11) Not be toxic if absorbed into circulation
12) Be cheap & easily available
13) Be safe & easy to use.
 The factors that determine the potency of disinfectants are :
a) Concentration of the substance
b) Time of action
c) pH of the medium
d) Temperature
e) Nature of the organisms.
 These substances act in various ways. The main mode of action are :
i. Protein coagulation
ii. Disruption of cell membrane, results in exposure of the cell contents to the
environment & leads to cell death
Agent
Mechanism of action
Phenolic compounds
1. Germicidal effect caused by alteration of 1. 89% solutions: Cauterization of
Phenol
protein
structure
Use
resulting
14
in
protein minor wounds.
denaturation.
2. Surface – active agent (surfactant) 2. 5% solutions: Disinfection.
precipitates cellular proteins and disrupts
cell membranes.(Phenol has been replaced 3. 0.5% to 1% solutions: Antiseptic
by better
disinfectants
that
are
less effect and relief of itching as it exerts
irritating, less toxic to tissues, and better a local anesthetic effect on sensory
Cresols
inhibitors of microorganisms.)
nerve endings.
1. Similar to phenol.
2% to 5% Lysol solutions used as
2. Poisonous and must be used externally.
disinfectants.
3. 50% solution of cresols in vegetable oil
known as Lysol.
Hexachlorophene
Germicidal activity similar to phenol.
1. Reduction of pathogenic organisms
(This agent is to be used with care, on skin; added to detergents, soaps,
especially of infants, because following lotions, and creams.
absorption it may cause neuro toxic 2. Effective against gram- positive
effects.)
organisms.
3. An antiseptic used topically.
Resorcinol
1. Germicidal activity similar to that of 1. Antiseptic.
phenol.
2. Keratolytic agent for softening or
2. Acts by precipitating cell protein.
Hexylresorcinol
Thymol
dissolving keratin in epidermis.
Germicidal activity similar to that of 1. Treatment of worm infections.
phenol.
2. Urinary antiseptic.
1. Related to the cresols.
1. Antifungal activity.
2. Treatment of hookworm infections.
Alcohols
2. more effective than phenol.
3. Mouthwashes and gargle solutions.
1. Lipid solvent.
Skin antiseptics:
Ethy1 – 50% to 70%.
Ethyl
CH3 CH2 OH
2.Denaturation and coagulation of proteins.
Isopropyl
(CH3) 2 CHOH
3. Wetting agent used in tinctures to
increase the wetting ability of other
15
Isopropyl - 75%.
chemicals.
4. Germicidal activity increases with
increasing molecular weight.
Halogens
1. Germicidal effect resulting from rapid 1. Water purification.
Chlorine compounds:
combination with proteins.
Sodium hypochlorite
2. Chlorine reacts with water to from dairy and restaurant industries.
(Dakin’s
fluid
2. sanitation of utensils of utensils in
): hypochlorous acid, which is bactericidal.
3. Chloramine, 0.1% to 2%
NaOCI
3. Oxidizing agent.
solutions, for wound irrigation and
Chloramine
4. Noncompetitively inhibits enzymes, dressings.
CH3 C6 H4 SO2 NNa CI
especially those dealing with glucose 4. Microbicidal.
metabolism, by reacting with SH and NH2
groups on the enzyme molecule.
Iodine compounds:
1. Mechanism of action is not entirely 1. Tinctures of iodine are used for
Tincture of iodine
known, but it is believed that it proteins.
Povidone
–
skin antisepsis.
iodine 2. Surface – active agent.
2. Treatment of goiter.
solution (Betadine)
3. Effective against spores, fungi, and
viruses.
Anionic agents
1. Nautral or alkaline salts of high 1. Cleansing agent.
molecular – weight acids. Common soaps
included in this group.
2. Exert their maximum activity in an acid 2. Sclerosing agent in treatment of
medium and are most effective gram- varicose
Tincture of green soap
Sodium
positive cells.
veins
and
internal
hemorrhoids.
tetradecyl 3. Same as all surface – active agents.
sulfate
4. Same as all surface – active agents.
Acids (H+)
1.Destruction
Alkali (OH-)
membrane.
of
cell
wall
and
cell Disinfection;
however,
of
little
practical value.
2. Coagulation of proteins.
Formaldehyde
or gas)
(liquid Alkylating agent causes reduction of 1. Room disinfection
enzymes.
2. Alcoholic solution for instrument
disinfection.
3. Specimen preservation.
16
Ethylene oxide
Alkylating agent causes reduction of Sterilization of heat – labile material.
enzymes.
Beta – propiolactone Alkylating agent causes reduction of 1. Sterilization of tissue for grafting.
(liquid or gas)
enzymes.
2. Destruction of hepatitis virus.
3.Room disinfection.
Basic dyes
Affinity for nucleic acids; interfere with Skin antiseptic.: Laboratory isolation
Crystal violet
reproduction in gram- positive organisms.
Heavy metals
1. Mercuric ion brings about precipitation 1. Inorganic mercurials are irritating
Mercury compounds;
of cellular proteins.
Inorganic:
2. Noncompetitive inhibition of specific adversely affected by organic matter,
Mercury bichloride
enzymes caused by reaction with sulfhydryl and they have on action on spores.
Mercurial ointments
group (SH) on enzymes of bacterial cells.
of gram – negative bacteria.
to
tissues,
toxic
systemically,
