UNIT- II

General Characteristics of microbes

 CLASSIFICATION OF MICRO-ORGANISMS

MICRO-ORGANISMS

1. Eukaryotes 2. Prokaryotes 3. Sub cellular

organisms
(Possess a true nucleus. (Primitive nucleus: Nuclear ( DNA or RNA)
Nuclear membrane present) membrane absent)

I. Fungi I. Bacteria I. Viruses

II. Protozoa(parasites)
Cocci
Bacilli: rod shaped bacteria
Mycoplasma
 ARANGEMENT

• Some bacteria show a typical arrangement or

grouping.
a) Staphylococci : Cocci arranged in irregular groups
resembling a bunch of grapes.
b) Streptococci/Strepto bacilli: Cocci or bacilli arranged in
chains
c) Diplococci: Cocci arranged in pairs.
d) Cuneiform arrangement: Arranged at angles to each other,
This gives V,L, N, M or W letter pattern.
Arrangements of cocci
 Motility
• Bacteria which have flagella exhibit motility.
• It is best in the liquid cultures.
• It can be demonstrated by two methods.
1.Hanging drop method.
2. Wet film method.
 1.Hanging drop method.
This is mainly used to test for the motility of
bacteria.
True motility can be identified by change in
place and direction of bacteria, where as false
motility is identified as only a change in
direction but not place.
PROCEDURE OF HANGING DROP METHOD
 Wet film method
• Wet films are used for bacteria like in change
fluid, Vibrio cholera in stool sample, ova, cysts
and larvae of intestinal parasites and vaginal
fluid.
 Types of motility bacteria
• 1. Slow stately motility
• 2. Darting motility
• 3. Tumbling motility
 Bacterial colonisation
• Bacterial are present all around us. They are
present in the air we breathe. In the water we
drink and I the food we eat.
• Sometimes they help human beings and
sometimes they become necessary to normal
human life.
• Bacterial are divided into groups according to
the nature of their activity.
 Pathogens
• These bacteria live in or on human beings and
cause damage (disease) to the host. These are
of serious concern.
 Commensals
• These organisms live harm lessly in or on the
human body.
• They never cause disease under any circum-
stances, and on the other hand, some of the
important substances required for the body.
(eg: lactobacilli in the intestine ,
B complex vitamins).
 Opportunistic pathogens
• Opportunistic pathogens are those which are
present in or on human host.
• They are harmless under ordinary conditions
when the host's immune system is normal. But
when the host's immune function is damaged
due to any reason, they take an up-per hand
and cause disease.
 Useful Bacteria
• These are useful to human existence in a
number of ways.
• Putrefaction and Decay: Bacteria, by this
process, convert nature's organic matter into
useful plant food like nitrates, phosphates and
sulphates of minerals.
• This is the natural means of decomposition of
animal waste and dead bodies after burial.
 Putrefaction and Decay
• Bacteria, by this process, convert nature's
organic matter into useful plant food like
phosphates and sulphates of minerals.
• This is the natural means of decomposition of
animal waste and dead bodies after burial.
 Parification of sewage and water
• The soil and water bacteria purify sewage
water by converting the organic substances in
sewage water into manure useful for plants.
Septic tanks work this way.
 Production of food for plants
• bacteria in the soil convert the nitrogen as
food for plants. These are called nitrogen
fixing bacteria.
• The nodules on the roots of plants like beans
and peas are nothing but multitudes of a
nitrogen fixing bacterium.
 Curdling of milk
• Milk into curd is facilitated by a bacterium
called Lactobacillus lactis.
• When milk is inoculated with curd containing
this organism and kept warm(37°c), the
organism utilises the sugar in milk and
produces acid which curdles the milk.
 Protection against intestinal infections
• Some useful bacteria colonise along the length
of the gut mucosa and synthesise B complex
vitamins.
• They also prevent colonisation with
pathogenic bacteria.
 BACTERIA COLONISING THE BODY
1. Skin
2. Respiratory tract
3. Gastro intestinal tract
4. Genito urinary tract
 Skin

• Healthy skin has a number of colonising bacteria.

