SOPs Medical Devices|SOPs Pharmacovigilance

1.0 Introduction:

Nuclease free water is non-DEPC treated, suitable for use in molecular biology applications where high quality of water is required and should be free from Nuclease, DNase, RNase, and protease contamination.

Water is one of the elemental solvents which is extensively used in preparing solutions. Nuclease free water is suitable for use in all kinds of molecular biology applications where high quality of water is required.

2.0 Application:

Nuclease free water is ideal for molecular biology applications and preparation of reagents where RNase, DNase, and Protease-free water is required. It is widely used for several fundamental procedures like PCR, gel electrophoresis, DNA sequencing etc.

3.0 Materials required:

Chemicals:

70% isopropanol alcohol

Nuclease free water

Consumable:

Sterile gloves

Sterile 125mL PETG bottles

Sterile 1L PETG bottles

Equipment:

Laminar air flow

4.0 The composition of Nuclease Free Water:

Molecular Biology Grade water is filtered through 0.2-micron filter and filled under sterile condition.

5.0 Manufacturing procedure:

 Clean the Laminar Hood with 70% alcohol. Turn on the UV for 30 minutes.

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1.0 Introduction:

DEPC Treated water is sterile filtered water treated with 0.1% Diethylpyrocarbonate (DEPC). It is produced specifically for molecular biology applications like DNA/RNA purification, protein applications and experiments involving RNA. DEPC inactivates RNases by carboxylation of specific amino acid side chains in the protein.

Water is one of the elemental solvents which is extensively used in preparing solutions DEPC treated water is suitable for use in all kinds of molecular biology applications where high quality of water is required. Each lot of this product is treated with DEPC and autoclaved.

2.0 Application:

DEPC Treated water is ideal for the preparation of reagents, rinsing glassware and plasticware, and other molecular biology applications where RNase, DNase, and Protease-free water is required. It is widely used for several fundamental procedures like PCR, gel electrophoresis, DNA sequencing etc.

3.0 Materials required:

3.1 Chemicals:

• DEPC transparent liquid form (≥ 95.5%).
• Nuclease free water

3.2 Consumable:

• Glass bottles 5L
• Measuring cylinder
• Beaker
• Pipette
• Sterile gloves
• Sterile 125mL PETG bottles

3.3 Equipment:

• Laminar air flow
• Auto clave
• Milli-Q Bio-Pack filter

1.0 Principle:

Acid fast satin is the differential staining techniques which was first developed by Ziehl and later on modified by Neelsen. So this method is also called Ziehl-Neelsen staining techniques. Neelsen in 1883 used Ziehl’s carbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques was developed.

The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups.

This method is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacterium, are resistant and can only be visualized by acid-fast staining.

When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the Mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal wall and enters cytoplasm. Then after all cell appears red. Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol) but the acid-fast cells are resistant due to the presence of large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing solution. The non-acid fast organism lacks the lipoidal material in their cell wall due to which they are easily decolorized, leaving the cells colorless. Then the smear is stained with counterstain, methylene blue. Only decolorized cells absorb the counter stain and take its color and appears blue while acid-fast cells retain the red color.

2.0 Application:

Acid Fast Stains-Kit is used for staining of acid-fast bacteria.

3.0 Composition:

1. Basic Fuchsin                                
2. Ethyl alcohol
3. Phenol
4. Hydrochloric acid
5.  Methylene blue 

4.0 Manufacturing procedure:

a) Carbol Fuchsin  

    Basic Fuchsin                                    0.3g

    Ethyl alcohol,95%                           10.0ml

    Phenol                                             5.000 ml

b) Acid Fast Decolorizer

Hydrochloric acid, concentrated          3.0 ml

 Ethyl alcohol                                     97.0

c) Methylene Blue

Methylene blue                                0.3g

Ethyl alcohol                                  30.0ml

5.0 Staining Procedure:

5.1 Material Required:

• Loop sterilization device,
•  Inoculating loop or needle, swabs, collection containers,
• Quality control organisms,
• Glass slides, coverslips,
• Slide warmer,

1.0 Introduction:

1X TE Buffer pH 7.4 - 8 is an extensively used buffer in Molecular Biology. Its principal application includes protection of DNA and RNA from degradation.

