Microbiology, Classification of Microbiology, Bacteria, Spores, Fungi, Viruses, Method of isolation of pure cultures, Staining techniques

1. Microbiology
Mr. Dipak B. Bari
KES’s College of Pharmacy,
Amalner

2. Microbiology: It is the study of living
organism that are microscopic in size.
Medical Microbiology: It is the study
of microscopic organism that infect
man, his reaction to such infections,
the way in which the disease is
produced & the method of diagnosis,
prevention & treatment of such
infectious disease.

3. Micro-organism: The causative agents are
very minute in size & not visible to naked
eyes is known as Micro-organisms. They are:
Bacteria, algae, fungi, rickettsias,
mycoplasms, viruses & protozoas.

4. History
Scientist Stated
In 1683 Antony Van
Leeuwenhock,
First accurately observed and described about various types of
bacteria.
In 1762 Plenicz Suggested that each disease was caused by a separate agent.
Jenner (1796) The technique of vaccination using cow pox material.
Louis Pasteur (1822-95) Who suggested that different fermentations were associated with
the activity of different kind of bacteria.
Ellerman and Bang. The role of virus infection in malignancy was isolate in 1908
Twort (1915) and
d'Herelle (1917)
Independently discovered a lytic phenomenon in bacterial culture.
In 1929 Fleming Discovered penicillin & this was the beginning of antibiotic era.
Domagk (1935) Started scientific chemotherapy with the discovery of protonsil.

5. • Microorganisms are a heterogeneous group of several
distinct classes of living beings.
• They are classified under the kingdom of protista which
is different than animal or plant kingdom.
• Depending on the cellular organisation and biochemistry,
the kingdom protista is divided into two groups —
prokaryote's and eukaryotes.
CLASSIFICATION OF MICROORGANISMS

6. Classification
Micro-organism
Having cellular organisation
Prokaryotes Eukaryotes
Having no cellular organisation
Viruses, Viroids,
prion
Bacteria, blue green
algae
Fungi, algae,
moulds, protozoa

8. BACTERIA
A bacterium is a unicellular prokaryotic
microorganism which does not contain
chlorophyll and does not multiply by true
branching (Except in the higher bacteria
such as Actinomycetes).
The unit of measurement used in
microbiology is micron (micro meter)

9. Shape of Bacteria
•Spirilla are rigid spiral shaped
bacteria.
•Spirochaetes are spiral
shaped cells look like coiled
hairs.
•Cocci are oval or spherical bacterial cells.
•Bacilli are rod shaped cells.
•Vibrios are comma shaped curved rods. These bacteria possess characteristic
vibratory motility.

10. • Actinomycetes are branching
filamentous bacteria. In the tissue
lesions they appear as if radiating
rays.
• Mycoplasm are bacteria that do not
have a cell wall & therefore their
morphology is not suitable. They
occur as round or oval bodies.

11. Arrangement of Bacteria
1. Bacteria arranged in group.
– Diplococci: Cocci arranged in pair. It is a round bacterium that typically
occurs in the form of two joined cells.
– Streptococci: Bacterium observed in chain form.
– Tetrade: It is a group of four.
– Sarcina: Bacterium (Cocci) observed in group of eight.
– Staphylococci: It is observed as a bunch of grapes.
2. Bacteria arranged in typical arrangement.
– Cluster: They arranged in chain.
– Diplobacilli: Group of two chain.

12. Anatomy of Bacteria
Capsule: slimy layer, consisting
of polysaccharide and water
surrounding many cells. Also
called slime coat, extracellular
layer, etc.
Cell Wall: rigid layer
surrounding the bacterial cell.
Made of peptidoglyan.

14. Anatomy of Bacteria
Cell Membrane: flexible, semi-
permeable barrier with lipid center
that controls diffusion in and out of
cell.
Cytoplasm: the fluid-filled space
inside the cell. Contains hundreds of
different enzymes, along with
ribosomes, DNA, RNA, and a "pool"
of millions of small molecules and
ions.

15. Anatomy of Bacteria
Ribosomes: particles made of
protein and RNA, sites of
protein assembly. Ribosomes may
occupy 25% of the volume of a
typical bacterial cell.
A plasmid: is a small,
extrachromosomal DNA molecule
within a cell that is physically
separated from chromosomal
DNA and can replicate
independently.

