4BBY1030 Cell Biology and Neuroscience

Formative Lab Report based on Practical 2

Introduction to Histological Staining

Date and time of Practical 2: 26/10/2021 at 9 am

Practical 2 Lead: Carl Hobbs

Introduction (max. 150 words)

Haematoxylin and eosin (H & E) were used to stain slides containing muscle and lung
tissues from a rat. Gomori rapid one step trichrome was used to stain kidney tissue. These
slides were then dehydrated and mounted. Mounting required a cover slip and the protective
glue: DPX.

The aim was to successfully carry out two histological staining procedures to produce three
clear tissue samples that can be observed under a microscope.

Materials and Methods

1) Haematoxylin & Eosin (H & E)

Technique

Use this stain for lung and muscle tissue.

1. Stain slide in Harris Alum Haematoxylin for 5 minutes

2. Quickly rinse the slide in beaker of water to remove excess haematoxylin
3. Differentiate in 0.5% acetic acid for 30 seconds
4. Quickly rinse in beaker of water
5. Place in Scott’s water (pH>7) until the sections ‘blue’ (about 2 minutes)
6. Quickly rinse in water
7. Stain in alcoholic eosin for 5 minutes
8. Dehydrate section by dipping twice in 70% ethanol. Do NOT wash in water or leave
the eosin in the alcohol (the eosin will be washed out)
9. Place in 90% ethanol for 20 seconds
10. Place in absolute ethanol for 2 minutes

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 11. Place in ‘Histoclear’. If the sections appear cloudy go back to step 9
12. Mount using a cover slip and DPX
2) Gomori Rapid One Step Trichrome

Technique

Using this stain on kidney tissue.

1. Stain in Harris Alum Haematoxylin for 5 minutes

2. Quickly rinse the slide in beaker of water to remove the excess haematoxylin
3. Differentiate in 0.5% acetic acid for 30 seconds
4. Quickly rinse in water
5. Place in Scott’s water (pH>7) until the sections ‘blue’ (about 2 minutes)
6. Quickly rinse in water
7. Stain in Trichrome stain for 10 minutes
8. Quickly rinse the slide in beaker of water to remove excess Trichrome solution
9. Two dips in 0.5% acetic acid
10. Quickly rinse in water
11. Dehydrate section by dipping twice in 70% ethanol
12. Place in 90% ethanol for 20 seconds
13. Place in absolute ethanol for 2 minutes
14. Place in ‘Histoclear’. If the sections appear cloudy go back to step 12
15. Mount using a cover slip and DPX

Have you been able to follow these procedures exactly or have you deviated from the
protocol and how? (max. 100 words)
Minor deviations in timing occurred as a result of insufficient use of stop clocks. Broken
buttons on these clocks meant that the timer would not start when expected to.

Figures (max. three images, drawn by you)

Fig. 1 Muscle tissue

Muscle tissue from a rat, showing the presence of muscle fibres containing nuclei and few
erythrocytes

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Fig. 2 Lung Tissue

Lung tissue from a rat, showing the presence of a bronchiole surrounded by muscle fibres
containing abundant erythrocytes and nuclei.

Fig. 3 Kidney Tissue

Kidney tissue from a rat, showing the presence of a glomerulus, closely surrounded by thick
muscle. The tissue contains abundant erythrocytes and nuclei.

Results (max. 200 words)

Despite the minor changes in timing, the experiment produced successful results.

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The muscle tissue slide (Fig.1) clearly shows the presence of striated and branched muscle
cells with centrally located nuclei. The surrounding regions show that the cells are encased
with collagen fibres. Erythrocytes can also be seen; these are less vivid.

The lung tissue (Fig.2) shows a bronchiole surrounded by muscle fibres. Eminent staining
also shows the presence of many erythrocytes and nuclei. The staining of the erythrocytes is
far clearer than those present in the muscle tissue.

The kidney tissue (Fig.3) shows the presence of a glomerulus, both containing and
surrounded by muscle. Many erythrocytes and nuclei can be seen.

Conclusions/Discussion (max. 150 words)

It may be concluded that the type of muscle stained in figure 1 is cardiac muscle, this is due
to the features that were present; branched, striated muscle cells and erythrocytes. The
reason why the erythrocytes were less vivid in the muscle tissue compared to the lung
tissue may be due to variations in the amount of time the specimens were present in each
stain.

This staining procedure may be applied to evaluate morphology, diagnose patients, and
even detect malignancies. However, contamination of the stain may easily occur which may
lead to loss of stain contrast and colour.

To take this line of research further, the kidney slides should be stained with H & E in order
to observe which staining method produced the greatest contrast and clearer stained
specimen.

Bibliography (optional, not more than three sources)