Saturday, July 25, 2009

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

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


  1. Hey Lok Pui!

    Sherman here!

    I realise that for the H&E staining, it has alot of steps involved.
    I've also noticed that certain steps involved dehydrating and rehydrating of the tissue sections yea? Like immersing in different graded conc of alcohol along the way.

    What is the purpose of it?
    Is it because some of the reagents are alcohol-based and water-based respectively?

    THANKS! =)

  2. Hi Lok Pui!
    the function of lithium carbonate is just to maintain the pH value 9-10 to stain the nucleus blue is it?


  3. * To Sherman

    There are 2 purposes for the slides to be immersed in different graded alcohol. For step 1-5, it is to dewax the slides whereby paraffin wax is being removed by Xylene. As Xylene is imiscible with water, different graded alcohol is used to remove Xylene as it is both soluble in Xylene and water. Furthermore, Haematoxylin is water based, so it must be immersed with water before staining.
    However, for step 16-24, it is to dehydrate and mount the slides. After staining, the slides needed to be mounted. But the mounting media (Depex) is a Xylene based media, so it must be introduced into different graded alcohol to remove water before going through Xylene and then lastly mounted with Depex.

    Hope u understand wat i'm saying..haha..can ask me again if u dun understand..

    * To Stella

    Its function is not to maintain the pH of 9-10 but act as an alkaline solution to blue the nucleus more effectively when they are placed in water.

    For addition information, other than lithium carbonate, ammonium hydroxide can also be used as an alkaline solution.

    U can ask me again if you dun understand..haha :)

    Lok Pui

  4. Hiya lok pui,

    I don't understand why when alkaline solution, the nucleus will be blue more effectively when placed in water. Can you explain the principle behind it? thanks! :D

    Zi Shuang

  5. hey lok pui!
    what type of tissues did you usually stain?


  6. hi lok pui,
    if you have diagnosed a tissue which is malignant,will the staining solutions in the auto-stainer machine be contaminated and affect the subequent tissues(which may not be malignant)that are going to be stain?

    happy histo-ting
    Yong Herng

  7. Lok Pui,

    Do you have any microscopic pictures on how a normal and malignant tissue differs in morphology and staining? Thank you.

    Li Yinliang Alex
    TG02 0704894E
    Group 8
    28 July 2009

  8. hi lok pui!
    after examine the stain under microscope,do you store the specimens for a day or two or just simply throw it away?

  9. Hi Lok Pui,

    what is photo-oxidation and how exactly does it affect the microscopic images of the slides?


    GROUP 10

  10. Hi .This is Jennifer. Erm for the staining right, isnt Haemetoxylin being used to stain so what is the reason of using Lithium Carbonate? Is there other function of Lithium Carbonate?

  11. hi lok pui, i understand the principle ler...please igmore the previous comment. Thanks! :)

    zi shuang

  12. hi can we do alcohol free h and e staining ?