Download microscopy technique-2

ERT107
MICROBIOLOGY
FOR BIOPROCESS
ENGINEERING
Pn Syazni Zainul Kamal
PPK Bioprocess
Chapter 2: Microscopy
Techniques
CO2 :
Ability to demonstrate practices in microscopy,
staining, sterilization, isolation and identification
of bacteria and fungi
Purpose of STAINING?
STAINING
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Microorganisms must be fixed and stained prior
examined under microscope to :
- increase visibility (increase contrast)
- accentuate specific morphological features
- preserve them for future study
Staining – coloring specimens with stain (dyes)
Preparing specimens for staining

Prior to staining, specimens need to be smear and fixed
which involve following steps :
making a thin film (smear) of the specimen on a slide
The smear is air dried
Fixation
Microorganism is killed & attached firmly to microscope
slide
(heat fixation)
passing the slide through a flame
(preserve overall morphology but not structure
within cell, bacteria, archae)
OR
(chemical fixation)
applying a chemicals (ethanol, acetic acid,
formaldehyde) to attach the specimen to the
slide
(used to protect overall morphology &
subcellular structure, larger microbes)
Preparing specimen for staining
Dyes
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make internal and external structures of cell
more visible by increasing contrast with
background

have two common features :
1) chromophore groups
 chemical groups with conjugated
double bonds
 give dye its color
2) ability to bind cells

Commonly used dyes (ionizable dyes):
1) Basic dyes
- methylene blue, basic fuchsin, crystal
violet
- have +ve charged groups, bind to –ve
charged eg nucleic acids, proteins, surface
of bacteria
2) acidic dyes
- eosin, rose bengal, acid fuchsin
- have –ve charged groups, bind to +ve
charged cell structure
Simple staining for microorganisms
1)
2)
3)
4)
Prepare bacterial fixed smear on slide
Place a slide on staining tray and flood the
smear with dyes using appropriate exposure
time
Gently wash the excess stain off with water
Blots the slide dry (do not wipe)
Simple staining to determine the size, shape,
arrangement of procaryotic cells
Differential staining
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Differential staining – a series of more than
one dye
distinguish organisms based on their staining
properties (gram staining, acid-fast staining)
Detect presence/absence of structures (flagella,
capsule, endospore)
(1)
Gram staining
- developed in 1884 by Christian Gram.
- widely used today
- divides bacteria into 2 classes ; gram positive
& gram negative
- result – gram positive (purple), gram negative
(pink)
Procedures for GRAM STAINING
(2) Acid-fast staining
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Commonly used to stain cells of the genera
Mycobacterium tuberculosis and Nocardia
(have cell wall with high lipid content)
Developed by Franz Ziehl and Friedrish
Neelsen
Ziehl-Neelsen acid fast staining
Acid-fast staining procedures :
1)
Flood the slide with the red primary stain,
carbolfuchsin, for several minutes while
warming it over steaming water.
Heat is used to drive the stain through the
waxy wall & the cell, where it remains
trapped.
2. Cool the slide
3. Decolorize the smear by rinsing it with a
solution of acid-alcohol.
*Result from acid-alcohol bleaching action:
- remove color from non acid-fast cells
- Acid-fast cells retain their red color because
the acid cannot penetrate the waxy wall.
4. Counterstain with methylene blue, which
stains only bleached, non-acid-fast cells.
5. Rinse slide with water to remove excess
methylene blue
6. Blot dry
3. Capsule staining
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Reveals the presence of capsules (made of
polysaccharides), B.anthracis
Dyes used : india ink, eosin, nigrosin (acidic
dyes)
Mix dye with cell & make a smear over the
slide
Air dried
Cell appear brighter again dark background
Acidic dyes stain the background & does not penetrate the capsule
The bacteria cell had been counterstained with basic dyes
4. Endospore staining
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Endospore - dormant, tough and temporarily nonreproductive structure produced inside the cytoplasm
by certain bacteria eg. Bacteria from genera Bacillus
and Clostridium.
Can survive over heat, extreme chemical and
desiccation
Spherical/elliptical/either smaller or larger than the
diameter of parent bacterium.
Not stained well by most dyes. Need a harsh treatment
to drive the dye into endospore
Schaeffer-Fulton
1) Smear the organism and heat fix to a slide
2) Place the slide over a steam bath and flood
with Malachite Green
3) Keep the stain over the bath for 3 - 5 minutes,
recovering the slide with Malachite Green if
some evaporates.
4) Dump the Malachite Green off and allow to
cool
5) Rinse the slide with water to remove excess
stain
6) Counterstain the smear with Safranin for 2
minutes
7) Rinse the slide with water to remove excess
stain
8) Blot dry the stain and view under a
microscope
Step Finished
Color of Vegetative
Cell
Color of Endospore
Smear
Colorless
Colorless
Malachite Green
Green
Green
Cool/Wash
Colorless
Green
Safranin
Safranin
Green
5. Flagella staining
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Provide information on the presence and distribution
pattern of flagella on procaryote cells
Procaryote flagella – fine, threadlike organelles of
locomotion that are so slender (10-30mm). Can only
be seen directly using EM
So, to observe using light microscope -increase
thickness of the flagella

