Microbiology, Classification of microbiology, bacteria, viruses, identification or isolation of bacteria, Staining of bacteria, protozoa, fungi

1. Microbiology
DR. RAMESH BHANDARI
ASSISTANT PROFESSOR,
DEPARTMENT OF PHARMACY PRACTICE,
KLE COLLEGE OF PHARMACY, BELGAUM

2. •Microbiology is a branch of Science
which deals with propagation, isolation
and identification of micro-organisms.
•It is the science of living organisms that
are not directly visible to naked eye but
only under the microscope.
•Medical microbiology deals with the
microorganisms which produce
infectious disease in man

3. Classification of Microorganisms
1) Prokaryotic:
i. Bacteria
ii. Rickettsias
iii. Mycoplasmas
2) Eukaryotic:
i. Algae
ii. Fungi
iii. Moulds
iv. Protozoa
Viruses

4. Prokaryotic Sl. No. Eukaryotic
Small cell size (0.2 – 2 micrometer) 01 Larger cell size (>10 micrometer)
Always unicellular 02 Often multicellular
No Nucleus 03 Always have nucleus
DNA is circular without proteins 04 DNA is linear and associated with histone
proteins
No Cytoskeleton 05 Always have cytoskeleton
Cell division by binary fission 06 Cell division by mitosis or meiosis
Cell wall made up of peptidoglycan 07 No cell wall, if present made up of
cellulose, chitin(fungi)
Ribosomes smaller size (70S) 08 Ribosomes larger size (80S)
Eg: Bacteria, mycoplasma 09 Eg: Algae, Fungi, Protozoa

5. Bacteria
• Bacteria is a unicellular microorganisms which
does not contain chlorophyll.
• The common pathogenic bacteria generally vary
in size from 0.2 to 1.5 micrometer in diameter
and 3 to 14 micrometer in length.
• They reproduce by an asexual process,
characteristically by simple cell division.
• They are present everywhere i.e. Air, water, soil,
internal and external parts of the human body,
animals and plants.

6. Classification of bacteria
1. Based on the shape of bacteria:
i. Cocci: Oval or spherical shaped
a) Cocci in pair: Diplococci
b) Cocci in group of 4:Tetrad cocci
c) Cocci in group of 8: Sarcina
d) Cocci in cluster: Staphylococci
e) Cocci in chain: streptococci
ii. Bacilli: Rod shaped eg: Brucella
iii. Vibrios: Comma shaped eg:Vibrio cholerae
iv. Spirilla: Spiral shaped , eg: Spirillum minus
v. Spirochaetes: spiral shaped (coiled hair) eg:Treponema Pallidum

7. 2. Based on gram staining:
i. Gram Positive bacteria: Streptococcus and
staphylococcus
ii. Gram negative bacteria: salmonella and
shigella
3. Based on need of oxygen or not:
a) Aerobic: Staphylococcus
b) Anaerobic: Clostridium botulinum

8. Structure of Bacterial cell

9. • Bacterial cell consists of cell wall made up of proteins
and carbohydrates.
• The cell wall is surrounded with a distinct sheath or
capsule.
• Inside the cell wall there is thin plasma membrane
formed by the cytoplasm.
• Cytoplasm includes ribosomes, mesosomes, granules,
vacuoles and the nuclear body.
• Some bacteria may contain whip like thread called
flagella which originate from cytoplasm. Flagella is
used for locomotion.
• Genetic material is present in the form nuclear
material composed of twisted and folded strands of
DNA.

10. Algae
• Green algae are found in fresh water either as free floating or
attached to some support.
• They are also found in rivers, ponds, ditches and other pools
of stagnant water.
• Some grows in the soil or surface of the soil or surface of the
tree or rock.
• Generally algae are green due to presence of chlorophyll
hence they can prepare their own food.
• Some algae are unicellular while as others are multicellular.
• Cell wall is composed of cellulose.
• A definite nucleus is present inside the cell.
• Reproduction may be sexual or asexual

12. Fungi
• Fungi are unicellular or multicellular micro-
organisms.
• They have rigid cell wall containing
polysaccharides.
• The cytoplasmic membranes contain
steroles.
• Reproduce sexually, asexually or by both
mechanism.

13. Morphology of fungi:
• Yeast is the simplest fungus and is unicellular
and grows by budding.
• Hypha is an elongated cell which is a tubular
thread like structure.
• Mycelium is a tangled mass of hyphae.
Fungi which forms mycelia are called moulds.
Mycelium which grows below the medium is
called vegetative mycelium.
• Mycelium which grows above the surface is
called aerial mycelium.

14. Classification of fungi
A. On the basis of morphology:
1) Yeasts: unicellular, spherical in shape and
reproduce by simple budding. Eg:
Cryptococcus neoformans.
2) Yeast like fungi: grow partly as yeast and
partly as elongated cell. Eg: Candida
albicans
3) Moulds: Filamentous fungi, forms true
mycelia. Eg: Dermatophytes.
4) Dimorphic fungi: Grows as filaments or as
yeasts depending on the condition of
growth.

