Transcript Red rose template

Bacteriology
revision
Nada Mohamed Ahmed ,
MT (ASCP)i
Types of Microscopes
Types of
Microscopes
Function
1. Compound Light study the cells that
Microscope
are between 1 and
100 µm.
2-Stereoscopes –
also known as
dissecting scopes
3. Electron
Microscopes
Gives a three
dimensional view
(3D) of an object
revealed many
organisms and sub
cellular structure
that were
impossible to
resolve with the
light microscope
Used for
(what we use
most often)
Bacterial
morphology
insects and leaves
Mitochondria ,golgi
apparatus
Parts of microscope:
Parts of
microscope
Eyepiece (ocular)
B-Body tube
(Head)
Stage clips
Nosepiece
Function
The lens the viewer looks through to see
the specimen. The eyepiece usually
contains a 10X or 15X power lens.
The body tube connects the eyepiece to
the objective lenses
Metal clips that hold the slide in place.
: A rotating piece that holds the objective
lenses
Objective lenses:
Arm(neck)
Stage
diaphragm:
Stage Control
One of the most important parts of a
compound microscope, as they are the
lenses closest to the specimen.
The arm connects the body tube to the
base of the microscope. Used to carry
The flat platform where the slide is placed.
Adjusts the amount of light that reaches
the specimen
These knobs move the stage left and right
or up and down.
Coarseadjustment- Brings the specimen into general focus.
Fine adjustment:
K-Fine adjustment: Fine tunes the focus
and increases the detail of the specimen.
Light source:
The hole in the middle of the stage that
allows light from the illuminator to reach
the specimen.
Base
The base supports the microscope used
to carry microscope
Types of culture media Based on
their consistency
media
Example
Solid media – contains
2% agar
•
Semi solid medium –
0.5% agar.
Motility medium
Liquid media – no
agar.
Nutrient broth
Nutrient agar, Blood
agar
Picture
Special media
Special media
Enriched media
Enrichment
media Media.
Liquid media
Substances contained
Example
Substances like blood,
serum, egg are added
to the basal medium
Blood agar,
is incorporated with
inhibitory substances to
suppress the unwanted
organism
Selenite F Broth
Alkaline Peptone Water
Chocolate agar
Selective media
solid media
The inhibitory substance is added
to a solid media.
Indicator media
These media contain an indicator
which changes its colour when a
bacterium grows in them.
Differential media
media which has substances
incorporated in it enabling it to
distinguish between bacteria.
Sugar media
Media containing any fermentable
substance.
Contain a small tube (Durham’s
tube) for the detection of gas by
the
bacteria.
Transport media
non nutrient soft agar gel
Media used for transporting the samples containing a reducing agent
Mac Conkey’s medium for gram
negative bacteria
TCBS – for V.cholerae
L J medium –
M.tuberculosis
Wilson and Blair medium –
S.typhi
Potassium tellurite medium –
Diphtheria bacilli
Blood agar
Mac Conkey’s medium
Christensen’s urease medium
Mac Conkey’s medium
Distinguish between lactose
fermenters & non lactose
fermenters.
Usual Sugar media
Stuart’s medium
Special
media
Enriched
media
Picture
Blood agar
Chocolate
agar
Enrichmen Selenite F
t media
Broth
Selective
media
Mac Conkey’s
medium
Indicator
media
Christensen’s
urease medium
Differentia Mac Conkey’s
l media
medium
Sugar
media
Usual Sugar
media
Transport Stuart’s
media
medium
Classification of stain (Based on function of stain:
Stain
Function
Example
Simple staining
To study morphology and
arrangement of bacteria.
only one dye is useddifferentiation among
bacteria is impossible
more than one dye is usedDifferentiation among
bacteria is possible
it provides more information
about the characteristics of
the cell wall (Thickness).
Crystal violet
, Methylene blue
, Basic fuschin,
Malachite green
More than one dye used Special structures are seen.
Capsule staining,
Spore staining.
Differential staining
Special staining –
more than one dye
used -Special
structures are seen.
Gram’s staining
, Acid-fast
staining.
Basic requirements for staining
Requirements
Function
Clean grease-free slide.
To hold the smear
Bacteria to be stained
Inoculating loops(wire loop - to transfer bacterial
Platinum loop)suspension to slide
Bunsen burner –
To sterilize inoculating loops
before and after smear
preparation.
Pencil marker – to mark
to mark
Picture
REAGENTS USED IN GRAM STAIN
Reagents
Function
Time
CRYSTAL VIOLET
• Primary stain
Violet colored, stains
all microorganisms
• Mordant
• Forms Crystal
violet iodine
complexes
30-60 sec
3. DECOLORIZER
• Acetone +
Methanol
•
Removes Crystal
violet iodine
complex from thin
peptidoglycan
layers
10-15 sec
4.Gram safranin;
•
•
•
Counter stain
Red colored
Stains thin walled
Gram negative
organism.
2 min
2. GRAM IODINE
bacteria.30-60 sec
Reagents used in Ziehl- Neelsen
Reagents
Function
Carbol Fuchsin stain
a lipid soluble, phenolic 10 minutes
compound,
which is able to
penetrate the cell wall
decolorizer
leave 15 seconds
acid alcohol
Loeffler’s Methylene
Blue stain
(Counter stain). This
stain adds blue color to
non-acid fast cells.
Time
1 minute
Staining principle
Staining
principle
Basic stain(+ve charge) –
To stain -ve charged molecules of bacteria
Mostly used because cell surface is –ve charge
As cell surface is –ve charged- Basic dyes mostly used.
SIMPLE STAINING
All bacteria in smear take stain and appear in colour of stain.
Capsule stain
Negative Staining
Capsule is not stain by Indian ink or Nigrossin instead the
background is stain dark (black)
Background leaving the capsule as unstained area (with
hollow) colorless area .
•
Gram staining is used to determine gram status to classify
bacteria broadly. It is based on the composition of their
cell wall
Mycobacteria, which do not stain well by Gram stain
, are stained with carbol fuchsin combined with
phenol. In the ‘hot’ Zn technique, the phenol-carbol
fuchsin stain is heated to enable the dye to
penetrate the waxy mycobacterial cell wall.
Gram staining
Ziehl-Neelsen stain