BUFFERS

The quality of fixation is influenced by pH and the type of ions present.

The choice of buffer is based on:

1. the buffering capacity in the desired pH range with the ability to maintain constant pH during fixation.

2. the side effects which vary with the tissue type:

a. suitable osmolarity so that cells and organelles neither swell nor shrink during fixation.

b. suitable ionic concentration so that materials are neither extracted nor precipitated during fixation.

c. the toxicity of the buffer.

Criteria of a good buffer:

1. pKa: usually between 6 and 8 desired for biological specimens.

2. Maximum solubility in water and minimum solubility in all other solvents.

3. Reduced ion effects.

4. Dissociation of buffer least influenced by buffer concentration, temperature and ionic composition.

5. Resistance to oxidation (stable).

6. Inexpensive and easy to prepare.

7. No reaction with fixation.

Common Buffers

I. Phosphate Buffer (Sorenson's buffer) pH 5.8-8

Advantages:

1. Most physiological of common buffers. Mimics certain components of extracellular fluids.

2. Non-toxic to cells.

3. pH changes little with temperature.

4. Stable for several weeks at 4 C.

Disadvantages:

1. Precipitates more likely to occur during fixation. Tends to form precipitates in presence of calcium ions. Precipitates uranyl acetate and tends to react with lead salts.

2. Becomes slowly contaminated with micro-organisms

Preparation of Buffer

Stock solutions:

0.2M dibasic sodium phosphate 1 liter

Na₂HPO₄*2H₂0 (MW = 178.05) 35.61 gm

Na₂HPO₄*7H₂0 (MW = 268.07) 53.65 gm

Na₂HPO₄*12H₂0 (MW = 358.14) 71.64 gm

+ ddH₂0 to make 1 liter

0.2M monobasic sodium phosphate 1 litter

NaH₂PO₄*H₂0 (MW = 138.01) 27.6 gm

NaH₂PO₄*2H₂0 (MW = 156.03) 31.21 gm

+ ddH₂0 to make 1 liter

Working buffer: 0.1M 100 ml

Mix X ml of 0.2M dibasic sodium phosphate with Y ml monobasic sodium phosphate. Dilute to 100 ml with ddH₂0 or dilute 1:1 with fixative.

pH (25 C) X ml Y ml

5.8 4.0 46.0

6.0 6.15 43.75

6.2 9.25 40.75

6.4 13.25 36.75

6.6 18.75 31.25

6.8 24.5 25.5

7.0 30.5 19.5

7.2 36.0 14.0

7.4 40.5 9.5

7.6 43.5 6.5

7.8 45.75 4.25

8.0 47.35 2.65

Osmolarity is adjusted by varying the molarity of phosphates or by the addition of sucrose, glucose or sodium chloride.

At pH 7.2:

0.10M = 226 mOs (milliosmoles)

0.05M = 118 mOs

0.075 = 180 mOs

0.15M = 350 mOs

II. Cacodylate Buffer (arsenate buffer) pH 5-7.4

Advantages:

1. Easy to prepare.

2. Stable during storage for long periods of time.

3. Does not support growth of microorganisms.

4. Precipitates usually do not occur. Precipitates do not occur at low concentrations of calcium.

Disadvantages:

1. Toxic. Contains arsenic.

2. Unpleasant smell.

Preparation of Buffer:

Stock solutions:

0.2M sodium cacodylate 1 liter

Na(CH₃)₂As0₂*3H₂0 (MW = 195.92) 42.8 gm

+ ddH₂0 to make 1 liter

0.2M HC1

Conc. HC1 (36-38%) 10 ml

ddH₂0 603 ml

Working buffer: 0.1M 100 ml

Adjust 50 ml of 0.2M sodium cacodylate to desired pH with 0.2M HC1. Dilute to 100 ml with ddH₂0 or dilute 1:1 with fixative.

pH 0.2M HC1 (ml)

6.4 18.3

6.6 13.3

6.8 9.3

7.0 6.3

7.2 4.2

7.4 2.7

Buffer may also be made with cacodylic acid.

Stock solutions:

0.2M cacodylic acid 1 liter

(CH₃)₂AsO₂H (MW = 138.0) 27.6 gm

+ ddH₂0 to make 1 liter

0.2M NaOH 100 ml

NaOH (MW = 40) 0.8 gm

+ ddH₂0 to make 100 ml

Working buffer: 0.1M

Adjust 50 ml of 0.2M cacodylic acid to desired pH with 0.2M NaOH. Dilute to 100 ml with ddH₂ or dilute 1:1 with fixative.

III. Veronal-acetate Buffer (Michaelis buffer)

Advantages:

Useful for block staining with uranyl acetate since precipitates do not form.

Disadvantages:

1. Reacts with aldehydes.

2. Poor buffer at physiological pH.

3. Supports growth of micro-organisms.

4. Contains barbiturate.

Preparation of Buffer:

Stock solution: 0.28M 100 ml

Sodium veronal (barbitone sodium)

C₈H₁₁0₃N₂Na (MW = 206.18) 2.89 gm

Sodium acetate (anhydrous)

CH₃C00Na (MW = 82.03) 1.15 gm

Sodium acetate (hydrated)

CH₃C00Na*3H₂0 (MW = 136.09) 1.90 gm

+ ddH₂H₂0 to make 100 ml

Solution is stable and may be stored for some months at 4 C.

