2. Microbiology
Microbiology is the study of living organisms that are
microscopic in size
Medical microbiology is the study of microscopic
organism that infect man, his reaction to such
infections, the way in which the disease is produced
and the method for diagnosis, prevention and
treatment of such infectious disease.
Microorganisms can be seen only with the help of
microscope.
Bacteria, fungi, algae, viruses, protozoa,
mycoplasms, rickettsias all are microorganisms.
3. Classification of microorganism
They are classified under kingdom protista.
Depending on the cellular organisation and
biochemistry, the kingdom protista is divided into two
groups- prokaryotes and eukaryotes.
MICROORGANISMS
HAVING CELLULAR ORGANISATION
(KINGDOM PROTISTA)
PROKARYOTES
Bacteria, Blue green
algae
EUKARYOTES
Fungi, Algae, slime,
moulds, protozoa
HAVING NO CELLULAR ORGANISATION
Viruses
Viriods
prion
4. Character Prokaryotes Eukaryotes
Cytoplasm
Cytoplasmic streaming Absent Present
Pinocytosis Absent Present
Mitochondria Absent Present
Lysosomes Absent Present
Golgi bodies Absent Present
Endoplasmic reticulum Absent Present
Nucleus
Nuclear membrane Absent Present
Nucleolus Absent Present
Deoxyribonucleoprotein Absent Present
Number of
chromosomes
One More than one
Mitotic division Absent Present
Chemical variations
Sterols Absent Present
Muramic acid Present Absent
5. Bacteria
A bacterium is a unicellular prokaryotic microorganism
which does not contain chlorophyll and does not
multiply by true branching.
The unit of measurement used is micron (micrometer).
6. Shape of Bacteria:
Bacteria are classified into varieties based on their
shapes:
a) Cocci are oval or spherical bacterial cells
b) Bacilli are rod shaped cells
c) Vibrios are comma shaped curved rods. These
bacteria possess characteristic vibratory motility
d) Spirilla are rigid spiral shaped bacteria
e) Spirochaetes are spiral shaped cells look like
coiled hairs
f) Actinomycetes are branching filamentous bacteria.
In the tissue lesions they appear as if radiating
rays.
g) Mycoplasmas are bacteria that do not have cell
wall and therefore their morphology is not stable.
They occur as round or oval bodies and as
filaments
7. Bacteria loose their cell wall and hence their shape can
change, due to defective formation of cell wall either
spontaneously or as a result of administration of drugs
such as penicillin. Such cells are called protoplasts,
spheroplasts or L forms
Arrangement of Bacteria:
Cocci are arrange in pairs(diplococci), in chains
(streptococci), groups of four(tetrads) or eight(sarcina) or
as bunch of grapes (staphylococci).
Bacilli can be arrange in chains (Anthrax bacilli) in
cluster, in groups of two(diplobacilli eg: pneumoniae)
and sometimes at angles to each other making a
cuneiform pattern(corynebacteria).
8.
9.
10. Anatomy of Bacteria:
It consists of an outer layer cell envelop which is
differentiated into an outer rigid cell wall and beneath it a
cytoplasmic membrane which is also called plasma
membrane.
Inside the cell envelop lies the protoplasm which contains
cytoplasm and cytoplasmic inclusions such as ribosomes,
mesosomes, granules, vacuoles and the nuclear body.
Sometimes a bacteria is enclosed into a viscid layed which
may be a loose layer or organised as a capsule.
Some bacteria also posses filamentous appendages at
their surface.
These appendages can be flagella(organ of locomotion)
and fimbriae (organ of adhesion)
Bacterial nucleus does not contain nuclear membrane or
nucleolus.
The bacterial chromosome is haploid and replicate by
simple fission
11. Spores:
Some bacteria(Bacilli and clostridium) have the ability
to form highly resistant resting stages called spores.
a single spore is formed by one bacterium and one
spore gives a single bacterial cell germination.