Staphylococci (aureus and albus) grow at all times in the
hair fol- licles and sweat ducts.
• The skin can not be made absolutely sterile even with
most through scrubbing and application of antiseptics.
• This is because, it is easy to remove bacteria from the
outer layers but it impossible to get rid of those in the
deeper layers.
• This is the reason why the operating team wears
protective gowns and rubber gloves during surgical
work.
 Respiratory Tract

• The inside of the nose has a mucous membrane.

• The bacteria in the air stick to this as the air is breathed in,
and later colonise. merous kinds of microorganisms.
• Commonly found bacteria are streptococci, staphylococci,
pneumococci, During normal respiratory mechanism, some of
these bacteria are exhaled.
• This is the reason of wearing a mask during surgical
procedures.
• Wearing a mask can also be considered as a protective
equipment while examining a patient with a lung infection like
tuberculosis, as the tubercle bacilli are shed in large amounts
during coughing and contaminate the air.
 Gastro Intestinal Tract
• The mouth contains a number of commensal bacteria.
• They line as saprophytes in the crevices of gums, spaces between
teeth and on the surface of the tongue.
• They feed on the left over food particles. Good oral and dental
hygiene can frequently remove these bacteria.
• Some of the commensals like streptococcus viridans group of
bacteria produce acid from carbohydrates.
• The acid damages the enamel of teeth resulting in dental caries.
Some of them form compact mass along with food particles and the
deposited salts at the junction of gums and teeth, causing plaques
(tartar).
• Therefore the importance of oral and dental hygiene has to be
greatly stressed.
 Genito Urinary Tract
• the coronal sulcus and the inner prepuce are lined with commensals,
predominantly staphylococci.
• The terminal one or two inches of the urethra is also lined by bacteria. In
females, the vagina normally contains lactobacilli which break down glycogen
in the vaginal secretions to reduce the vaginal pH.
• This occurs in women of the sexually active age group, under the influence of
female hormones. Acid pH is most suitable for the spermatozoa to travel
across the vagina into the fallopian tubes.
• Before puberty and after menopause, the pH of the vagina is alkaline. Other
than lactobacilli, the vagina and streptococci. Under conditions of unsterile
labour and inefficient care of the mother after delivery.
• the anaerobic bacteria travel into the uterus. The raw area in the uterus and
presence of blood clots favour the growth of these bacteria, which cause
sepsis. This is the reason for puerperal fever. This is why sterile procedures are
compulsory at and after child birth.
 Blood
• Blood is normally sterile. However, bacteria temporarily gain
access into the blood of perfectly healthy persons, after escaping
through capillaries from local areas of colonisation.
• These are quickly removed from the blood by phagocytosis,
which is an important activity of blood macrophages. Bacteria
may also get into the blood if aseptic precautions are not
followed during venepuncture (introducing needle into the vein).
• If after unsterile venepuncture, blood is collected for transfusion,
the bacteria multiply in the blood bags even under refrigerator
conditions.
• If such blood is transfused it may result in fever and rigors after
transfusion.
 GROWTH AND NUTRITION OF MICROBES AND
INFLUENCE OF TEMPERATURE AND MOISTURE

• The interval between two cell divisions, or the

time taken by the bacterium to produce two
daughter cells is called generation time or
population doubling time.
• For most of the bacteria, the generation time
is 20 minutes.
• For Mycobacterium tuberculosis, it is 20 hours
and for Mycobacterium leprae, it is as long as
20 days.
 BACTERIAL GROWTH CURVE

