Basic Science, Microbial Control & Lab Safety

Lesson focus - Classify cells and bacteria, describe bacterial and viral structures, explain viral multiplication, compare microbial control methods, and apply lab and clinical safety practices.

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Basic Science

Classification of Cells

Bacterial Shape

Bacterial Spatial Arrangement

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Bacterial Structure and Function

Main Structures

• Cellular envelope
  • Cytoplasmic membrane
  • Periplasmic space
  • Cell wall/peptidoglycan
  • Outer membrane
  • Cell capsule
• Cytoplasm
• Appendages
  • Fimbria/pili
  • Flagella

Cellular Envelope

Cytoplasmic Membrane

• Also called the inner membrane
• Found in Gram-negative and Gram-positive bacteria
• Provides selective permeability
• Innervated with essential proteins

Periplasmic Space

• Located between the peptidoglycan and cytoplasmic membrane
• Gel-like substance with enzymes that detoxify harmful substances
• Secures nutrients
• Larger in Gram-negative bacteria

Cell Wall / Peptidoglycan

• Gives the bacterial cell its shape
• Thicker in Gram-positive bacteria

Outer Membrane

• Found in Gram-negative bacteria
• Provides selective permeability through porins
• Adds a barrier against the outside environment

Cell Capsule / Slime Layer

• Located outside the outer membrane
• Found only in select Gram-negative bacteria
• Adds protection
• Uses porins for selective permeability
• Helps resist phagocytosis
• Allows attachment to surfaces

Cytoplasm

• Gel-like substance
• Contains organelles and DNA/RNA

Appendages

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Viruses

Key Terms

• Virus - submicroscopic infectious agent that replicates only inside living cells.
• Viruses are unable to reproduce genetic material on their own.
• Capsid - shell that encloses viral genetic material.
• Spikes - help the virus attach to host cell membrane receptors.
• Virion - complete virus particle with a genetic core and protein coat; the extracellular infectious form.

Multiplication of Bacteriophage

1. Adsorption / attachment

   • Tail fibers or spikes bind to specific receptors on the bacterial cell surface.
   • Viral tropism means the virus can attach only to specific binding sites.
   • Tropism may be cellular, tissue, or host specific.

2. Penetration

   • Virus injects nucleic acid through the bacterial cell wall into the host cell cytoplasm.
   • Some viruses use a tubelike projection.
   • Some viruses use digestive enzymes to break down the host cell wall.
   • Multiple cytoplasms can fuse together to form syncytia, which are formed by several cells combining after viral infection.

3. Assembly

   • Phage DNA directs the host cell to synthesize viral components.

4. Maturation

   • Viral components assemble into virions.

5. Lysis

   • Host cell lyses and new virions are released.

Multiplication of Animal Virus

1. Adsorption
2. Penetration - host cell engulfs the virus by endocytosis.
3. Uncoating - capsid is broken down, releasing the viral genome.
4. Replication / macromolecular synthesis - virus uses the host cell to replicate genetic material.
5. Assembly - capsid forms.
6. Release - mature virions exit host cells by lysis or budding.

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Tools for Study and Control of Microorganisms

Microbial Control: What to Consider

• Can the site or item be treated without damage?
• Environmental conditions:
  • Can the item be exposed to heat?
  • Does the item contain biofilms?
  • Does the item need to be cleaned before disinfecting?
• Susceptibility or resistance of the microbe

Endospores

Endospores are dormant, highly resistant structures formed by vegetative bacterial cells when unfavorable environments are present.

Terminology for Microbial Control

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Physical Methods of Control

Temperature

Thermal Death Point

• Describes heat resistance among different microbes.

Dry Heat

• Object is exposed to direct flame.
• Requires high temperatures and long exposure times.
• Not all items can be exposed to direct flame.
• Useful when an item cannot be exposed to moisture.

Moist Heat

• Uses lower temperatures and shorter periods of time than dry heat.
• Autoclave uses steam under pressure.
• Inactivates fungi, bacteria, viruses, and some but not all spores.
• Equipment must be cleaned first.
• Efficient, quick, and cheap.

Refrigeration and Freezing

• Halts the growth of many pathogens.
• Slows metabolism.

Osmotic Pressure

• Uses a hypertonic solution to dehydrate the cell.
• Interrupts metabolism and growth capability.

Radiation

• Damages DNA.
• Causes mutations and cell death.
• Most radiation does not penetrate deeply.
• Primarily used to disinfect surfaces, air, and fluids.

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Chemical Methods of Control

Disinfectants and Antiseptics

• Spores may survive.
• Consider how much bacteria must be killed and whether normal flora may be affected.

Factors Influencing Antimicrobial Effectiveness

• Population size
• Population composition
• Contact time
• Concentration of chemical agent
• Temperature
• Organic matter

Antimicrobial Agents

Glutaraldehyde is used to clean bronchoscopes and requires a 20-minute contact time.

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Lab Techniques

Gram Staining

• One of the most common staining techniques.
• Determines whether bacteria are Gram-positive or Gram-negative.

Gram Staining Process

1. Cells are stained with crystal violet.
2. Cells are decolorized with alcohol.
3. Cells are counterstained with safranin, which is red.