16. Anatomy of Bacteria
Cell Chromosome: The DNA
of a cell, normally a single
circular molecule that is
tightly supercoiled and packed
inside the cell. Actively
dividing cells may contain 2 or
even 4 copies of this
chromosome, replicated and
ready for dividing up among
future daughter cells.

17. Anatomy of Bacteria
Flagella: are microscopic hair-
like structures involved in the
locomotion of a cell.
Pili are shorter and thinner
than flagella.

18. SPORES
 Some bacteria (Bacilli and Clostridium) have the ability to form highly resistant resting
stages called spores.
 In bacteria spore formation is a method of survival but not for reproduction.
 A single spore is formed by one bacterium and one spore gives a single bacterial cell on
germination.
 They formed within bacteria so called as Endospores.
 They are resistance to chemical & heat. They are destroyed by autoclaving at 1200 c for 15
min.
 Sporulation helps bacteria to survive for long period under unfavorable environment.
 A fully developed spore is oval or spherical in shape & attached to parent cell.

19. Disease produced by Bacteria
Name of the Bacteria Diseases produced
Staphylococci Localized suppurative lesion
Streptococci Rheumatic fever
Pneumococci Pneumonia, bronchitis & Meningitis.
Meningococci Meningitis or Cerebrospinal fever
Gonococci Gonorrhea
Corynebacterium diphtheria Diphtheria
Bacillus anthracis Anthrax
Clostridium tetani Tetanus
E. Coli UTI, gastroenteritis

20. Fungi
• Fungi are eukaryotic protists. They have rigid cell wall containing
polysachrides (chitin).
• Cytoplasmic membarane of fungi contains sterols.
• Their nuclei contain nuclear membrane & paired chromosomes.
• They multiply by sexually, asexually or by both mechanism.
• They can be unicellular or multicellular. Elongation of the fungal cell
produces a thin cellular structure called hypha.
• A tangled mass of hyphae constitute the mycelium.
• The fungi which produces mycelia are called Filamentous fungi or
Moulds.

21. Schematic diagram of fungi

22. Type of Fungi
Yeast
They are unicellular fungi. Their cells are spherical or ellipsoidal in
shape, they multiply by budding. Ex. Cryptococcus neoformans.
1
2
3
4
5
Yeast like fungi
They partly grow as yeast & partly as elongated cell like hyphae
called psuedomycellium. Ex. Candida albicans.
Moulds
They are also called as Filamentous fungi. They multiply by formation
of spores. Ex. Micosporum, Trichophyton, & Epidermophyton.
Dimorphic fungi
They occur as filaments or yeast. Ex. Blastomyces dermatitidis,

23. Systematic classification of fungi
They are classified according to their sexual spore formation as:
1. Lower fungi
1. Phycomycetes
2. Higher fungi
1. Ascomycetes
2. Basidiomycetes
3. Fungi imperfecti (Deuteromucetes/Hyphomycetes)

24. Phycomycetes
1. These are lower fungi.
2. These fungi have non-separated
hyphae.
3. They form endogenous asexual spores
called sporangiospores which are
contained in a sac like structure
known as sporangia.
4. Some phycomycetes also produce
sexual spores called Oospores in some
fungi and Zygospores in other ones.

25. The other three classes of fungi i.e.
Ascomycetes, Basidiomycetes and fungi
imperfecti are higher fungi.
1. These fungi have separated hyphae.
2. They form exogenous asexual spores called
Conidia.
Higher fungi

26. • Ascomycet:
These fungi form sexual spores
called ascospores within a sac
or ascus.
Yeasts and filamentous fungi
are included in this class.

27. • Basidiomycetes:
These fungi form sexual spores
known as bisidiospores on a
basidium or base.

28. • Fungi imperfecti:
This class consisting of
fungi whose sexual
phases have not been
identified.
Most of the fungi of
medical importance
belong to this class.