15. B. Systematic classification of fungi:
1) Lower fungi: form asexual
(sporangiospores) or sexual
spores (oospores and zygospores).
Eg: Phycomycetes
2) Higher fungi: Form asexual
(conidia) and sexual spores.
Eg: Ascomycetes
Basidiomycetes
Fungus imperfecti.

16. Viruses
• Viruses are the simplest and smallest
organisms.
• They are much smaller than the bacteria varying
size from 20 nm to 300nm.
• These can be seen under electron microscope.
• Virus is composed of nucleic acid DNA or RNA
but never both.
• Viruses have no metabolic machinery of their
own.
• They cannot synthesize their own protein and
nucleic acid. So they are strictly parasitic in
nature.

17. Classification of Viruses
A. On the basis of shape:
1) Spherical shape: eg: Influenza virus
2) Rod shaped: eg: tobacco virus
3) Bullet shaped: eg: Rabies virus
4) Brick shaped: eg: pox virus

18. B. On the basis of RNA or DNA:
1) DNA viruses: Poxviridae
Papoviridae
Parvoviridae
Herpesviridae
Hepadenoviridae
Adenoviridae
2) RNA viruses: Picornoviridae
Paramyxoviridae
Togaviridae
Arenoviridae
Buniaviridae
Orthomyxoviridae
Rhabdoviridae

19. Protozoa
• Protozoa are single celled or unicellular
organisms which are microscopic in size.
Classification of Protozoa:
1. Rhizopoda: Moves with the help of
Pseudopodia. Eg: Entamoeba
2. Mastigophora: Which have flagella. Eg:
Trypanosoma
3. Sporozoa: Which exhibit no movement. Eg:
Malarial parasite
4. Ciliata: Which have cilia. Eg: Balantidium Coli

20. Isolation of Bacteria:
1) Streak Culture Method
2) Pour Plate culture Method
3) Enrichment and selective media
4) Aerobic and anaerobic condition
5) Separation of vegetative and spore forming
bacteria
6) Separation of motile and non motile Bacteria
7) Animal Inoculation
8) Filtration

21. Streak Culture Method
• It is also known as surface culture method.
• Specimen to be cultured is taken in a platinum loop
then it is spread on to the surface of well dried plate
containing culture media.
• The inoculum is spread over the plate in series of
parallel lines.
• Then incubate it at 370C.
• After incubating the plate, it can be seen that more
growth can be seen at the original site of inoculation
and it becomes progressively thinner.
• Final series of streak contains well separated colonies
of Bacteria.

22. Pour Plate Culture Method
• The specimen containing the bacteria is first diluted in
tubes of agar medium a number of times.
• The inoculums is added to the medium and shaken
thoroughly to distribute the inoculum.
• The inoculum material is poured into previously cleaned
and sterilized petriplates.
• Then Incubate it of 370C for 24 hours.
• Within this time the bacterial colonies develop.

23. Isolation of Viruses:
1) Animal Inoculation:Viruses can be isolated
by inoculating them in animals like monkeys,
mice, guinea pigs and rabbits.
2) Inoculation into embryonated eggs: In
embryonated eggs, viruses can be inoculated
in :
Chorioallantoic Membrane
Yolk Sac
Amniotic Sac
3) Tissue Culture: Viruses can be cultivated in
bits of tissues, organs or cell culture like
PRIMARY, DIPLOID, CONTINUOUS CELL
CULTURE

24. Isolation of Fungi
• Fungi can be isolated by growing in
Sabourad’s Glucose agar which contains:
Peptone 40g
Dextrose 20g
Sodium Chloride 10g
Agar 2g
Water 100ml at pH 5.4

25. Isolation of Protozoa
• Amoeba – Bock and Drbohlav’s diphasic
medium.
This medium contains egg slant
with sterile serum or liver extract. Sterile
rice powder is added to this before
inoculation with feces. Incubate it at
370Cfor 48 hours
• Leishmania andTrypanosomes: Novy,
Macneal and Nicolle (NNN) Medium. It
consists of 2 parts of salt and 1 part of
defibrinated rabbits blood.

26. Staining of Bacteria
Stains are dyes or reagents used for
colouring bacteria or other
microorganisms in order to observe them
clearly and specifically under the
microscope. Eg: CrystalViolet, Methylene
blue, fuschin and Safranin.
Staining in which bacteria are in the
living state is called as vital staining.
If the bacteria are killed during staining,
the process is called supravital staining.

