Bacterial

identification
Microscopy
 Types of Microscopes
 Light-field microscope – uses ordinary (visible)
light as its light source
- can view living and non living objects
 Dark-field microscope – uses visible light as its
source but the ordinary condenser is changed to
a darkfield condenser which provides a dark
background, objects viewed appear brightly
illuminated against a dark background
- used to observe spirochetes such as
Treponema pallidum
 Fluorescent Microscope – uses UV
light to illuminate the object but does
not pass the objectives, uses
fluorescent dye (auramine)
- when this light strikes the object it
emits a certain kind of light (neon)
 Phase Contrast microscope – used to observe living
microbes without staining
 Electron microscope – uses an electron beam as a
source of illumination and magnets instead of lenses
to focus the beam
- the specimen undergoes lyophilization (freeze-dry
technique) and is mounted in resin, the image is seen
on a fluorescent screen
- makes use of metallic stains like phosphotungsten
dye
- 1)Transmission EM – 2 dimensional image
2) Scanning EM – 3 dimensional image
Light field
Dark field
Transmission EM
Scanning EM
Microscope
 Parts of a compound
microscope
 Objectives– lenses that are used to
enlarge the object
1) Ocular or Eyepiece
2) Scanner (4x or 5x)
3) Low Power Objective (10x)
4) High Power Objective (40x)
5) Oil Immersion Objective (100x)
 Body tube
 Draw tube
 Dust shield
 Revolving nosepiece
 Arm
 Stage
 Stage clips
 Irisdiaphragm
 Condenser
 Mirror
 Coarse adjustment
 Fine adjustment
 Base
 Inclination joint
Staining
 Simple staining
 Differential staining
 Special staining
 Differential Staining
1) Gram Staining – uses the following
a) crystal violet
b) Gram’s iodine
c) ethyl alcohol
d) safranin
2) Acid Fast staining
a) Carbolfuschin
b) acid alcohol
c) methylene blue
 Zielnielsen
 Kinyoun
Gram Positive
bacteria
Corynebacterium
diphtheriae
Clostridium tetani
Staphylococcus aureus
Streptococcus
pneumoniae
Streptococcus pyogenes
Gram Negative
Bacteria
Bacteroides fragilis
Escherichia coli
 Special staining
 Cell wall
– dyar
 Capsule
– Tyler, Hiss, Anthony
 Spore staining
– Heat and acetic acid
– Schaeffer and fulton
– Dorner’s
 Flagella
– Gray’s, leiffson’s
 Metachromatic granules
– Neisser’s , Albert
Cultural method
 Nutritional requirements
 Lithothrophs
( autotrophs )
 Organothrophs ( heterotrophs )
 Oxygen requirements
 Aerobes
– Obligate
– facultative
 Anaerobe
– Obligate
– facultative
 microaerophillic
 Thermal requirement
 Cryophillic
 Mesophillic
 Thermophillic
 Thermoduric
 pH requirement
 Acidophillic
 basophillic
 Characteristics of culture
media
 Pure
 Mixed
 stock
Classification ( consistency)
 Liquid
– BHI, NA, thioglycolate
 Semi solid
– SIM
 Solid
– BAP, CAP, EMB
 Classification
( composition )
 Synthetic
 Non synthetic
 Classification ( Use )
 Simple
– NA/broth, BHI
 Enrichment
– BAP, CAP
 Differential
– BAP
 Selective
– EMB, SSA
 Special
– Thayer martin, Petragnani
 Bacterial growth curve
 Lag
 Logarithmic
 Stationary
 Decline phase
 Biochemical reaction
 CHO fermentation
 Catalase reaction
 Oxidase reaction
 Hydrolysis of urea
 Nitrate reduction
 Liquefaction of gelatin
 Serological identification
 Widal
 Weil felix
 TPHA
 VDRL
 FTA-ABS
 HI
 ASOT
 FAT
 CFT
 Animal inoculation