Readkit 2 6 3 0

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BUFFERS

Files for Babel, version 2.8.0; Filename, size File type Python version Upload date Hashes; Filename, size Babel-2.8.0-py2.py3-none-any.whl (8.6 MB) File type Wheel Python version py2.py3 Upload date Dec 31, 2019 Hashes View. Looking at the list of g(x) points, I find (0, 2), so g(0) = 2, and I need now to find f(2). Looking at the list of f(x) points, I find (2, –3), so f(2) = –3. Then: (f o g)(0) = f(g(0)) = f(2) = –3 (ii) The second part works the same way: (f o g)(–1) = f(g(–1)) = f(–2) = 3.

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

The choice of buffer isbased on:

1. thebuffering capacity in the desired pH range with the ability to maintainconstant pH during fixation.

2. theside effects which vary with the tissue type:

a. suitableosmolarity so that cells and organelles neither swell nor shrink duringfixation.

b. suitableionic concentration so that materials are neither extracted nor precipitatedduring fixation.

c. thetoxicity of the buffer.

Criteria of a good buffer:

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

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

3. Reducedion effects.

4. Dissociationof buffer least influenced by buffer concentration, temperature and ioniccomposition.

5. Resistanceto oxidation (stable).

6. Inexpensiveand easy to prepare.

7. Noreaction with fixation.

Common Buffers

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

Advantages:

1. Mostphysiological of common buffers. Mimics certain components of extracellularfluids.

2. Non-toxicto cells.

3. pHchanges little with temperature.

4. Stablefor several weeks at 4 C.

Disadvantages:

1. Precipitatesmore likely to occur during fixation. Tends to form precipitates in presenceof calcium ions. Precipitates uranyl acetate and tends to react with leadsalts.

2. Becomesslowly contaminated with micro-organisms

Preparation of Buffer

Stock solutions:

0.2M dibasic sodiumphosphate 1 liter

Na₂HPO₄*2H₂0 (MW= 178.05) 35.61 gm

or

Na₂HPO₄*7H₂0 (MW= 268.07) 53.65 gm

or

Na₂HPO₄*12H₂0 (MW= 358.14) 71.64 gm

+ ddH₂0 tomake 1liter

0.2M monobasic sodiumphosphate 1 litter

NaH₂PO₄*H₂0 (MW= 138.01) 27.6 gm

or

NaH₂PO₄*2H₂0 (MW= 156.03) 31.21 gm

+ ddH₂0 to make 1liter

Working buffer: 0.1M 100 ml

Mix X mlof 0.2M dibasic sodium phosphate with Y ml monobasic sodium phosphate. Diluteto 100 ml with ddH₂0 or dilute 1:1 with fixative.

pH (25 C) Xml 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

Osmolarityis adjusted by varying the molarity of phosphates or by the addition ofsucrose, glucose or sodium chloride.

At pH 7.2:

0.10M = 226 mOs (milliosmoles)

0.05M = 118 mOs

0.075 = 180 mOs

2.6 Naruto Vs Bleach

0.15M = 350 mOs

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

Advantages:

1. Easyto prepare.

2. Stableduring storage for long periods of time.

3. Doesnot support growth of microorganisms.

4. Precipitatesusually do not occur. Precipitates do not occur at low concentrations ofcalcium.

Disadvantages:

1. Toxic.Contains arsenic.

2. Unpleasantsmell.

Preparation of Buffer:

Stock solutions:

0.2M sodium cacodylate 1 liter

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

+ ddH₂0 tomake 1liter

0.2M HC1

Conc. HC1 (36-38%) 10ml

ddH₂0 603ml

Working buffer: 0.1M 100ml

Adjust50 ml of 0.2M sodium cacodylate to desired pH with 0.2M HC1. Dilute to 100 mlwith ddH₂0 or dilute 1:1 with fixative.

pH 0.2MHC1 (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 madewith cacodylic acid.

Stocksolutions:

0.2Mcacodylic acid 1liter

(CH₃)₂AsO₂H (MW= 138.0) 27.6 gm

+ ddH₂0 tomake 1liter

0.2M NaOH 100 ml

NaOH (MW= 40) 0.8 gm

+ ddH₂0 tomake 100ml

Working buffer: 0.1M

Adjust50 ml of 0.2M cacodylic acid to desired pH with 0.2M NaOH. Dilute to 100 mlwith ddH₂ or dilute 1:1 with fixative.

