FDE 203

EXPERIMENT 1
Microscopy Techniques; Wet Mount,
Simple Staining and Negative Staining

Microscope

Basically consist of;

A light source to illuminate specimen
A condenser lens to direct the light
to the specimen
A stage to put specimen
An objective lens
to collect the light which hits the
specimen and direct it to the
detector
to magnify the image obtained
from the specimen
A detector to see the specimen

Types of Microscope

Bright
field

There are several

microscopes which
provides different
abilities and
magnifying ratios with
several modifications on
the basic principle.

Dark
field

Electron
Types of
Microscope

Fluorescent
3

Phase
Contrast

Types of Microscope

Resolution is the ability to see 2 points separately. Resolution is

best when two objects are seen as distinct even though they
are very close to each other

Brightfield Microscope
Uses light to visualize specimens The major drawback of this

The

light is focused on the

specimen by a condenser

The specimen are visualized by

using ocular lenses

The

image appears dark

against a bright background

system is the absence of

contrast between the specimen
and the background
So, most of the observations

done by brightfield microscope

are nonviable and stained.

Brightfield Microscope

Brightfield Microscope
The magnification of a microscope

depends on the type of objective

lenses used with the ocular lens
In a classical light microscope; 4X,
10X, 40X and 100X objective
lenses exist
Total magnification is calculated by
multiplying the magnification rate
of objective lens and the ocular
lens
Total
7

magnification = (objective
lens) x (ocular lens)

Darkfield Microscope
Have similar principle with brightfield microscope
In this case, the condenser is modified not to illuminate the specimen directly
The condenser directs the light obliquely so that the light is deflected or

scattered from the specimen

This provides a bright

appearance against a dark

background
Living specimen can be observed
easier with darkfield microscope
than brightfield microscope
8

Phase Contrast Microscope

This

microscope
provides A portion of the light is refracted
observation of living specimens
due to slight variations in density
without staining
and thickness of the cellular
components
The
special
objective
and
condenser make visible cellular The special optics convert the
components which differ only
difference between transmitted
slightly in their refractive index
and refracted light and provide
a significant variation in the
When the light is transmitted
intensity of light and thereby
through a specimen, its refractive
producing a discernible image of
index differs from the background
the structure under study.
9

Phase Contrast Microscope

10

Fluorescent Microscope
This

microscope is used most

frequently to visualize specimens
that are chemically tagged with a
fluorescent dye

UV light is absorbed by the

fluorescent label and the energy is
re-emitted with a different
wavelength in the visible range
and tags are seen against a dark
In this case, UV light is used as the background
source of illumination
This microscope is generally used
The ocular lens is fitted with a filter for the detection of antigenthat permits the longer UV antibody reactions
wavelengths to pass while blocking
the shorter ones
11

Fluorescent Microscope

12

Electron Microscope
This microscope provides a magnification up to 1 million X which permits

visualization of submicroscopic cellular particles and viral agents

In this microscope, electron beams are used to illuminate the specimen

instead of light
Focusing is done by electromagnets instead of optics
There are 2 types of electron microscope
Transmission Electron Microscope (TEM) intracellular structures
Scanning Electron Microscope (SEM) 3D image
13

The Aim of The Experiment

oTo learn use and care of the light microscope

oTo learn specimen preparation techniques for light

microscope
Wet mount
Simple staining

Negative staining
14

Specimen Preparation
Techniques For Light Microscope

15

Wet Mount Technique

It is the basic specimen preparation

technique
No stains or fixation are used in this
technique, so the microorganisms stay
alive.
Motility of microorganisms can be observed under light
16

microscope.
Microorganisms are observed colorless

Simple Staining
Observing microorganisms is very difficult by wet

mount technique because the lack of contrast between

the specimen and the background
So, stains are used to provide a contrast between
the specimen and the background to observe
microorganisms easily
Basic dyes such as methylene blue, crystal violet etc.
are used for staining the microorganisms
17

Simple Staining
Basic dyes with positive charge bind to negatively charged

microorganism membrane
Heat fixation is also important to fix microorganisms on the slide
during the staining procedure
This causes microorganisms to die during the process, so motility
of the microorganisms can not be observed by this technique

18

Negative Staining
This technique has the same principle with the simple staining
In this technique, acidic stains such as nigrosin are used with

negative charge to dye background and not to dye the

negatively charged microorganism membrane
This provides a bright image against dark background

19

In This Experiment???
In this experiment, two bacterial strains, Staphylococcus aureus and
Bacillus subtilis, will be observed under the Brightfield microscope
using:

20

Wet Mount Technique

Simple Staining

Negative Staining