 Lag Phase
 Log Phase
 Stationary Phase
 Phase of Decline
 Lag Phase

• Initially for some time after the seeding, the

bacterial counts remain the same.
• This phase is called the lag phase.
• During this phase the cell reaches maxi- mum
size.
 Log Phase
• During this phase, there is enormous speed in
the increase of bacterial count which rises
exponentially.
• This phase is called log phase or exponential
phase. During this phase, all bacteria are
young and active and all their properties are
best expressed.
• This phase occurs between 4-6 hrs of
inoculation.
 Stationary Phase
• The bacterial counts remain stable for some
time. This is because the number of dividing
cells is equal to those that die because of lack
of nutrients in the medium and accumulation
of toxic byproducts of metabolism.
• This is called stationary phase.
 Phase of Decline
• The bacterial count falls due to cell death.
Autolytic enzymes are produced by some
bacteria (eg: Pneumococci)during this phase.
Phase
 BACTERIAL NUTRITION
• Basing on the nutritional requirements,
bacteria are classified as autotrophs which can
synthesise all their compounds, and
heterotrophs which depend upon preformed
organic compounds.
• Most of the pathogenic bacteria are
heterotrophs.
 The nutrients required by bacteria vary
greatly.
• i) Substances like glucose, amino acids,
• ii) Inorganic salts: Phosphates, chlorides, and
sulphates of sodium, potassium, etc.
• iii) Some organic compounds in minute
quantities. These are called bacterial vitamins.
eg: Vitamin B1, riboflavin, nicotinic acid,
pyridoxine, B12 etc.
 ENVIRONMENTAL FACTORS AFFECTING
GROWTH
• These are water, oxygen, carbon dioxide, tens
perature and pH. Most of the pathogenic
bacte ria grow best at 37°C (similar to body
temperature and at pH 7.4 (pH of human
blood).
 1. Energy requirement
• Some bacteria derive energy from sunlight
(phototrophs), some derive energy from
chemical schemotrophs)
• some bacteria can synthesise their own food
( autotrophs), and some bacteria cannot
synthesise their own food (heterotrophs).
• Pathogenic bacteria causing human disease
are heterotrophs.
 2. Oxygen requirement
• oxygen requirement
• Required (aerobes) required in low quantity
(microaerophilic) not required (anaerobes)
• compulsory(obligate aerobes) be can survive
in both aerobic/anaerobic conditions
(facultative anerobes) most of the pathogenic
bacteria belong to the facultative anaerobic
group..
 3. Temperature
• Pathogenic bacteria ideally require a tem- perature of
37°c, though most bacteria have a temperature range of
25 to 40°c.
• The called mesophilic bacteria.
• Some bacteria which prefer temperatures below 20 care
called psychrophilic bacte ria.
• They are usually present in spoilt refrigerated food.Some
bacteria prefer very high tempera- tures of upto 80 c.
• They are called thermophilic bacteria.
• They usually pose a problem in sterilisation.
 Conti…
• Under moist conditions, most of the
bacteria of human importance die at
temperature of 60c, and bacterial spores die
at 120c
 4. Moisture
• Water is an essential component of bacterial
protoplasm.
• Dry heat is lethal to bacteria, as it dehydrates them.
But bacterial spores have thick walls which resist
dessication.
• There fore they can survive in Body fluids: body
fluids can be understood in dry conditions for years.
• If we need to preserve bacteria as in vaccine
preparations or research.
 BLOOD AND BODY FLUIDS (NORMAL
MICROBIAL FLORA OF THE BODY)
• In a healthy person, there is a microbial
population called 'microbio’ at various places
in the.
• They colonise mucosal surfaces. These are
called 'normal microbial flora of the
body''resident flora, or 'commensals'.
• About 30 to 40 such families of bacteria,
millions in numbers, live in and on human
body.
 Role of normal bacterial flora
• They synthesise B complex vitamins and
vitamin K
• They prevent pathogenic bacteria from
colonising
• they provide immunity
• they prevent inflammatory diseases of thegut.
Laboratory methods for identifications of
microorganisms
• Depending upon the types of infection and
the system involved the nurse collects the
clinical sample from the patient.
• Blood, CSF, Sputum, urine, fluids etc..
 Laboratory methods
I. Direct smear
II. Culture
III. Motility
IV. Biochemical Properties
V. Slide Agglutination
VI. Antibiotic Sensitivity Testing
 Direct Smear
I. Direct Smear: A portion of the specimen is
picked up with the bacteriological loop and is
spread into a smear on a slide.
It is then stained suitably. The causative organ
ism can be seen sometimes along with pus
cells. This procedure is useful for fluids like
pus CSF etc.
 Culture