III. Veronal-acetate Buffer (Michaelisbuffer)

Advantages:

Usefulfor block staining with uranyl acetate since precipitates do not form.

Disadvantages:

1. Reactswith aldehydes.

2. Poorbuffer at physiological pH.

3. Supportsgrowth of micro-organisms.

4. Containsbarbiturate.

Preparationof Buffer:

Stocksolution: 0.28M 100ml

Sodiumveronal (barbitone sodium)

C₈H₁₁0₃N₂Na (MW= 206.18) 2.89 gm

Sodium acetate(anhydrous)

CH₃C00Na (MW= 82.03) 1.15 gm

or

Sodium acetate(hydrated)

CH₃C00Na*3H₂0 (MW= 136.09) 1.90 gm

+ ddH₂H₂0to make 100ml

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

Workingbuffer:

Veronal acetate stocksolution 5 ml

ddH₂0 15ml

Add 0.1 HC1 gradually todesired pH.

Solution cannot bestored.

Supports growth ofbacteria and molds even at 4 C.

Crystallizes in absenceof osmium tetroxide.

IV. Collidine Buffer pH7.25-7.74

Advantages:

1. Maximumbuffering capacity about 7.4.

2. Stableindefinitely at room temperature.

3. Usefulfor fixation of large tissue blocks. Aids penetration of fixative due toextractive effects (see disadvantage 1).

Disadvantages:

1. Notsuitable as buffer during primary fixation with osmium tetroxide due toconsiderable extraction of tissue components.

2. Useleads to lysis of cytoplasmic matrix and extensive membrane destruction whenused with paraformaldehyde fixatives.

3. Usegives poorer results with glutaraldehyde than those obtained with phosphate or cacodylatebuffer.

Preparation of Buffer:

Stock solution: 0.4M 100ml

Pure s-collidine 5.34gm

2,4,6(CH₃)₃(C₂H₅N) (MW= 121.18)

+ ddH₂0 tomake 100ml

Working buffer: 0.2M 100ml

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

pH 1NHC1 (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. Goodbuffering capacity at higher pH required for some tissues and some cytochemicalprocedures.

2. 'Moreor less' physiologically inert.

Disadvantages:

1. pHchanges with temperature. Must be measured at desired temperature.

2. pHmust be measured with certain type of electrode.

Preparation of Buffer:

A. TrisBuffer pH 7.1-8.9

Stock solution 0.2M 1liter

Tris(hydroxymethyl)aminomethane 24.2gm

H₂NC(CH₂0H)₃ (MW= 121.13)

+ ddH₂0 tomake 1liter

Workingbuffer: 0.1M 100ml

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

B. Tris-maleateBuffer pH 5.8-8.2

Stock solution: 0.2M liter

Tris(hydroxymethyl)aminomethane 24.2gm

Maleic acid 23.2gm

HO₂CCH:CHCO₂H (MW= 116.07)

+ ddH₂0 tomake 1liter

or

Trizima-maleate (MW= 237.2) 47.4 gm

+ ddH₂0 tomake 1liter

Working buffer: 0.2M 100ml

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Adjust50 ml of stock solution to desired pH with 0.1M NaOH. Dilute to 100 ml with ddH₂0.

VI. Special Buffers Used forCytochemical Reactions.

A. AcetateBuffer (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 toacetic acid to give desired pH. Dilute with ddH₂0 to desired molarity.

B. BorateBuffer 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 boraxsolution until desired pH is reached. Dilute to desired molarity with ddH₂0.

C. CitrateBuffer (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 andsodium citrate to give desired pH. Dilute with ddH₂0 to desired molarity.

D. DimethylglutarateBuffer pH 3.2-7.6

Dimethylglutaric acid 0.1M= 16.02 gm/1

C₇H₁₂0₄(MW = 160.2)

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

E. SuccinateBuffer pH 3.8-6

Pictures to gif 1 4 0 x. Succinic acid 0.2M= 23/62 g/1

C₄H₆0₂ (MW= 118.09)

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

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

Stock solution: 0.2M 1 liter

Maleic acid (MW =121.14) 23.2 gm

+ ddH₂0 tomake 1liter

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

G. ImidazoleBuffer pH 6.2-7.8

Imidazole 0.2M= 13.62/1

C₃H₄N₂ (MW= 68.08)

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

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H. AMPdBuffer pH 7.8-9.7

Readkit 2 6 3 0 0

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

C₄H₁₁NO₂ (MW= 105.14)

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