29. Laboratory Diagnosis of Fungi
 The diagnosis of fungal infections is made by morphological
study of the material taken from the lesion, under a
microscope.
 The most common specimen i.e. skin scrapings are treated with
10% potassium hydroxide and then are examined under
microscope as wet mount.
 Small bits of fungus colonies may be taken on a slide and
mounted in lacto-phenol cotton blue for microscopic study.
 Slide culture technique is very useful for the morphological
study of the fungus. The periodic acid Schiff (PAS) and
methanamine silver strains are valuable methods for the
demonstration of fungal elements in tissue sections.

30. Culture of Fungus
The commonest culture media for the fungi are:
1. Sabouraud's glucose agar
2. Czapek—Dox medium and
3. Cornmeal agar
To prevent bacterial contamination, antibiotics are
added in the media. Similarly cycloheximide (actidione)
incorporation in the medium inhibits many contaminant
moulds.
Cultures are routinely incubated in parallel at room
temperature (22°C) for weeks or at 37°C for days.
Identification of the fungus is based on its morphology
and colony.

31. Growth Characters
• Growth characters, useful for identification are the rapidity of
growth, morphology as well as the colour and pigmentation of the
colony.
• The morphology of hyphae, spores etc. is studied on slide cultures.
• The diameter of hyphae, presence or absence of septa and other
special structures are also helpful in the diagnosis.

32. Commonly seen hyphae on the basis of their
structures are:
1. Spiral hyphae: They are spring like helical coils.
2. Racquet hyphae: These localised swellings are
formed by twisted hyphae and look like tennis
racquets.
3. Favic Chandlier hyphae: In these types of
hyphae numerous short branches appear at the
end of the hyphae.

33. The morphology of the asexual spores is also of diagnostic value.
1. Microconidia are small single celled spores.
2. Macroconidia are large, single or multicellular spores.
3. Blastospores are formed by budding as in yeast.
4. Arthrospores are formed along the mycelium by segmentation and
condensation of hyphae.
5. Chlamydospores are thick walled resting spores formed by rounding up
and thickening of the segments of the hyphae.

34. Fungal infection (Mycoses)
Superficial fungal infections:
Superficial fungal infections are the common infections. The fungi
causing the superficial (but chronic) diseases are specialised
saprophytes which can digest keratin of the skin including nails and
hairs.
Superficial mycoses comprise the various types of tinea and
ringworm affecting the skin, hair and nails.
The causative fungi of superficial infections are dermatophytes,
candida albicans, pityrosporum orbiculare, cladosporium, and
dermatophytes.

35. Fungal infection (Mycoses)
Subcutaneous fungal infections:
(Subcutaneous mycosis) are
caused by fungi such as
rhinospordium, fonsecaea,
madurella nocardia, sporotrichum
etc.

36. Deep seated fungal infections (Systemic mycoses):
Mycoses are caused by fungi, which are mostly soil saprophytes. Systemic mycoses vary in
severity ranging from asymptomatic infections to fatal diseases.
The causative agents of systemic mycoses include actinomycetes (produces Madura foot),
rhinosporidium seeberi (produce rhinosporidiosis), crypto-coccus neoformans (produce
cryptococcosis), dimorphic fungi-blastomyces dermatitidis (produce blastomycoses) and
histoplasma capsulatum (produce histoplasmoses).

37. Opportunistic fungal infections:
 These infections occur in patients suffering from debilitating diseases such
as cancer, AIDS or diabetes.
 The use of drug s/a immunosuppressive agents, steroids, broad spectrum
antibiotics & x-ray exposure make the body vulnerable to these infections.
 The opportunistic infection caused by mucor, penicillium, rhizopus &
aspegillus.

38. Mycotic poisoning
Many fungi can produce poisoning. It can be of two types mycetism &
mycotoxicosis.
1. Mycetism: In this type, a fungus which is eaten, produce toxic effects. Ex.
Mushroom produce GIT disturbances, dermatitis or even death.
2. Mycotoxicosis: In this type poison produced by fungi itself & contaminate
food material & produce their toxic effects. Ex. Aflataxin which produced by
Aspergillus flavus presents in mouldy food s/a ground nuts, corn & peas. It
will produce carcinogenic effect in body.