• The sample is inoculated into suitable media

which are selected depending on the nature
of the disease.
• The inoculated media are incubated at the
required temperature (37°C for most of the
bacteria) and time (usually 18-24 hours)
• Inoculation and incubation depend upon the
type of the medium and the oxygen
requirement of the organism.
 Conti…
• After incubation under optimal conditions,
bacterium divides repeatedly over a period of
18-24 hrs.
• All the daughter cells (progeny) remain
together to be visible to the naked eye, in the
form of a colony which ranges in size between
1 mm-5mm. depending on the organism.
 Conti…
• The morphological features of the colony like
size, shape, etc., are typical for each
bacterium.
• One of the identifying markers of a bacterium
is study of the colony morphology.
• Some bacteria produce pigments, and the
colonies assume the colour of the pigment.
eg. Staphylococcus aureus produces golden
yellow pigment.
Motility • A hanging drop
preparation of
bacteria is done
and motility is
studied.
 Biochemical Properties
• A few of the biochemical properties of the
isolate are test , like sugar fermentation,
production of enzymes or production of
certain by products of metabolism.
 Slide Agglutination
• After all the above properties are tested, a few
colonies of the organism are emulsified in saline
and a drop of high titre serum (antibody to that
organism in a high concentration) is added.
Clumping of the organism is made visible in a few
seconds.
• This is the procedure for confirmation of the
organism.
• This is especially useful for salmonella, shigella and
vibrios.
 Antibiotic Sensitivity Testing
• Bacteria isolated from clinical specimen
exhibit great variation in their sensitivity to
antibiotics.
• it is compulsory to test the isolate for its.
 Diffusion Tests

• The organism is inoculated on the surface of

the agar plate to which filter paper discs with
absorbed antibiotics are applied.
• The antibiotic diffuses around and reacts with
the organisms. Sensitivity is measured by a
circular zone of inhibition.
• Examples of diffusion tests are Kirby- Bauer's
method. Stokes method, etc.
 Dilution Tests
• The antibiotic is serially diluted in a nutrient
broth, to which a standard volume of bacterial
in Sensitivity is measured by the highest
dilution of the antibiotic.
• which can kill or inhibit the growth of bacteria,
evidenced by clear broth without turbidity.
 Kirby-Bauer's method
• Kirby-Bauer's method is a commonly used
method in the laboratory for antibiotic
sensitivity testing.
 Procedure
• The organism is inoculated into broth and a
growth in log phase is obtained after 4-6 hrs.
• This suspension is then inoculated as a law
culture inoculum on the surface. It is allowed
to dry for 1/2-1h at 37°C.
 Conti…
• Various antibiotic discs are applied on the plate (a
maximum of 6 on a plate of 10 cm diameter) at equal
distances.
• The plate is then incubated upright for 18-24 hrs.
• A incubation, various zones of clearance appear
around the antibiotic. which the organism is sensitive.
• The diameters of these zones are measured and the
sensitivity pattern is determined b comparison of the
zones with those obtained for the control organism
which similarly processed in the laboratory.
 Culture media
Introduction
Culture media are required to grow the
organisms from infected material to
identify the causative agent.
The basic constituents of culture media.
1. water
2. Electrolyte
3. Peptone
4. Meat extract
5. Agar
 culture
• IS the term given to microorganisms that are
cultivated in the lab for the purpose of
identifying and studying them.
 Media
• Is the term given to the combination of
ingredients that will support the growth and
cultivation of microorganisms by providing all
the essential nutrients for the growth in order
to cultivate these microorganisms in large
number to study.
 Definition of culture media
An Artifical media contains basic requirements
needed for microorganisms growth. Used for
recognition and identification of
microorganisms.
 The basic constituents of culture media.

The basic constituents of culture media.

