PLAN

1) Microscopy
2) Cytologic & Histologic technics
3) Cell culture

⚠️ Things to know before the Exam:

1) MICROSCOPY

Light microscope (original):

- used in medicine to analyze biopsy and complete diagnosis

- study cell structure ( membrane - cytoplasm - nucleus )
- light microscope : 3 components: basement - handle - optic tube:
1 light source : lamp, illuminate the image from below
3 glass lenses:
- Condenser: focalize the lightning on the specimen
- objective: magnify image: 4x / 10 x / 100 x
- ocular: magnify image 10 x

Epifluorescent microscope: it is an original microscope with an UV lamp at the top

( fluorescent labeled molecules )
Confocal microscope: an improved fluorescent microscope ( the image is better without
background noise )) Instead of UV lamp: laser, pinhole, and photomultiplicateur.

Reversed or inverted microscope: the objective is under the stage and lightning in the top (it
shows us a black and white image ) + (transform the amplitude differences of the cell
structures into different phases). Ex: cellular divisions.

● The coloration of a cell can lead to its death. That's why we can use the reversed
microscope.

Electron microscope (TEM) : shows ultrastructure or organelles: magnify image 1 000 000 x
=> sans couleur

3 differences with the LM:

- Electron beam source instead of photo lamp.
- 3 magnetic bands: condenser, objective, ocular.
- vacuum pump: prevent electron deviation by magnetic fields.

Scanning electron microscope (SEM): For cell topography ( 3D images ).

- Electron beam
- electron detector
- TV screen

2) CYTOLOGIC & HISTOLOGIC TECHNICS

Biopsy Processing for LM

2 types of processing for different types of tissues:

Cytology: Concerns free cells like blood, bone marrow, vaginal squamous cells, ascites fluid,
cerebrospinal fluid, etc.

The technique (is the same for all the cytology tissues):

- Put sampling blood (drop) on the slide

- - spread the sample on the slide
- - by shaking the slide (air drying)
- - stain with MGG (May Grunwald Giemsa) 1min /10min) : the stain kills the cells.
 For vaginal smear there is a better stain that MGG =
papanicolaou

For oral smear there is a better stain that MGG ( but

MGG can be used) = Toluidine blue

To have a more concentrated amount of cells you can put the sample in a centrifuge: ex:
PRP => Plasma riche en plaquettes

Histological processing : (liver, skin, muscle, bone, so on....) Require thin sections before
microscopic observation

1- Carottage
2- Endoscopy
3- Removed organ
4- Sectioning the tissue
The 4 main steps of histological processing: Fixation – Embedding ( Inclusion ) –
Sectioning ( microtomy ) – Staining.

Fixation: Preserve the tissue in a life-like ( approximately in the state it was in the body ) +
You must be fast to avoid cell damage.

BOIN MIXTURE

➔ 2 types of fixers:

- Chemical fixers ( ex: alcohol, acids, FORMALIN…) But in medicine, we use the
Bouin mixture.

- Physical fixers: liquid nitrogen and dry ice.

First, we start with the chemical fixers, and then we use the physical fixers.

Inclusion: Makes tissue hard to facilitate thin sections + Changes the intracell water solid
substance -> paraffin = dehydration with an automaton.

⚠️ During freeze fixation, no inclusion because the sample becomes hard.

Sectioning:

We use a machine named microtome

Cryostat is a microtome inside a freezer. A Revoir!!!

• Thin sections: 3-4μm

• Allow photons to cross tissue
• Microtome (or cryostat for frozen tissue) with stainless steel knife
• Sections are extended and stick on slides with gelatinous or albuminous water overheating
plate.

● Rehydratation step before the standing: reintroduce intracell water.

Staining: Contraste
Staining for diagnosis in medicine

Standard stains:

Hematein-eosine (HE)

Hematein-eosine safranine (HES)

Masson’s Trichrome
Special stains : ( stain 1 molecule and detect 1 disease )

Pearl stain: iron spots in liver ( hemosiderosis

disease )

PAS ( periodic Acid Schiff ) stain: glycogen in

undifferentiated tumors ( glandular origin )

Red oil staining ( red lipids ) : parathyroid pathology,

fatty vacuoles in adenomas.

TEM Histological Techniques (like LM techniques with a few differences)

Fixation: with glutaraldehyde ( proteins ) and osmium tetroxide (04S4) ( lipids )

Inclusion: Synthetic resins, harder than paraffin and transparent to electron ( Epoxy resins,
Araldite)

Section: ultra-thin section ( 50-80nm), ultramicrotome with a glass or diamond knife,

sections metallic grids.

Contrasting: heavy metal salts ( osmium, uranium) dense to electrons

(Images in black and white: chromatin in black, cytoplasm in grey or white.)

Medical using:

1) Karyotype: individual collection chromosomes

2) Therapeutic effects of drugs: Marketing authorization for medicine ( in vitro essay
animal essay and human clinical essay )
3) AMP ( Assisted Medical Procreation ): In vitro fertilization
4) VACCINATION

Culture conditions :

- Temperature: +37 degrees

- PH: 7 to 7.4 (neutral)
- Sterility: laminar flow hood, germicidal UV lamps in the room.
- Culture vessels: plastic ( single use ), Petri dishes, flasks
tube, and so on…
- Culture Mediums: solid or liquid ( MEM, RPMI1640)
- Water, mineral salts, vitamins,
- Culture media composition: amino acids or proteins, glucose,
antibiotics, fetal calf serum (FCS) and growth factors.

Main steps of cell culture:

=> Under culture hood:
- Remove fragments of living tissue (liver, muscle, skin,...)
- Mechanical dissociation with scalpel or scissors
- Trypsination: enzyme dissociation, trypsin or collagenase
- Cell counting under the reverse microscope
- + Culture medium ( 10^6 cells / 10ml medium)

=> In Incubator

=> Each morning: Cell counting and Cell transplanting in others flasks when cell number >
10^6cells/10ml medium 55 AMP Cell culture animation = WE USE A REVERSED
MICROSCOPE.