Procedures :
1) coated with mordants eg. Tannic acid &
potassium alum (increase diameter)
2) stain with pararosaniline or basic fuschin
(change colour & increase contrast)
Electron Microscopy
Electron microscopy

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Light microscope resolution limit of about
0.2µm.
Limits to detailed studies of many
microorganisms.
Eg. Viruses & internal structure of
microorganisms
shortest wavelength of visible light is about 400
nm

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Electrons have wavelengths between 0.01 nm
and 0.001 nm; thus, their resolving power is
much greater, and they typically magnify
objects 10,000× to 100,000×.
EM – use beam of electron to illuminate and
create magnified images of specimen
There are two general types of electron
microscope:

Transmission Electron Microscopes (TEM)

Scanning Electron Microscopes (SEM)
TEM

A TEM generates a beam of electrons that passes
through a thinly sliced & dehydrated specimen,
through magnetic fields that manipulate and focus the
beam, and then onto a fluorescent screen that changes
the electron’s energy into visible light.

Column of the TEM must be vacuum
- electrons are deflected by collisions with air
molecules
TEM
TEM
specimen must be very thin (20 to 100nm)
(solid matter easily absorb and deflect electron)
- fixation of specimen using glutaraldehyde
- specimen need to be dehydrated
- embedded in plastic
- slice thinly
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Preparing sample for TEM
1)
fixation of specimen using chemicals eg
glutaraldehyde or osmium tetraoxide (stabilize cell
structure)
Dehydrated with organic solvent eg. Acetone or
ethanol
Specimen soaked in unpolymerized, liquid epoxy
plastic, hardened and form solid block.
Cut to a thickness of 100nm with diamond knife
mounted in a slicing machine (ultramicrotome)
Soak thin section with heavy metal eg lead citrate or
uranyl acetate (bind to cell & make it more electron
opaque, increase contrast)
Place thin section on copper grid n ready to view
2)
3)
4)
5)
6)
Other Preparation Methods
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negative stain
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heavy metals (phosphotungstic acid/ uranyl
acetate) do not penetrate the specimen but render
dark background
used for study of viruses, bacterial gas vacuoles
shadowing
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coating specimen with a thin film of a heavy metal
(platinum) only on one side
useful for viral morphology, flagella, DNA
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freeze-etching
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freeze specimen using liquid nitrogen
then fracture along lines of greatest weakness (e.g.,
membranes)
Shadowed & coated using platinum & carbon
allows for 3-D observation of shapes of
intracellular structures
SEM
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uses electrons reflected from the surface of a
specimen to create detailed image
produces a realistic 3-dimensional image of
specimen’s surface features
SEM use magnetic lenses to focus a beam of primary
electrons
Primary electrons are scanned across the metal-coated
surface of a specimen – produced secondary electrons
Secondary electrons are collected by a detector &
their signal amplified and displayed on a monitor
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1)
2)
3)
4)
Preparing sample for SEM
Fixed specimen using chemicals
Dehydrated using aseton & ethanol
Dried sample in critical point drying for
24hrs (to preserve surface structure and
prevent collapse of the cells)
Mounted and coated specimen with a thin
layer of metal eg platinum or gold
SEM or TEM?
ISOLATION
Isolation of microorganisms
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Natural habitat, microorganism grow in complex,
mixed populations with many spp.
Need a pure culture to study and characterize an
individual species
Pure culture =contain one type of microorganisms
Techniques to prepare pure culture:
-spread plate
-streak plate
-Pour plate
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Spread plate
Small volume of diluted microbial mixture
(30-300 cells) transferred to the center of agar
plate.
Spread evenly over the surface with sterile
bent-glass rod.
The dispersed cells develop into isolated
colonies
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Pour plate
Original sample is diluted several times –
reduce microbial population
Small vol. of diluted sample mixed with
melted agar
After agar had hardened, each cell is fixed in
place and form individual colony
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Streak plate
An inoculum is spread across the surface of an
agar plate in a sequential pattern of streak (as
indicated by the numbers n arrows)
The loop is sterilized between streaks
In streak 2,3 and 4, bacteria are picked up from
previous streak, diluting the number of cells
each time
These develop into separate colonies
Microbial Growth on Solid
Surfaces
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colony characteristics that develop when
microorganisms are grown on agar surfaces aid
in identification
Colony – a macroscopically visible cluster of
microorganisms
differences in growth rate from edges to center
is due to
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oxygen, nutrients, and toxic products
cells may be dead in some areas
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1. Form – The form refers to the shape of
the colony.
2. Elevation – This describes the “side view”
of a colony.
3. Margin – The margin or edge of a colony
(or any growth) may be an important
characterisic in identifying an organisms.