2. Mercury compounds are mainly
used as disinfectants of laboratory
materials.
Organic mercurials:
1. Similar to those of inorganic mercurials, 1. Less toxic, less irritating ; used
Mercurochrome
but in proper concentrations are useful mainly foe skin asepsis.
(merbromin)
antiseptics.
Merthiolate(thimerosal) 2.Much
2. Do not kill spores.
less
irritating
than
Merbak(acetomeroctol)
inorganicmercurials.
Silver compounds;
1. Precipitate cellular proteins.
Silver nitrate
2. Interfere with metabolic activities of throat and eyes.
Asepsis of mucous membrane of
microbial cells.
3. Inorganic salts are germicidal.
Surface – active agents
1. Lower surface tension and aid in Weak action against fungi, acid – fast
Wetting agents:
mechanical removal of bacteria and soil.
Emulsifiers, soaps, and 2. if active portion of the agent carries a
detergents
negative electric charge, it is called and
anionic surface- active agent. If active
portion of the agent carries a positive
electric, it is called a cationic surface –
active agent.
3. Exert bactericidal activity by interfering
with or by depressing metabolic activates
17
microorganisma, spores, and viruses.
of microorganisms.
4. Disrupt cell membranes.
5. Alter cell permeability.
Cationic agents:
1. Lower
surface tension because of 1. Bactericidal, fungicidal; inactive
Quaternary ammonium kerratolytic, detergent, and emulsifying against spores and viruses.
compounds
properties.
2. Asepsis of intact skin
Benzalkonium chloride
2. Their germicidal activities are reduced 3. Disinfectant foe operation – room
by soaps.
equipment.
4. Dairy and restaurant sanitization.
Testing of disinfectants :
 Testing of antimicrobial agents often begins with an initial screening test to see if they are
effective and at what concentration.
 The best known disinfectant screening test is the phenol coefficient test in which the
potency of a disinfectant is compared with that of phenol.
 A serial dilutions of phenol and the experimental disinfectants are inoculated with the test
bacteria such as Salmonella typhi and Staphylococcus aureus, then placed in a 20 or 37oC
water bath.
 These inoculated disinfectant tubes are subcultured to fresh medium at 5 mins interval
and incubated for 2 or more days.
 The highest dilutions that kill the bacteria after 10 mins exposure, but not after 5 mins are
used to calculate the phenol coefficient.
 The reciprocal of the appropriate test disinfectant dilution is divided by that for phenol to
obtain the coefficient. A value >1 means the disinfectant is more effective than phenol.
 To estimate the disinfectant effectiveness more realistically, other tests are often used. A
use dilution test can be done. Stainless steel cyclinders are contaminated with specific
bacterial species under controlled conditions.
 The cyclinders are dried, immersed in the test disinfectants for 10 mins, transferred to
culture media and incubated for 2 days.
 The disinfectant concentration that kills the organisms in the sample with a 95% level of
confidence is determined. In use testing techniques, allows a more accurate determination
of the proper disinfectant concentration for a particular situation.
18
TYPES OF MEDIA:
INTRODUCTION TO CULTURE TECHNIQUES:-
(Section – C)
→Micro organisms are ubiquitous in nature that is, it is present every where in soil, water, air,
hot springs, volcanoes, antartics, in plants, animals, humans, etc.
→They can be cultured by performing various techniques.
→Firstly, we have to isolate the microorganisms from any source by collecting the sample.
→If air, the media should be exposed to air. All living organisms require nutrition for their
survival.
→The nutrition, which we supply artificially, is called media.
→It should contain carbon, nitrogen, minerals, etc., in the form of micronutrients and
macronutrients.
→The media is prepared, sterilized and transferred to the required apparatus. These works
should be done aseptically to avoid contamination. There are various techniques for culturing
and preserving the microorganisms.
2) HOW TO PREPARE A CULTURE MEDIA? WHAT ARE THE TWO CLASSES OF
(Section – B)
CULTURE MEDIA?
→In a general sense, the nutrient requirements of all organisms are the same, i.e., all
organisms require the macro and micro nutrients.
→ Certain organisms require certain nutrients in specific forms.
→The proportion may vary for the growth of different microorganisms.
Two classes of culture media are used in microbiology laboratory.
They are:
 Chemically defined or synthetic media.
 Undefined or complex media.
1. Chemically defined (or) synthetic media:Synthetic media are prepared by adding precise amounts of pure inorganic or organic
chemicals to distilled water. Therefore, the exact chemical composition of a definite medium is
known.
2. Undefined (or) complex media:→Complex media employ crude digests of substances such as casein (milk protein), beef, soy
beans, yeast cells etc.
→They are commercially available in powered form and can be weighed out and added to
