Lab Hygiene Masterclass: Disinfecting Surfaces and Equipment Like a Pro

 

Introduction

10% bleach is recommended for disinfecting laboratory equipment and surfaces. For example, if you suspect nucleic acid (DNA/RNA) contamination of pipettes or surfaces, a 10% bleach solution can be made and sprayed on surfaces for 10 min before removing and following up with 70% ethanol.

Other Common Disinfectant Options Include:

• 70% isopropyl or ethanol alcohol for non-corrosive and rapid disinfection.

• Commercial disinfectants approved for lab use (check manufacturer’s guidelines).

Outline

1. Various Names for Bleach

2. Why Bleach is an Effective Disinfectant

3. How to Prepare 10% Bleach for Disinfection

4. Why 70% Ethanol is an Effective Disinfectant

5. Combining Bleach and Ethanol in a Cleaning Regimen

Bleach

Bleach is known by various names depending on its composition, usage, or the context in which it is referred. Here are the common names for bleach:

 General Names

• Bleach: The most common name used in household and industrial contexts.

• Chlorine bleach: Refers specifically to sodium hypochlorite solutions.

• Non-chlorine bleach: Used for alternatives like hydrogen peroxide-based bleaches.

Chemical Names

• Sodium hypochlorite (NaOCl): The active ingredient in liquid chlorine bleach.

• Calcium hypochlorite (Ca(ClO)₂): A solid form of bleach often used in pool sanitization.

• Sodium dichloroisocyanurate (NaDCC): A dry, granular form used for disinfection.

• Potassium hypochlorite (KOCl): A less common alternative to sodium hypochlorite.

Scientific and Industrial Names

• Hypochlorite solution: A generic term for sodium or calcium hypochlorite solutions.

• Liquid chlorine: An industrial term for sodium hypochlorite in high concentrations.

• Sodium oxychloride: Another term for sodium hypochlorite.

Informal and Regional Names

• Liquid bleach: Common term for sodium hypochlorite solutions in many households.

• Disinfectant bleach: Refers to bleach solutions marketed for sanitation and cleaning.

• Jik® or Powerzone:  Popular bleach brand in some African countries.

Why Bleach is an Effective Disinfectant

Bleach is  the most widely used disinfectant from the chlorine family. They are typically sold commercially at a concentration that is between 5-8.25%. When preparing bleach, you assume the commercial concentration to be 100% (although it is not) and dilute it 1 in 10.

Bleach is a good choice because it has:

• Broad-spectrum efficacy: Bleach is effective against a wide range of microorganisms, including bacteria, viruses, and fungi, which can harbor or transfer nucleic acids.

• Readily available: Bleach is inexpensive and easily accessible in most laboratories.

• Proven effectiveness: Numerous studies have demonstrated the effectiveness of bleach in decontaminating surfaces and equipment contaminated with nucleic acids.

How to Prepare 10% Sodium Hypochlorite for Disinfection 

To prepare a 10% bleach solution, you need the stock sodium hypochlorite solution to be around 5%–6% sodium hypochlorite.

A 10% bleach solution means 10% of the final volume should be the stock bleach (sodium hypochlorite solution) and 90% should be water.

Steps:

1. Determine the volume of the final solution you need. For example, if you want 100 milliliters (100 mL) of 10% bleach:
   

2. Use 10 mL of stock sodium hypochlorite solution.

3. Add 90 mL of water.

4. Mix well to ensure the solution is homogeneous.

Considerations

Fresh Preparation: Prepare bleach solutions fresh daily as sodium hypochlorite degrades over time, especially when exposed to light or heat.

Safety: Use gloves and work in a well-ventilated area when preparing and using bleach solutions.

70% Ethanol as a Disinfectant

70% ethanol or isopropyl alcohol is a highly effective disinfectant and can kill a wide range of organisms. However, its efficacy depends on factors such as exposure time, concentration, and the type of microorganism. Here's a breakdown of what 70% alcohol can kill:

Bacteria

• Gram-positive bacteria (e.g., Staphylococcus aureus, Streptococcus pneumoniae): Alcohol disrupts their lipid membranes, leading to cell death.

• Gram-negative bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa): Alcohol penetrates their thinner peptidoglycan layers effectively.