2.0 Description:

TE buffer is composed of Tris, a buffering agent and EDTA, a chelating agent. EDTA prevents the degradation of DNA and RNA by chelating divalent metal ions which are required for nuclease activity. The Tris buffering agent and EDTA metal chelating properties help protect DNA and RNA. Based on nuclease studies the pH should be adjusted to 7.5 for RNA and 8.0 for DNA as the respective DNA and RNA nucleases are supposed to be less active at these pH values, but pH 8.0 can be used for storage of both DNA and RNA.

3.0 Application:

TE buffer is mainly used in storing DNA. Genomic and plasmid DNA can be stored in TE Buffer at 4°C for short-term use, or -20°c to -80°C for long-term storage. Repeated freeze-thaw cycles should be avoided. Moreover, Tris-EDTA buffer disrupts protein cross-links and therefore is useful in unmasking antigens and epitopes in formalin-fixed and paraffin-embedded tissue sections. This buffer is also used in immuno-histochemical detection of some proteins as it enhances the staining intensity of antibodies.

1.0 Principle:

Trichrome Stain Set is a complete set of reagents recommended for use in the Wheatley Trichrome Stain for detection and identification of intestinal protozoa

A diagnosis of intestinal parasitic infections caused by protozoan organisms is confirmed by identification of trophozoites and cysts in fecal specimens. Because smaller protozoans often go undetected in direct wet mount and concentration methods, the identification of intestinal protozoa depends on examination of a permanent stained smear. The Trichrome stain provides excellent detail and contrast with preserved specimens. Trichrome stain was originally developed by Gomori for staining tissue sections and cytological smears.1 In 1951, Wheatley modified Gomori’s technique by addition of fixation and dehydration steps resulting in a simple and rapid staining procedure for intestinal amoebas and flagellates

Chromotrope 2R has an affinity for chromatin material. Nuclear chromatin, chromatoid bodies, karyosomes, parasite eggs and larvae, bacteria, and ingested erythrocytes stain red to purple-red. Light green and fast green dyes stain the cytoplasm of preserved cysts, trophozoites, and cellular constituents blue-green. The Trichrome Stain results in excellent contrast and visualization of cellular details that aid in the identification of protozoa.

2.0 Application:

.Trichrome kit is indicated for staining connective tissue. It stains gametes, nuclei, nerve fibres, neuroglias, collagen, keratin and intracellular fibres. It can also be used to obtain a negative image of the Golgi apparatus.

3.0 Composition:

1. Wheatley Trichrome Stain:

Phosphotungstic Acid                7.0 g

Chromotrope 2R                        6.0 g

Light Green SF                          1.5 g

Fast Green FCF                         1.5 g

Glacial Acetic Acid                   10.0 mL

Demineralized Water                1000.0 mL

1. Xylene S:
2. Ethanol 90%
3. Acid Ethanol 90%:

Ethanol 90%             995.5 mlL

Acetic Acid 0.5%       4.5 mL

1. Lugol's iodine

Iodine                       5.0 gm

Potassium iodide     10.0 gm

Distilled water        100.0 mL

4.0 Staining Procedure:

1.  Material Required:

• Loop sterilization device,
•  Inoculating loop or needle, swabs, collection containers,
• Quality control organisms,
• Glass slides, coverslips,
• Slide warmer,
• Microscope
• 100% alcohol,

 5.0 Procedure

1. Preparation of Smear: Stool specimens preserved in PVA should be allowed to fix at least 30 minutes. Fresh specimens received in the laboratory should be mixed with PVA (1-part feces to 3 parts fixative) and allowed to fix for 30 minutes.
2. Thoroughly mix the specimen and the PVA. Pour a small amount of the mixture onto a paper towel to absorb excess fixative. Allow the fixative to soak into the paper towel for 3 minutes before preparing slides.
3.   With an applicator stick, pipette, or brush transfer some of the stool material from the paper towel to 2 clean glass slides. Spread the mixture to the edges of the slide so the specimen will adhere to the slide during staining. The amount of material applied to the slide should be thin enough that newsprint can be read through the smear.