Spores which are formed inside the bacterial cells are
called as endospores.
Bacterial spores are resistant to chemicals and heat.
Spores of all medically important bacteria are
destroyed by autoclaving at 120°C for 15 minutes.
Sporulation helps the bacteria to survive for long
period under unfavourable conditions.
Spores germinate into bacterial cells when transferred
to favourable conditions
12. It is oval or spherical in shape and is attached to the
parent cell
It consists of a nuclear body, surrounded by spore
wall in the form of a delicate membrane.
Outer to the spore wall lies a thick spore cortex ,
which in turn is enclosed in a multilayered tough
spore coat.
Spores of some bacteria have an additional outer
covering called exosporium which have ridges and
grooves .
14. Fungi
Fungi are eukaryotic protists.
They have rigid cell wall containing polysaccharides-
chitin, mannan and others.
The cytoplasmic membrane of fungi contains sterols.
The nuclei contain nuclear membrane and paired
chromosomes.
Fungi multiply sexually, asexually or both.
They can be unicellular or multicellular.
Elongation of the fungi cells produces a thin tubular
structure called hypha.
A tangled mass of hyphae is called mycelium
Fungi which can make mycelia are called filamentous
fungi or moulds
15. Types of fungi :
Fungi are divided into 4 on the basis of cell morphology.
Yeasts:
Are unicellular fungi
Their cells are spherical or ellisoidal in shape.
They multiply by budding.
The pathogenic yeast is crytococcus neoformans.
Yeast like fungi:
They partly grow as fungi and partly as elongated cells
like hyphae called pseudomycelium
Eg: candida albicans
Moulds / filamentous fungi:
Can produce true mycelia
Multiply by spore formation
Eg: micosporum, trichophyton, epidermophyton
16. Dimorphic fungi:
They occur as filaments or as yeasts.
In tissues or in culture at 37°C they occur as yeast,
while in the soil and in culture at 22°Cthey appear as
moulds.
Eg: Blastomyces dermatitidis, Histoplasma capsulatum
etc
17. Systematic classification of fungi:
The systematic classification is based on the sexual
spore formation.
1. Lower fungi: Phycomycetes
2. Higher fungi:
Ascomycetes,
Basidiomycetes
Fungi imperfecti (Deuteromycetes or
Hypomycetes)
Phycomycetes:
They are lower fungi
These fungi have non segmented hyphae.
They form endogenous asexual spores called
sporangiospores which are contained in a sac like
structure called sporangia
Some of them produce sexual spores called Oospores
18. Higher fungi:
These fungi have separated hyphae
They form exogenous asexual spores called Conidia.
Ascomycetes:
These fungi form sexual spores called ascospores
within a sac or ascus
Yeast and filamentous fungi are included in this class
Basidiomycetes:
These fungi form sexual spores known as
basidiospores on a basidium or base.
Fungi imperfecti:
Sexual phase have not been identified.
Fungi of medical importance
19. Comparison of Fungi and
Bacteria
Sl
no
Characters Fungi Bacteria
1 Diameter 4μ 1μ
2 Nucleus Eukaryotic Prokaryotic
3 Cytoplasm Present Absent
4 Cell membrane Sterols present Sterols absent
5 Cell wall content Chitin(polysaccharide) peptidoglycan
6 Spores Sexual and asexual
spores for reproduction
Endospores for
survival and not for
reproduction
7 Thermal
dimorphism
Present in some Absent
20. Laboratory Diagnosis of Fungi:
The diagnosis is made by morphological study of the
materials taken from the lesions.
Skin scrapings are treated with 10% potassium
hydroxide and then examined under microscope as
wet mount.
For microscopic study, a small bit of fungus colony is
taken on a slide and mounted in lactophenol cotton
blue.
Slide culture technique is used for morphological study
of fungus
The periodic acid schiff (PAS) and methanamine silver
strains are valuable methods for the demonstration of
fungal elements in tissue sections
21. Culture of fungus:
The common culture media of fungi are:
1. sabouraud’s glucose agar
2. Czapek – Dox medium
3. Cornmeal agar
To prevent bacterial contamination, antibiotics are
added in the media.