• Mycobacteria (e.g., Mycobacterium tuberculosis): 70% alcohol is effective, but longer contact time is needed due to their waxy cell wall.

Viruses

• Enveloped viruses: Alcohol dissolves the lipid envelope, inactivating the virus. Examples include:

• Influenza virus

• SARS-CoV-2 (the virus causing COVID-19)

• HIV

• Herpes simplex virus (HSV)

• Non-enveloped (naked) viruses: 70% alcohol is less effective or ineffective against non-enveloped viruses, such as:

• Norovirus

• Hepatitis A virus

• Rotavirus

Fungi

• Yeasts: Effective against organisms like Candida albicans.

• Molds: Alcohol can kill spores of many fungi but may require longer exposure times.

Spores

• Bacterial spores (e.g., Clostridium difficile, Bacillus spp.):

• Alcohol is generally ineffective against spores because they have tough, resistant structures that alcohol cannot penetrate.

Protozoa

• Effective against vegetative forms (active, feeding stage), but not against cysts (dormant, resistant stage). Examples:

• Vegetative Giardia lamblia can be killed, but cysts may survive.

Why 70% Alcohol Is Effective

• Mechanism of Action:

• Alcohol denatures proteins and disrupts lipid membranes.

• Optimal Concentration:

• 70% alcohol is more effective than higher concentrations (e.g., 95%) because water assists in protein denaturation and slows evaporation, allowing better penetration of cell walls.

Organisms That Are Resistant

• Endospores (e.g., Clostridium botulinum, Bacillus anthracis).

• Non-enveloped viruses (e.g., Norovirus).

• Some protozoan cysts.

How to Prepare 70% Ethanol for Disinfection 

To prepare a 70% bleach solution, you need the stock ethanol solution to be around 100%.

A 70% ethanol solution means 70% of the final volume should be the stock ethanol and 30% should be water.

Steps:

1. Determine the volume of the final solution you need. For example, if you want 100 millilitres (100 mL) of 70% bleach:
   

2. Use 70 mL of stock ethanol solution.

3. Add 30 mL of water.

4. Mix well to ensure the solution is homogeneous.

Considerations

70% ethanol or isopropyl alcohol effectively kills most bacteria, enveloped viruses, yeasts, and molds, but is not effective against bacterial spores, certain non-enveloped viruses, and protozoan cysts. Always use alcohol with adequate exposure time (at least 30 seconds to a few minutes) to maximise its efficacy.

Some cleaning and disinfection protocols use both bleach (sodium hypochlorite) and alcohol (ethanol or isopropanol) in combination or sequentially to maximize microbial elimination. 

Combining Bleach and Ethanol in a Cleaning Regimen

1. First Clean with Bleach (10% dilution)

• Kills spores, hardy bacteria, and viruses.

• However, bleach leaves residue, which may be corrosive or irritating.

2. Follow with Alcohol (70%)

• Helps remove bleach residue.

• Enhances drying and prevents damage to sensitive equipment.

This is because each disinfectant has strengths and limitations, and using both ensures broader-spectrum effectiveness.

NB: Do NOT mix bleach and alcohol directly – this can create toxic chloroform gas, which is highly dangerous.

Conclusion

Use Bleach (10%) when killing all microbes, including spores, is necessary (e.g., spill cleanup, surfaces in biosafety labs).

Use 70% Alcohol for quick disinfection of non-porous surfaces and electronic equipment where bleach may cause damage.

Bibliography

1. CDC (Centers for Disease Control and Prevention). (2023, November 28). Chemical Disinfectants [Web page]. Retrieved from https://www.cdc.gov/infection-control/hcp/disinfection-sterilization/chemical-disinfectants.html
   
   

2. World Health Organization (WHO). (2014). Infection prevention and control of epidemic- and pandemic-prone acute respiratory infections in health care. Geneva: World Health Organization. 

3. Gallandat, K., Kolus, R. C., Julian, T. R., & Lantagne, D. S. (2020). A systematic review of chlorine-based surface disinfection efficacy to inform recommendations for low-resource outbreak settings. American Journal of Infection Control, 49(1), 90–103. 

BIOENGINEERING : Databases for Obtaining Nucleotide Sequences for Expression Vectors.