culture media.
19
→Disadvantage of using complex media is the loss of control over the precise nutrient
specifications of the medium.
→Culture media can be prepared for use either in a liquid state or in a gel (semi solid) state.
→A liquid culture medium is converted to the semi solid state by the addition of a gelling
agent.
→It is manufactured from certain seaweeds and is not a nutrient for most microorganisms.
→Microbial cells grow on it and form visible masses called colonies.
WHAT ARE THE VARIOUS PRODUCERS (OR) TECHNIQUES AVAILABLE FOR
THE PRESERVATION OF CULTURES? EXPLAIN IT.
(Section – C)
→It is very important to preserve the microorganisms to conserve all the characteristics of the
species as they were at the time of preservation.
There are basically two major methods for preservation of cultures:
 Methods in which organisms are in a state of continuous metabolism.
 Methods in which organisms are in a state of suspended metabolism.
1. Organisms in continuous metabolism state:→It depends on repeated sub culturing of organisms onto fresh nutrient medium when the
formerly used medium gets dried or is used up.
→This includes the following methods:
A. PERIODIC TRANSFER TO FRESH MEDIA:→All micro labs preserve microorganisms on agar slants.
→The slants are incubated for 24 hours or more and then stored in a refrigerator.
→These cultures are periodically transferred to fresh media.
→The time interval at which transfers are made varies with the microorganisms and the
conditions of growth.
B. OVERLAYING CULTURES WITH MINERAL OIL:→The agar slants are inoculated and incubated until good growth appears.
→They are then covered with sterile mineral oil to a depth of 1 cm above the slant surface.
→This method has the unique advantage that one can remove some of the growth under the
oil with a transfer needle.
→Some species have been preserved satisfactorily for 15 – 20 years by this method.
C. STORAGE IN STERILE SOIL:→This method is widely used for preserving spore forming bacteria and fungi.
20
→Spore suspensions are added to sterile soil (soil is sterilized for 2-3 hours at intervals of 1-2
days) and the mixture is dried at room temperatureerature and stored in refrigerator.
→ Bacterial cultures are maintained by this procedure have been found viable for 70-80 years.
D. SALINE SUSPENSION:→Sodium chloride in high concentration is frequently used as an inhibitor of bacterial growth.
→Bacteria are suspended in 1% salt solution in screw cap test tubes to prevent evaporation.
→The tubes are stored at room temperature.
2. Organisms in suspended metabolism state:This depends on some sort of drying or preservation at low temperature to a level at
which the metabolism is slowed down or stopped, but revival of culture is possible. It includes:
A. DRYING IN VACCUM:→The organisms are dried over calcium chloride in a vacuum are then stored in the
refrigerator.
→The organisms survive longer than when air-dried.
B. LYOPHILIZATION:-[FREEZE DRYING]
→Lyophilization is the most effective procedure for the preservation of cultures.
→ Many species of bacteria are preserved by this technique have remained viable and
unchanged for more than 20 years.
→In this process the cells are dried rapidly, while they are frozen.
→The bacterial; suspension is placed in small vials, which are then immersed in a mixture of
dry ice and alcohol at a temperature of –780C.
→The vials are immediately connected to a high vacuum line.
→ This dries the organisms while still frozen.
→Finally, the vials are sealed off in a vacuum with a small flame.
→These cultures can then be stored for several years at 40C.
→This method is also employed for the preservation of sera, toxins, enzymes and other
biological materials.
→When needed, it is necessary to break open the vial aseptically, add a suitable sterile
medium and after incubation make further transfers.
Advantage: Reduces the danger of contamination.
 Requirement of a small storage area.
C. USE OF LIQUID NITROGEN:21
→In this method, the cultures are frozen to -1960C with a protective agent (glycerol or
dimethyl sulfoxide) in sealed vials.
→The frozen cultures are kept in liquid nitrogen refrigerator.
→This process has been successful with many specimens, which cannot be maintained by
lyophilization.
D. STORAGE IN SILICA GEL:→Both bacteria and yeas can be stored in silica gel powder at low temperature for a period of
1-2 years.
→In this method, finely powdered, heat sterilized and cooled silica powder is mixed and
stored at low temperature.
→The basic principle in this technique is quick desiccation at low temperature which allows
the cells to remain viable for a longer period.
4) WHAT ARE THE DIFFERENT TECHNIQUES USED FOR OBTAINING MIXED
(Section – B)
AND PURE CULTURES? EXPLAIN.
A number of techniques have been employed for the isolation of microorganisms from
natural environment. The different techniques involved are:
1. Streak plate method:
Media is prepared and sterilized.