Gram Staining Interpretation

Acid-Fast Staining

• Smears are stained with carbol fuchsin and decolorized.
• Smears are counterstained with methylene blue.
• Cells that retain red carbol fuchsin stain are acid-fast bacteria.
• Useful in identifying tuberculosis.

Agar Differentiation

• Promotes the growth of specific organisms while killing off others.
• Bacteria may respond differently to agar by showing certain colors in response to specific media or nutrients.

Kirby-Bauer Disk Diffusion

• Susceptibility test used to evaluate disinfectants and antibiotics.
• Culture is spread over agar.
• Paper disks with specific concentrations of antibiotics or disinfectants are evenly spaced on agar.
• The inoculated plate is incubated.
• If the antibiotic or disinfectant affects growth, a halo forms around the disk.
• The halo is the zone of inhibition.

Minimal Inhibitory Concentration Method

• Culture is added to a series of test tubes with decreasing concentrations of a specific antibiotic.
• Tubes are incubated and evaluated by turbidity, or cloudiness.
• The goal is to identify the lowest concentration of drug that inhibits visible growth.
• That value is the minimal inhibitory concentration, or MIC.

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Lab and Clinical Safety

Biosafety

• Occupational hazards are risks accepted as consequences of a particular occupation.
• Healthcare workers are exposed to biological agents and diseases every day.

Handwashing

Proper handwashing is the simplest and most effective method of control to prevent infectious disease spread.

1. Wash with approved soap and warm running water.
2. Rub vigorously for at least 20 seconds.
3. Pay attention to the backs of hands, between fingers, wrists, and nails.
4. Rinse well under running water.
5. Dry hands with a single-use towel.
6. Turn off water with a paper towel to prevent recontamination.

Eyewash and Safety Showers

• Must be accessible within 10 seconds after exposure to hazardous materials.
• Must be checked regularly for function.

Refrigerators and Freezers

• Temperatures must be checked regularly.
• Must be labeled for lab purposes and not for food storage.
• Stored material must be sealed, capped, secure, and properly labeled.
• Waste and expired materials must be removed regularly.

Disposal of Hazardous Waste

Sharps Safety

• Always use caution when handling sharps.
• Never recap needles.
• Do not point needles toward any part of the body or toward others you do not intend to stick.
• Do not remove needles from disposable syringes by hand.
• Do not bend, break, or manipulate used needles by hand.

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Protective Gear

Gloves

• Used for all patients in all settings, all of the time.

Respiratory Protection

Clothing

• Change clothing if visibly soiled.
• Follow approved lab guidelines, which may vary by setting such as the operating room.
• Wear closed-toe, puncture-resistant shoes.
• Additional protection, such as gowns, may be needed for contact precautions or splashing risk.
• Remove soiled gown or clothing quickly.
• Perform hand hygiene to avoid transfer of microorganisms.

Eye Protection

• Safety glasses with side shields may be used.
• Full face shield may be needed to protect mucous membranes.
• Use eye protection when sprays of blood, body fluids, secretions, or excretions are likely to splash.

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Patient Care and Equipment Care

Private Rooms

Private rooms are used for:

• Patients who contaminate the environment under transmission-based precautions
• Patients who do not or cannot assist with appropriate hygiene

Limit transport to essential purposes only. The patient should wear a mask during transport.

Reusable Equipment

• Do not use reusable equipment for another patient unless it has been properly cleaned.
• Dedicate noncritical equipment to a single patient or patient cohort if possible.
• Handle contaminated equipment in a way that minimizes cross-contamination.
• Clean, disinfect, or sterilize equipment according to the manufacturer.

Vent Circuits and VAP Protocols

• Ensure circuits are clean and free from rainout.
• Use HEPA filters inside and outside the machine.
• Protect the ventilator from spreading pathogens.
• Place filters between the machine and external circuit, proximal to any humidification.
• Minimize breaking the circuit.
• Exchange the circuit only if visibly soiled or damaged.
• Routinely drain tubing condensation away from the patient and discard it.

Accoutrements

• Covered suction catheters help reduce ETT contamination.
• HMEs should be changed every 4 days or if visibly soiled.

Humidifiers

• Fill with sterile water.
• Use prefilled disposable units if available.
• Discard remaining fluid, clean, and dry before refilling.
• May be used for 30 days.

Oxygen Tubing and Oxygen Delivery Devices

• Discard when visibly soiled.

Bronchoscopes

• Require meticulous cleaning according to the manufacturer.
• Improper cleaning can transmit TB and Pseudomonas aeruginosa.

Bag-Mask Ventilation

• Use a one-way valve or disposable mask.
• Dispose of mask/valve after single-patient use.

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Handling Specimens

• Prevent contamination of the external surface of the container.
• Disinfect the external surface and place the specimen in an impervious bag.
• Prevent leaking by ensuring the integrity of the seal or cap.
• Label all specimens.

Specimen Labels Include

• Patient information
• Time of collection
• Location of site withdrawn when appropriate, such as right radial or left lower lobe

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Review

• Classification of cells
• Bacterial structure and function
• Viruses
• Physical and chemical methods of control
• Lab techniques
• Lab and clinical safety