39. Viruses
• These are much smaller than bacteria.
• They do not have cellular organisation.
• They contain only one type of nucleic acid either RNA or DNA.
• They do not contain enzymes which are necessary for synthesis of proteins &
nucleic acids.
• They are dependent on the host cells for replication.
• They are obligate intracellular parasites and are not affected by usual
antibiotics.
• The intracellular infectious virus particle is called VIRION.

40. Size:
They are vary in sizes. The largest virus s/a
poxvirus is about 300 nm in size. While smallest
viruses that causing mouth & food diseases are
about 20 nm in sizes.

41. Shape & structure
• Viruses are vary in shape.
• Most animal viruses are spherical.
• The rabies virus is bullet shape,
• The pox virus is brick shaped,
• And tobacco virus is rod shaped.
• Bacteria viruses are having a complex
morphology.

42. VIRIONS
• The virion consists of a nucleic acid. Core
surrounded by a protein covering called
capsid. The capsid together with the
nucleic acid is called the nucleocapsid.
• The capsid is formed by a large number
of capsomeres made up of polypeptide
molecules. The main function of capsid is
to introduce viral genome into host cells
by adsorbing readily to cell surfaces.

43. VIRIONS
• Virons may be enveloped or non-enveloped
(naked). The envelope or outer covering of
viruses is derived from the host cell membrane
at the time of budding of the virus and consists
of lipoprotein.
• The protein subunits projecting like spikes on
the surface of the envelope are called peplomer.
• A virus may have more than one type of
peplomer. Envelopes confer chemical, antigenic
and biological properties on viruses.

44. Classification of viruses
Viruses are classified into two main groups depending on
the presence of nucleic acid in them.
1.The Deoxyriboviruses (DNA Viruses)
2.The Riboviruses (RNA Viruses)

45. The Deoxyriboviruses or DNA viruses:
• These viruses contain DNA in them.
• The examples of these viruses are
poxvirus, Herpes virus, Adenovirus,
Papilloma virus, Polyoma virus, Parvovirus,
Human hepatitis type B virus and related
viruses.

46. The Riboviruses or RNA viruses:
These viruses contain RNA in them.
The RNA viruses include Enteroviruses (Polio and
Coxasackie virus), Rhinovirus, Influenza virus,
mumps virus, para influenza virus, measles virus,
pneumovirus, rubella virus, cholera virus, bunya
virus, chandipura virus, rabies virus, reovirus,
tongue virus, rota virus, corona virus and Retrovirus
(AIDS virus or HIV).

47. ISOLATION OF MICROORGANISMS
To arrive at a proper diagnosis of various infectious
diseases it is necessary to know the type and nature
of the causative microorganism.
For this purpose specimen of the possible infected
material are collected.
Mostly these specimens sent to laboratory are swabs
(from infected area), pus, sputum, urine, stool, blood,
cerebrospinal fluids, pleural fluids, peritoneal fluid,
and aspiration material.

48. • The specimen should be collected before starting the
antimicrobial treatment otherwise the specimen may
become sterile.
• The material should be collected from the site most
likely to be infected by the suspected microorganisms.
– For example in respiratory tract infection sputum should
be examined.
– In diarrhea stool should be examined.
– Similarly pus from suppurative lesion; swab from infected
areas (nose, throat or conjunctiva) and fresh discharge
from urethra are to be examined.
– Similarly cerebrospinal fluid (CSF) in meningitis and blood
in enteric fever is studied for the presence of
microorganisms.

49. • Stage of disease is also an important factor for collecting specimen. For example 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 of the sample is another factor contributing to successful
isolation of the causative agent.
• For example in urinary tract infection the first morning sample of urine is the best
for culture. Similarly sputum and conjunctival swabs should be collected in the
morning.
Stages of Disease & Timing of Specimen Collection:

50. Containers for specimen collection
• The specimens should be collected in sufficient quantity in sterilized 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 the sterilized test tube immediately after taking the sample.
• Sputum should be collected in petridish.
• CSF is collected in sterilized vials.
• Similarly blood for culture is collected in blood culture bottle.
• After collection the specimens should be delivered to laboratory without any delay to
avoid the overgrowth of commensal organisms. Sufficient clinical informations should
always be given with the specimens.