Working buffer:

Veronal acetate stock solution 5 ml

ddH₂0 15 ml

Add 0.1 HC1 gradually to desired pH.

Solution cannot be stored.

Supports growth of bacteria and molds even at 4 C.

Crystallizes in absence of osmium tetroxide.

IV. Collidine Buffer pH 7.25-7.74

Advantages:

1. Maximum buffering capacity about 7.4.

2. Stable indefinitely at room temperature.

3. Useful for fixation of large tissue blocks. Aids penetration of fixative due to extractive effects (see disadvantage 1).

Disadvantages:

1. Not suitable as buffer during primary fixation with osmium tetroxide due to considerable extraction of tissue components.

2. Use leads to lysis of cytoplasmic matrix and extensive membrane destruction when used with paraformaldehyde fixatives.

3. Use gives poorer results with glutaraldehyde than those obtained with phosphate or cacodylate buffer.

Preparation of Buffer:

Stock solution: 0.4M 100 ml

Pure s-collidine 5.34 gm

2,4,6(CH₃)₃(C₂H₅N) (MW = 121.18)

+ ddH₂0 to make 100 ml

Working buffer: 0.2M 100 ml

Adjust 50 ml of s-collidine stock solution to desired pH with 1N HC1. Dilute to 100 ml with ddH₂0.

pH 1N HC1 (ml)

7.25 22

7.33 20

7.41 18

7.5 16

7.59 14

7.67 12

7.74 10

V. Tris buffer

Advantages:

1. Good buffering capacity at higher pH required for some tissues and some cytochemical procedures.

2. "More or less" physiologically inert.

Disadvantages:

1. pH changes with temperature. Must be measured at desired temperature.

2. pH must be measured with certain type of electrode.

Preparation of Buffer:

A. Tris Buffer pH 7.1-8.9

Stock solution 0.2M 1 liter

Tris(hydroxymethyl)aminomethane 24.2 gm

H₂NC(CH₂0H)₃ (MW = 121.13)

+ ddH₂0 to make 1 liter

Working buffer: 0.1M 100 ml

Adjust pH of 50 ml of stock solution with 0.1M NaOH. Dilute to 100 ml with ddH₂0.

B. Tris-maleate Buffer pH 5.8-8.2

Stock solution: 0.2M liter

Tris(hydroxymethyl)aminomethane 24.2 gm

Maleic acid 23.2 gm

HO₂CCH:CHCO₂H (MW = 116.07)

+ ddH₂0 to make 1 liter

Trizima-maleate (MW = 237.2) 47.4 gm

+ ddH₂0 to make 1 liter

Working buffer: 0.2M 100 ml

Adjust 50 ml of stock solution to desired pH with 0.1M NaOH. Dilute to 100 ml with ddH₂0.

VI. Special Buffers Used for Cytochemical Reactions.

A. Acetate Buffer (sodium acetate-acetic acid buffer) pH 4-5.6

Sodium acetate 0.2M = 27.2 gm/1

CH₃CO₂Na*3H₂0 (MW - 136.09)

Acetic acid 0.2M

CH₃COOH (MW = 60)

Add sodium acetate to acetic acid to give desired pH. Dilute with ddH₂0 to desired molarity.

B. Borate Buffer pH 7.4-9.2

Borax (sodium tetraborate) 0.2M = 76.2 gm/ml

Na₂B₄0₇*120H₂0 (MW = 381.37)

Boric acid 0.2M = 12.37 gm/1

H₃BO₃ (MW = 61.83)

Add boric acid to borax solution until desired pH is reached. Dilute to desired molarity with ddH₂0.

C. Citrate Buffer (sodium citrate-citric acid buffer) pH 3-6.2

Sodium citrate 0.2M = 58.8 gm/1

Na₃C₆H₅0₇*H₂0 (MW = 294.12)

Citric acid 0.2M = 42.02 gm/1

C₆H₈0₇*H₂0 (MW = 210.14)

Mix citric acid and sodium citrate to give desired pH. Dilute with ddH₂0 to desired molarity.

D. Dimethylglutarate Buffer pH 3.2-7.6

Dimethylglutaric acid 0.1M = 16.02 gm/1

C₇H₁₂0₄ (MW = 160.2)

Add 0.2N NaOH to give desired pH. Dilute with ddH₂0 to desired molarity.

E. Succinate Buffer pH 3.8-6

Succinic acid 0.2M = 23/62 g/1

C₄H₆0₂ (MW = 118.09)

Add 0.2M NaOH to desired pH. Dilute with ddH₂0 to desired molarity.

F. Maleate Buffer (sodium hydrogen maleate buffer) pH 5.2-6.8

Stock solution: 0.2M 1 liter

Maleic acid (MW = 121.14) 23.2 gm

+ ddH₂0 to make 1 liter

Adjust pH with 0.1M Na0H. Dilute with ddH₂0 to desired molarity.

G. Imidazole Buffer pH 6.2-7.8

Imidazole 0.2M = 13.62/1

C₃H₄N₂ (MW = 68.08)

Adjust 0.2N HC1 to imidazole solution until desired pH is reached. Dilute to desired molarity with ddH₂0.

H. AMPd Buffer pH 7.8-9.7

2-amino-methyl-1,3-propanediol 0.2M = 21.03 gm/1

C₄H₁₁NO₂ (MW = 105.14)

Add 0.2M HC1 until desired pH is reached. Dilute with ddH₂0 to desired molarity.