Cycloheximide incorporation in the medium inhibits
many contaminant moulds
Cultures are incubated at room temperature (22°C)
for weeks or at 37°C for days.
22. Growth characters:
Growth characters useful for identification are the
rapidity of growth, morphology as well as the color of
pigmentation of the colony.
The diameter of hyphae, presence or absence of
septa and other special structures are also helpful in
the diagnosis
Spiral hyphae: they are spring like helical coils
Racquet hyphae: these localised swellings are
formed by twisted hyphae and look like tennis
racquets.
Favic chandlier hyphae: contain numerous long
branches at the end of hyphae.
23. The morphology of asexual spores is also of
diagnostic value.
Microconidia are small single shaped spores
Macroconidia are large, single or multicellular spore
Blastospores are formed by budding as in yeast
Arthospores are formed along the mycelium by
segmentation and condensation of hyphae
Chlamydospores are thick walled resting spores
formed by rounding up and thickening of the
segments of the hyphae.
Fungal infection (mycoses):
4 types: Superficial,
Sub cutaneous,
Deep seated (systematic)
Opportunistic
24. Superficial fungal infections:
The fungi causing the disease are specialised
saprohytes which can digest keratin of the skin
including nails & hairs.
It include various types of tinea and ringworm affecting
the skin, hair and nails.
Causative fungi for superficial infections are
dermatophytes, candida albicans, pityrosporum
orbiculare, cladosporium, piedraia hortai.
Subcutaneous fungal infections(Subcutaneous mycosis):
Caused by fungi such as rhinospordium, fonsecaea,
madurella norcardia, sporotrichum etc.
25. Deep seated fungal infections(systematic mycosis):
Are caused by soil saprophytes.
severity ranges from asymptomatic infections to fatal
diseases
The causative agents include actinomycetes (produces
madura foot), rhinosporidium seeberi (produce
rhinosporidiosis), cryptococcus neoformans
(cryptococcosis), dimorphic fungi- blastomyces dermatitidis
(blastomycoses) and histoplasma capsulatum (produce
histoplasmoses)
Opportunistic fungal infections:
Occur in patients suffering from diseases such as cancer,
AIDS or diabetes.
The use of drugs like immunosuppresive agents, steroids,
board spectrum antibiotics and exposure to X-rays makes
the body vulnerable to these infections.
Caused by mucor, penicillium, rhizopus and aspergillus
26. Mycotic poisoning:
Many fungi can produce poisoning.
Are of 2 types: mycetism and mycotoxicosis
Mycetism :
Fungi which is eaten for itself and produces toxic
effects.
Eg: some mushrooms after ingestion causes toxic
effects such as GIT disturbances, dermatitis or even
death.
Mycotoxicosis :
The toxins produced by fungi ie, mycotoxins
contaminate the food material and produce side
effects.
Eg: aflatoxin produced by Aspergillus flavus present in
mouldy foods such as ground nuts, corn and peas can
produce carcinogenic effects in humans
Ergotism:
27. Viruses
These microorganisms are much smaller than
bacteria.
They do not have a cellular organisation.
They contain only one type of nucleic acid , either
DNA or RNA.
They do not contain enzymes necessary for protein
and nucleic acid synthesis.
Viruses are dependent on the host cell for
replication.
They are obligate intracellular parasites that are not
affected by the usual antibiotics.
The extracellular infectious virus particle is called
virion
29. Viruses produce diseases like AIDS, cancer, rabies or
yellow fever etc.
The viral diseases can be sporadic like mumps,
endemic such as measles or pandemic such as
influenza.
Size:
Viruses vary in size
The largest virus such as pox virus is 300 nm in size.
The smallest viruses such as those causing foot and
mouth diseases are 20 nm in size .