Poured in petridish and allowed to solidify.

Streak plates are prepared by streaking a small amount of the diluted sample over the
surface of the solid medium using nichrome wire inoculation loop.

The plate is then incubated at proper temperature.

There are different types of streaking. They are:
(diagram)
 Simple or continuous streaking.
 T-streak.
 Quadrant streaking.
 Radiant streaking.
→ This gives the mixed culture.
→ Isolate colonies may be present in the plate.
→ From the isolated colonies, a little amount of the culture is transferred to a tube of medium
22
this becomes a pure culture.
2. Spread plate method: A serially diluted inoculum is transferred over the surface of the solid medium in
petridish.
 It is then spreaded uniformly with a sterile bent glass rod (or) L – rod (or) spreader, to
separate aggregates of cells in the sample which on incubation give isolated colonies of
mixed culture.
 A portion of a colony transferred to a tube of medium becomes a pure culture.
Growth from each culture should be checked microscopically and culturally to verify that
it is a pure culture.
3. Pour plate method: The principle of the pour plate technique is dilution of the specimen in the tubes of
liquefied or melted agar medium. The inoculum is serially diluted.
 An aliquot of diluted inoculum is added to sterile Petri plate followed by the addition of
melted and cooled nutrient agar medium.
 Then mix the sample with medium by slowly rotating the plate.
 After solidification, the plates are incubated.
 A decrease in number of the colonies can be observed resulting in the form of isolated
colonies.
 To get pure culture Ex: only spore formers, the sample can be first exposed to 850C
temperature for 5 minutes, so that the non spore formers will be destroyed and the spore
formers alone will grow on incubation because they are heat resistant.
4. Serial dilution method: An organism that predominates in a mixed culture may be obtained as a pure culture by
serial dilution technique.
 A mixed culture can be serially diluted in the test tubes of the sterile medium.
 A drawback in this technique is that one can never be sure that the last dilution tube will
always contain a pure culture.
 Lister originally described this technique.
 In this technique, mixed culture can be obtained mainly.
5. Enrichment culture technique:An enrichment culture provides a specially designed cultural environment by
incorporating a specific nutrient in the medium and by modifying the conditions of incubation.
23
This will favour the growth of the other types. The procedure increases the proportion of the
desired micro organism by the application of natural selection.
Example: - 1. Blood agar.
2. Chocolate agar.
6. Use of selective media:Selective media contains specific chemicals, which suppresses or kills unwanted types of
microorganisms, while allows the desired type to multiply.
Example: crystal violet, brilliant green inhibit gram-positive bacteria and thus help in the
isolation of gram-negative bacteria.
7. Use of differential media:These media contain dyes, reagents or chemicals that allow the observer to distinguish
between types of bacterial colonies, after incubation, on the basis of colony characteristics.
Example: on eosin methylene blue agar, E. coli and related species produce colonies with
a brilliant green metallic sheen. On the same medium Enterobacter aerogenes and related species
produce pink colonies with dark centers.
5) CULTIVATION OF ANAEROBES? / TECHNIQUS FOR CUTIVATION OF
ANAEROBES? (Section B)
FOR ANAEROBES:The cultivation of anaerobes is a complex process, because anaerobes may be killed by
exposure to oxygen. Special media called “reducing media” must be used. These media contain
ingredients such as sodium thioglycolate that chemically combine with dissolved oxygen and
deplete the oxygen of the culture medium. Media, during cultivation should be stored in
ordinarily, tightly capped test tubes. These media are heated shortly before use, so that any
absorbed oxygen is driven off.
Anaerobic containers used in the laboratory are the Brewers plate, Spray plate, Brewers
jar and the Gas Pak anaerobic jar.
1. Brewer’s plate:
This is the simplest method.