1. water
2. Electrolyte
3. Peptone
4. Meat extract
5. Agar
 Types of media
I. Based on classified in many ways.
a) Liquid media
b) Semisolid media
c) Solid media
II. On the basis of presence of molecular oxygen
and reducing substances in the media.
a) Aerobic media
b) Anaerobia media
Conti…
III. Based on nutritional factors.
a) Simple media
b) Complex media
c) Synthetic media
d) Special media
1. Enriched media
2. Enrichment media
3. Selective media
4. Differential media
5. Indicator media
6. Transport media
7. Sugar media
 Methods of Culture
I. Streak culture
II. Lawn culture
III. Stroke culture
IV. Stab culture
V. Pour plate culture
VI. Liquid culture
 Streak culture

It is the routine method employed for

bacterial isolation in pure culture.
A platinum or nichrome loop of 2-4 mm
internal diameter is used.
 Lawn culture

This types of culture method is employed in

antibiotic sensitivity testing.
This method is bacteriophage typing.
it may also be employed for preparation of
bacterial antigens and vaccines.
Where a large amount of bacterial growth is
required.
 Stroke culture

Stroke culture ids done in tubes containing

and is employed for providing a pure growth
of the bacterium for slide agglutination and
other diagnostic tests.
commonly used agar slop is nutrient agar slope.
 Stab culture

• Stab Culture is performed by a straight wire

charged with culture material (bacteria) by
puncturing deep inside the
• This Technique is employed to demonstrate
gelatin liquefaction oxygen requirement of the
bacterium and to maintain stock culture for
preservation of bacteria.
 Pour plate culture

• Tubes containing 15 ML of agar medium in

each are melted and kept to cool in water
bath at 45-50 C.
• This inoculum to be tested is diluted in serial
dilutions one ml of each diluted inoculum is
added to each tube of molten agar.
• Mixed well and the contents of the tube
poured into a sterile petridish and allowed to
solidify.
 Liquid culture

Liquid culture in test tubes screw-capped

bottles or flasks may be inoculated by
touching with a charged loop or by adding the
inoculum with adopted for blood culture and
for sterility test.
 Culture and media preparation
• It is a possible to grow bacteria in the
laboratory by supplying the necessary
nutrients and other conditions for growth.
• According to their nutritional needs.
• Bacteria are classified as fastidious organisms
which repuire rich sources of Nutrition like
serum, blood,egg,milk etc..
 Simple Media

• These contain simple sources of nutrition

which are just required to growth non
fastidious bacteria.
• Eg. Coli, Pseudomonas etc…
• A Number of special culture media have been
employed for the cultivation of V.Cholerae.
 Enriched Media
• These are solid media which contain
additional source of nutrition to support the
growth of fastidious organismas.
• 1) Blood agar
• 2) Chocolate agar
• 3) Loeffler’S serum slope
 This contains 5-10% blood
1) Blood agar added to nutrient agar.
Fastidious bacteria grow on
this medium and produce
colonies with a zone of
clearing around.
If the clearing is complete, it is
called B haemolysis and if
the clearing is partial, the
zone is green and is called
haemolysis.
Chocolate
Agar
This is heated
blood agar.
It is more
nutritious and
supports more
fastidious
bacteria like
Haemophilus
influenzae.
Loeffler’s
serum slope
It contains 20%
serum.
It supports the
growth of
corynebacterium
diphtheriae.
 Enrichment Media
• These are liquid media containing an
ingredient which prevents the growth of
unwanted bacteria and enhances the growth
of required bacterium in the clinical sample.
• These media are useful for isolation of
pathogenes from samples like blood ,sputum
and stool.
 Selective Media
• These are solid media containing a selective
agent .
• Which inhibits the growth of unwanted
bacteria and enhances the growth of required
bacteria.
 Differential Media
• These are useful to differentiate between two
groups of organisms based on the nature of
their colonies on the media.
 Differential Media
• These are useful to differentiate between two
groups of organisms based on the nature of
their colonies on the media.
 Transport media
• When the laboratory is situated from the
place of sample collection immediate
processing of the sample is not possible.
 Anaerobic media
• These media contain a reducing agent.
• Which absorbs all the dissolved oxygen in the
medium.
• Such media support the growth of anaerobic
organisms.
Thank you