Shape and Structure:
The viruses vary in shape.
Most viruses are spherical.
The rabies virus is bullet shaped, the pox virus is brick
shaped and the tobacco virus is rod shaped.
30.
31. Virion:
Virion consists of nucleic acid.
Core surrounded by a protein covering called
capsule. The capsule together with the nucleic acid
is called nucleocapsid.
The capsid is formed by a large number of
capsomers made up of polypeptide molecules.
The main function of capsid is to introduce viral
genome into the host cells by adsorbing readily to
the cell surfaces.
Two types of symmetry are seen in the capsid, ie,
icosachedral (cubical) and helical
Virions may be enveloped or non-enveloped
(naked).
The envelop or outer covering of the viruses is
derived from the host cell membrane at the time of
budding of virus and consists of lipoprotein.
32. The protein subunits projecting likes spikes on the
surface of the envelope are called peplomer.
A virus may have more than one peplomer. Envelopes
confer chemical, antigenic and biological properties on
viruses.
33.
34. Classification of viruses:
Classified into two based on the presence of nucleic
acid present.
(1)The Deoxyriboviruses or DNA viruses:
These viruses contain DNA in them.
Eg: poxvirus, Herpes virus, Adenovirus, Papilloma
virus, Human Hepatitis Type B virus etc
(2) The Riboviruses or RNA viruses:
These viruses contain RNA in them.
Include Enteroviruses, Rhinovirus, Influenza virus,
Mumps virus, Cholera virus, Rabies virus, Corona
virus, Retrovirus etc
36. Isolation of microorganisms
For a proper diagnosis of infectious diseases , it is
necessary to know the type and nature of causative
microorganism.
For this the specimen of the infected material are
collected.
Mostly used specimens are swabs, pus, sputum,
urine, stool , blood, cerebrospinal fluids, pleural
fluids and aspiration material.
The specimen should be collected before starting
the antimicrobial treatment otherwise the specimen
may become sterile.
The specimen should be collected from the site
most likely to be infected by the microorganism
37. Eg: sputum is examined in respiratory tract
infections, stool in diarrhoea , cerebrospinal fluid in
meningitis and blood in enteric fever is studied for the
presence of microbes.
Stage of disease is an important factor for specimen
collection.
Eg: in the early stage of enteric fever blood culture is
done, in the second week widal test is done and in
third week stool culture is performed.
Timing of collection is also important for the
successful isolation of causative microorganism
Eg: In UTI the first morning sample of urine is best for
culture.Sputum and conjunctival swabs should be
collected in the morning
38. The specimen should be collected in sufficient
quantity in suitable containers.
Urine sample is collected in sterile test tube.
Swab from eye, throat, rectum or vagina should be
collected by sterile swab stick and that too should be
placed into sterilized test tube immediately after
taking the sample.
Sputum should be collected in petridish.
Cerebrospinal fluid is collected in sterilized vials.
Blood for culture is collected in blood culture bottles.
After collection the specimen should be delivered to
laboratory without any delay to avoid the overgrowth
of organisms.
Sufficient clinical information should always be given
with the specimen
39. Methods of isolating pure
cultures
Surface planting method: the distinct colonies of
bacteria are picked out for further purification and
examination
Enriched selective and indicator media are used
widely for isolation of bacteria from specimens.
Pretreatment of specimens with appropriate
bactericidal substances destroys the unwanted
bacteria and pure culture can be obtained. Tubercle
bacilli are isolated by this method from sputum
Obligate aerobes and anaerobes can be separated
by cultivating the specimen in aerobic and
anaerobic conditions
40. Separation of bacteria with different optimum
temperatures can be done by incubating the material
at different temperatures. Thermophilic bacteria grow
at 60°C and Neisseria catarrhalis can be grown at
22°C.
By heating at 80°C the vegetative bacteria can be
eliminate from the mixture of vegetative and spore
forming bacteria.
Motile and non-motile bacteria can be separated by
using the Craigie’s tube- a tube containing semisolid
agar.