27. • Following are the various techniques used for
staining:
1) Simple staining
2) Differential staining:
a) Gram Staining
b) Acid fast staining
c) Ziehl-Neelsen Method
d) Staining of spores
e) Staining of capsules
3) Negative staining
4) Impregnation methods

28. Simple Staining
• When the staining solution contains only one
dye dissolved in either dilute alcohol or water
then the stains are known as simple stain and
the process is known as simple staining.
• It is also known as monochrome staining.
• The dye commonly used are crystal violet,
methylene blue, fuschin and safranin.
• It is used to study the shape, size, motility and
other morphological characteristics of
microorganisms.

29. Differential Staining
•In differential staining methods
more than one dye is used.
•These techniques are used to study
the morphological characteristics of
bacterial cells, spores and capsules.

30. Gram Staining Method
This is the most commonly used technique
for differential staining of bacteria.
It is first used by Christian Gram in 1884.
All bacteria stained by this technique can be
grouped as gram positive or gram negative.
Reagents used:
Gentian violet 0.5g in 100 ml water solution
Iodine 1g and potassium Iodide 2g in 100 ml
distilled water solution
Basic Fuschin 0.1g in 10 ml alcohol and
dissolves in 100 ml of distilled water.

31. Procedure:
Prepare a thin film smear of a test bacterium on a clean
slide.
Heat fix the film by passing through bunsen flame for 2-3
minutes or dip the film in alcohol if heat is contraindicated.
Apply gentian violet stain and allow it for 1 minute.Then
wash with water.
Apply gram’s iodine solution and leave it for 1 minute.
Wash the slide with alcohol or acetone in order to
decolourise the slide for not more than 5 seconds.
Wash the slide with water.
Counter stain with dilute carbol fuschin for 1 minute and
wash the slide under tap water and examine it with in
running water.

32. •Those bacteria which cannot be
decolorized with alcohol or acetone
and retain violet colour are known as
gram positive bacteria.
•Those bacteria which are decolorized
by alcohol or acetone and stains red
due to fuschin solution are called as
Gram negative bacteria.

33. Acid Fast Stain
• This method is first developed by Paul Ehrlich
in 1882 for differentiating the microorganisms.
• In this method dyes like malachite green and
methylene blue are used.
• When the smears are treated with these dyes
and washed with acids and alcohols they are
not decolorised and retain the stain of the dye.
• Such bacteria which are not decolorised are
known as acid fast bacteria but the bacteria
which lose the stain and get decolorised are
known as non-acid fast bacteria.

34. Ziehl – Neelsen Method
• This method is used for differentiating acid
fast bacteria like mycobacterium tuberculosis
and mycobacterium leprae.
• The reagents used for ziehl-Neelsen staining
are:
Ziehl-Neelsen’s carbol Fuchsin solution
(Basic fuschin, Absolute alcohol, 5% Phenol
in water)
Sulphuric Acid 20% solution
Alcohol 95%
Counterstain methylene blue or malachite
green.

35. Procedure
• Prepare a smear of the sputum on a slide fix it by
passing through bunsen’s flame.
• Cover the slide with strong carbol fuschin in solution
and heat until steam rises. Allow it for 5 minutes.
• Wash the smear with water.
• Cover the slide with 20% sulphuric acid for one minute
and remove the excess of the acid.
• Wash the slide with water till the colour of the smear
ceases come out.
• Counterstain the slide with methylene blue or dilute
malachite green for 30 seconds.
• Wash the slide thoroughly with water, dry it and see it
under microscope.

36. The slide will appear pink
coloured, rod shaped tubercle
bacilli scattered in the film. Non-
acid fast organisms will appear as
blue or green.

37. Staining of spores
• Prepare thin smear on glass slide and heat
fix.
• Apply primary stain, malachite green and
heat fixed to steaming.
• Wash the slide under running tap water.
• Counter stain with safranin for 4o seconds.
• Wash the slide under running tap water, dry
the slide and examine under oil immersion
lens.
The spores are stained green in colour.

38. Staining of Capsules
• Prepare a thin smear of bacterial culture.
• Stain slide with 1% crystal violet solution
for one minute.
• Wash gently with 20% copper sulphate.
• Examine under oil immersion lens.
Capsular zone is seen as a clear space
between the refractile portion and dark
background of the dye.

39. Staining of Protozoa
• Wheatley trichome stain: Simpler and
quickest method.
Smear is fixed in schaudinn’s solution.
Taken successively in different
concentrations of alcohol.
It is stained with trichome stain for 5 to 10
minutes.
Differntiated in acid alcohol.
Dryed and seen under microscope
• Iron Haemotoxylin stain

40. Staining of fungi
• A drop of 95% of alcohol is applied on the
slide.
• Fungus is kept on the slide.
• Allow the alcohol evaporate.
• Then add a drop of lactophenol cotton blue
solution.
• Apply a coverslip without allowing air
bubbles to form.
• Now see the slide under the microscope.

41. REFERENCE
• Ashok K Gupta. Handbook of Health Education and
Community Pharmacy. CBS Publishers &
Distributors Pvt. Ltd. 1st Edition.