51. Methods of Isolating Pure Cultures
Streak Plates Method
 A plate of solid medium (nutrient agar) is allowed to dry in an incubator for
about 30 min to dry the surface.
 Then by using bent wire which has been sterilized by heating directly on the
flame, is dipped in an inoculum.
 With this wire the inoculum is streaked across the surface of the agar medium
so that individual cells become separated from each other.
 These plates are incubated at 37°c for about 18-24 hrs, after which individual
colonies can be observed on the agar surface.

53. • Spread Plates Method
A drop of diluted sample of culture specimen is placed on the
surface of an agar medium, and this drop is spread over the
entire surface using a sterile bent glass rod.
These plates are incubated at 37°c for about 18-24 hrs, after
which individual colonies can be observed on the agar surface.

55. Pour Plates Method:
In this method the initial suspension of the culture is diluted to a
concentration of about 100 microbes/cm3.
This diluted specimen (1ml) is pipetted out in the empty petridishes and
mixed with nutrient agar by moving gently in the directions as shown in the
figure.
The temperature of agar is not allowed to exceed 45°c to avoid damage to
the microorganisms. After solidification the plates are incubated.
In this procedure the colonies will grow both on and below the surface,
because some of the cells are trapped within the agar medium when it
solidifies.

57. Single cell isolation by Micromanipulator:
i) The hanging drop preparation of specimen is prepared.
ii) The needle of micromanipulator is inserted in hanging drop.
iii) By pointed tip of needle of micromanipulator single isolated cell
of bacterium is lifted and taken out.
iv) Needle tip with isolated single cell is dipped in sterile growth
medium and this tube is incubated at appropriate incubation
conditions.

58. Single cell isolation by Micromanipulator

59. Staining techniques
The simple stain can be used as a quick and easy way to determine cell shape,
size, and arrangements of bacteria.
Since the surface of most bacterial cells and cytoplasm is negatively charged,
these positively charged stains adhere readily to the cell surface.
The principle of simple staining is based on the principle of producing a marked
contrast between the organism and its surrounding, by the use of basic stain.
Procedure:
Smear is fixed, stain is put, stain is allowed to react for 30 sec to 3 min, wash smear
with stream of cool water, dry and examine under oil immersion lens
It is used to study morphology, size, shape of microbes

60. Differential Staining:
In this method more than one stain is employed. In some method the
stains are applied separately, while in other method they are mixed and
applied in one application.
These procedures show differences between the cells or parts of a cell
and can be used for of identification.
The two most important differential stains used by bacteriologists are
Gram stain and Acid Fast stain.

61. 1. Gram’s Staining Method
The Gram’s stain technique was developed by Danish
Bacteriologist Hans Christian Gram in 1884.
It is one of the most useful staining methods
because it classifies bacteria into two large groups
namely Gram positive and Gram negative.
The organisms that retain the colour of the primary
stain are called Gram positive and those that do not
retain the primary stain when decolorised and take
on the colour of the counter stain are called Gram
negative.

62. 2. Acid fast
It is the differential staining techniques which was first developed by
Ziehl and later on modified by Neelsen.
So this method is also called Ziehl-Neelsen staining techniques.
The Ziehl-Neelsen stain is a type of differential bacteriological staining
method used to identify acid-fast organisms, mainly Mycobacteria
tuberculosis and M. Lepra

63. 1. Prepare a smear and add carbol fuchsin
2. Wash and add 20% sulfuric acid,
3. Wash and add methylene blue as counter stain.
4. Wash, dry and observe under microscope.
5. Acid fast bacteria- appear pink or red
6. Non acid fast bacteria appears- blue, green

64. Staining of spirochetes
 They are stain by Fonanta method.
 Ammonical silver nitrate stain is
used which increase the apparent
dimension of the spirochetes &
these microorganism appear as
opaque black spiral hairs against
light back round.

65. Staining of Ricketsiae
• They are gram negative, but
they does no stained well with
grams stain.
• They are stained well with
Giensa or Casteneda method

66. Staining of yeast & fungi
• Fixed smear of yeast can be stained with crystal violet or methylene
blue.
• Put to set 30 seconds.
• Put a drop on a slide.
• Cover with the cover glass.
• Lactophenol cotton blue is the excellent for staining fungi.