H & E Staining :)

Hi everyone!

Finally its my turn to post this week! I am attached to histopathology lab for my attachment (same as Zi Shuang). As what she said last week, our lab is separated into various areas, such as embedding, shaving, trimming, staining and sorting. For my case, I have been doing staining for my first posting. I find that staining is a very challenging task as it needs alot of concentration and the capability of multi-tasking because we are required to do heating, H&E staining and special stains at the same time. If we are lucky enough, we also have to do different types of special stains manually. For the past 5 weeks, I have done mainly H & E Stain and Special Stains using machine. However, I have also done manual special stains such as Alcian Blue, Mucicarmine, Ziehl Neelsen, PAS, PASD, Perl's Iron, CAB, VB and Orcein stains. As we have been doing H & E staining manually during our HTech practical, I will introduce to all of you on the H & E staining machine.

Subject title: Lab Technique

Topic: H&E Staining

Principle: The principle of H & E staining is to demonstrate different parts of the tissue components in contrasting colours, such as pink, blue, deep red, orange red and etc. Haematoxylin can be oxidized to Haematein by using natural oxidation or chemical oxidation. It requires a mordant, which is usually a metal, to enable Haematein dye to be well demonstrated on the acidic nucleus to give a blue colour. Eosin has the ability to distinguish between the cytoplasm of different types of tissues, connective tissue fibers and matrices.

Steps Involved: Slides are firstly collected from the trimming and fishing area and placed on the edge of the hotplate for air dry. The tissue sections are fixed on the slides by heating on the hot plate for 3 mins. The slides are placed in the staining rack and transferred to the H&E staining machine where dewaxing and staining are being carried out. After they are stained, they are directly transferred to the auto mounting machine for mounting.


Picture of H & E staining machine


Picture of Auto-mounting machine


Picture of Hot plate


Picture of Staining rack

Result:
Nucleus: Blue
Cytoplasm: Pink
Muscle fibers: Deep pinky red
Collagen: Pale pinky red
Fibrin: Deep red


Picture of slides produced

Procedures for H & E staining machine

1 Xylene 2 mins
2 Xylene 2 mins
3 Absolute alcohol 1 min
4 95 % alcohol 1 min
5 70 % alcohol 30 secs
6 Running water 30 secs
7 Haematoxylin 3 ½ mins
8 Haematoxylin 3 ½ mins
9 Running water 30 secs
10 0.5 % acid alcohol 2 dips
11 Running water 30 secs
12 Lithium carbonate 30 secs
13 Running water 30 secs
14 Blue in running water 3 mins
15 Eosin 25 secs
16 70 % alcohol 1 min
17 95 % alcohol 1 min
18 Absolute alcohol 1 min
19 Absolute alcohol 1 min
20 Absolute alcohol 1 min
21 Absolute alcohol 1 min
22 Xylene 1 min
23 Xylene 1 min
24 Xylene 1 min
25 Depex -

Some points to be noted:
To ensure that the slides are well demonstrated and differentiated, a control must be stained to assure that the machine is at its tip top condition.
Lithium carbonate must be replaced when it had turned purplish in colour. This is because Lithium carbonate needs to maintain a pH value of 9-10 to stain the nucleus blue, so if the pH value drops it will affect the demonstration of the nucleus.
Slides must be ensured that they are facing out of the staining rack so that the slides are mounted at the correct side.
Haematoxylin and Eosin should also be filtered before use and stored in the dark area to reduce photo-oxidation, debris and dust which will affect the quality of staining.

Clinical interpretation: The H & E slides can be used to diagnose the presence of tumors by looking at the abnormality in size and shape of the respective tissue cells.

I will stop here from now. If you have any doubts, pls feel free to ask me any questions! :)

Lok Pui
(0704138G)