By inoculation into appropriate animals the pathogenic
bacteria are isolated from a mixture, for example
anthrax bacilli are inoculated into guinea pig or mice.
By using selective filters, bacteria of different sizes
may be separated.
By single cell method using micromanipulators, a
41. Staining techniques
After the isolation of causative microorganism, the
morphological detail is studied.
For morphological study the bacteria are stained
properly.
By staining the bacteria become clearly visible and
can be identified.
Smear preparation:
First step in staining procedure.
A loopful of liquid culture or fluid specimen or a
section of bacterial colony is taken and spread as a
thin film over the slide.
The smear is dried in air and heat fixed by passing
through a flame gently.
42. Simple staining of bacteria:
Apply the stain to be used over the prepared smear.
Crystal violet, methylene blue, fuchsin or safranin .
Allow the stain to react for 30 seconds to 3 minutes
depending on the stain used.
Wash the smear with gentle stream of cold water.
Dry between bibulous paper and examine under oil
immersion lens of the compound microscope.
Special staining procedures:
Gram’s staining methods
The most important staining method for bacteria
The shape, size and structural details of
microorganisms are made visible.
Helps to group organisms into gram positive and
gram negative
43. Steps involved:
1. Prepare a thin film and dry it.
2. Stain the prepared smear with methyl violet for one
minute. Wash off the excess stain with Gram’s iodine
solution.
3. Cover the whole slide with fresh iodine solution for
one minute.
4. The smear is then decolorised with spirit.
5. Wash the smear quickly with running tap water.
6. Cover the smear with dilute carbol fuchsin for 30
seconds.
7. Wash it with tap water and then dry it in air
8. Examine the slide under oil immersion lens
44. Gram positive bacteria retain the violet colour of methyl
violet.
Gram negative bacteria are decolorized by spirit, alcohol
or acetone and are stained with a counter stain like
carbol fuchsin which imparts a pink color to them.
Examples for Gram positive bacteria :
Staphylococci, Corynebacterium diphtheria, Bacillus
anthracis, Clostridium tetani , Clostridium welchi ,
Streptococci, Pneumonococci etc
Examples for Gram negative bacteria :
Gonococci, Meningococci, E.coli, salmonella typhi etc
45. Acid Fast Staining:
The acid fast stain is a differential stain used to
identify acid fast organism such as members of the
genus Mycobacterium.
The technique was discovered by Ehrlich who
observed that after staining with aniline dyes, tubercle
bacilli resist decolourisation with acids.
Ziehl Neelsen method:
Prepare a smear of the mucoid part of the sputum on
a slide and fix it.
Put strong carbol fuchsin over the smear for five
minutes. Wash the smear with water.
Put 20% sulphuric acid for one minute.
Wash the slide again with water.
46. Now put methylene blue for 30 seconds.
Wash the slide with water.
Dry the slide and observe it under oil immersion lens.
The pink coloured, rod shaped tubercle bacilli will be
seen scattered in the sputum of open cases of
tuberculosis.
The acid fast organism are stained pink or bright red.
These organisms after being stained with carbol
fuchsin do not loose their red colour when washed
with acids
Staining of Spirochaetes:
Spirochaetes are stained by fontana method.
Ammonia silver nitrate stain is used which increases
the apparent dimensions of the spirochetes and
these microorganisms are seen as opaque black
spiral hairs against a light back ground
47. Staining of Rickettsiae:
Rickettsiae are gram negative but they are not stained
well wiith gram’s stain.
They are stained with Giemsa or casteneda method
Staining of Yeast and Fungi:
Fixed smear of yeast can be stained with crystal violet
or methylene blue.
These dyes are put for 30 seconds to one minute.
Wet mounts of yeast can be stained effectively with
methylene blue or Gram’s iodine.
The cell can be emulsified in a drop of the either stain
and covered with a cover glass.
Lactophenol cotton blue is excellent for staining fungi.