The plate is filled with sodium thioglycollate agar and inoculated with the anaerobic
specimen.

The lid is designed with a deep lip to make contact with the surface of the agar when it is
placed on the plate.
24

This prevents the oxygen from making contact with the center portion of the culture.
2. Spray plate:
A spray plate looks like a petridish with a deep base divided into 2 compartments.

Potassium hydroxide is placed on one side and pyrogallol is placed on the other side.

Then inoculated agar containing lid is placed on top of the base and sealed.

Gently shake the plate; so that the KOH and pyrogallol are mixed in the base thereby
oxygen will be removed from the plate.

Bacteria will then be able to grow on the inverted agar surface.
3. Brewer’s jar:
Is complex and unsafe than Brewer’s plate.

Cultures are placed inside and the lid is sealed.

Mixture of hydrogen, carbon-di-oxide and water is flushed through the jar.

Repeated for 2-3 times.

The remaining oxygen is removed by electrically heating a catalyst in the lid for about 10
times before placing in an incubator.
4. Gas Pak anaerobic jar:- (Section A)

A convenient method.

A disposable envelope containing chemicals capable of producing hydrgen and carbon-dioxide is chemically activated inside the jar.

Hydrogen gas reacts with oxygen to form water.

As the oxygen disappears, a mist or fog will appear on the inner wall of the jar and the lid
will warm.

An disposable anaerobic indicator inside the jar will change colour.

Anaerobic conditions is generated with in 25 minutes and then placed in incubator.
5. Anaerobic growth chamber / Anaerobic glow box:
Anaerobic glow box, which is small, transparent chamber, equipped with air locks and
filled with inert gases.

The technicians are able to change or manipulate the equipment by inserting their hands
in airtight rubber fitted gloves to the wall of the chamber.
WHAT ARE THE VARIOUS METHODS BY WHICH THE MICROBIAL GROWTH
CAN BE DETERMINED? (Section B)
25
Enumeration of microorganisms is required for
 Accessing the rate of microbial population.
 Determining the portability of water.
 Determining the suitability of food for human consumption.
 Evaluating the necessity for antimicrobial therapy.
 For industrial, research & medical reasons
 The enumeration methods are as follows:
1. Total Cell count:
In this method both living & dead cells are counted. Following technique are used for the
total cell count.
a) Dry slide technique/ direct microscopic count
b) Petroff – Hauser counting chamber
c) Electronic cell counter (coulter counters)
2. Viable cell point:
To determine the number of living cells in natural environment like milk, water etc,
Following techniques are used for viable cell count.
 Standard plate count
 Membrane filter technique
 Dip-Slide culture technique
 Most probable number method
3. Biomass Determination:
 Turbidometric methods
 Measurements of dry (or) wet weight of biomass

Estimation of chemical constituent of cell. Eg: N2
1. Total cell count:
Petroff – Hauser counting chamber:
 The culture is not dried stained
 A special slide cover glass is used to contain a sample of culture of known volume.
 The slide has a grid (or) graph etched on its surface to make counting easier more
accurate.
 When the count is made the total is multiplied by the dilution factor of the sample to
determine no of cells /mm of original sample.
 Haemocytometers are used for counting blood cells which are larger than this.
26
Advantages:
 Rapid direct microscopic counting can be mode
 Minimum equipments & requirements are needed
 Morphology of microbes can also be studied
Disadvantages:
 It gives a total cell count which includes the viable & movable cells
 Accuracy declines due to clumping of cells.
2. Viable cell count:
Membranes Filter technique:
 Can be used when number of cells in is too low.
 Sample is drawn thro membrane filters
(Eg : cellulose acetate filters ) of known pore size.
 Cells are separated from the medium
 Filters are removed aseptically from the assembly placed on the surface of soft nutrient
agar.
 If the nutrient penetrates the filter, each cell which develop to from colony, which can be
counted.
Disadvantage:
If the culture suspension has particular matter or is very turbid it would log the filters.
3. Biomass Determination:
Turbidometric methods:
 Jurbidometric measurements are based on the principle that the number of tiny particles
in a suspension is inversely proportional to the percentage of transmittance of light.
 Turbidity is measured in optical density (O.D) units that are inversely proportional to the
percentage of transmittance (%)
 The photo spectrometer is used to measure. It consists of a light source, a chamber in
which the specimen is placed a photoreceptor to measure the amount of transmitted light.
 If turbidity is high, the percentage of transmissions is low while the O.D high.
27