 

Introduction

In a previous tutorial, we looked at the rules that dictate whether a protein is amenable for expression based on size, location and solubility.  This form of information can be accessed from protein databases such as UniProt or the highly curated Swiss-prot. Once you have determined that a protein is likely to be expressed, and identified a suitable expression system, you will need the sequence of nucleotides required to produce the protein. Protein expression systems include e. coli (the most widely used), bacillus subtilis, yeast (Saccharomyces cerevisiae), insect cells (via baculovirus) and mammalian cells. Yeast, insect or mammalian expression systems are needed, where the protein of interest requires complex post-translational modifications for functionality.

In this tutorial, we're going to look at where you can get the nucleotide sequences required for expressing your protein of interest, in an expression system.

Outline

1. Databases for nucleotide sequences

2. Downloading the sequences

3. Steps After Downloading the Nucleotide Sequences
   
   

Databases for Nucleotide Sequences

When seeking to express proteins in an expression system such as e.coli, yeast, or mammalian cells,  you need the Coding Sequences (CDS) for that protein. The CDS is the nucleotide sequence encoding the protein of interest. It excludes regions of a gene known as the 5’ and 3’ untranslated regions (UTRs). Introns likewise, are excluded from CDS sequences. 

On the go? Watch the tutorial here:

NCBI (National Center for Biotechnology Information)
Website: https://www.ncbi.nlm.nih.gov

Steps:

1. Search for the gene of interest in the NCBI Gene database.

2. Look for the "mRNA and Protein" section.

3. Download the CDS (coding sequence) in FASTA format.
   
   

2. Ensembl
Website: https://www.ensembl.org

Steps:

1. Search for your gene of interest in Ensembl.

2. Navigate to the "Gene" page and select the transcript of interest.

3. Export the CDS as a FASTA file.
   
   

3. UniProt

Website: https://www.uniprot.org

Steps:

1. Search for your protein of interest.

2. Check the "Sequence" section to find CDS-related data or links to nucleotide sequences.

3. Export the sequence in FASTA format.

4. JGI Genome Portal
Website: https://genome.jgi.doe.gov

Steps:

1. Search for genes in specific microbial, fungal, or plant genomes.

2. Download annotated CDS sequences.

5. PlasmoDB
Website: https://plasmodb.org

Steps:

1. Search for your gene of interest in the search bar using the gene name, ID, or keyword.

2. Click on the relevant gene from the search results to view its detailed information.

3. Navigate to the "Sequences" tab on the gene's detail page.
   
   

4. Choose the sequence type you need:

1. Genomic DNA: Includes exons and introns.

2. CDS (Coding Sequence): The protein-coding exons, spliced and concatenated. No introns or untranslated regions.

3. Protein Sequence: The translated amino acid sequence.
   
   

5. Select FASTA format and click "Download" to save the sequence.

There are numerous additional databases for downloading organism specific sequences. For example, TAIR (The Arabidopsis Information Resource) etc.

Steps After Downloading the Nucleotide Sequences

You may improve the amount of soluble product or the efficiency with which your protein is translated, by optimizing the codons. This may be essential in heterologous systems. For example, if you are expressing a human protein in e. coli. Codon optimisation involves replacing certain codons in the gene with more commonly used codons, provided that they are synonymous. This ensures that the sequence that you have, corresponds with the most highly used codons in your host organism. You can use a tool such as GenScript’s Codon Optimisation Tool.

Website: https://www.genscript.com/tools

Steps:

1. Input the desired protein or organism-specific gene.

2. Optimize the CDS for your expression system.
   
   Use Case: Optimized sequences for custom expression.

Once you have the sequence, you will need to design primers to amplify the gene from its native environment. To do this, total RNA is often isolated and converted to something called complementary DNA, after enrichment for messenger RNA (mRNA). Once the complementary DNA is obtained (cDNA), PCR can be performed to isolate the gene of interest.

The most common reason for failure to detect a gene from a cDNA prep, is that the gene is poorly represented in the cDNA. Adding more cDNA to the PCR may work. Alternatively, you may extract the cDNA from a different tissue, where applicable.   If all fails, or where it is not possible to amplify a gene from cDNA, there are companies that can synthesise the gene for a price.  The gene sequence is designed and chemically synthesized in vitro.