BIOL 251 EXAM 2 STUDY GUIDE

learn these for the exam tmr!!!!

BSL-1

Nonpathogenic strains of E. coli, B. subtilis

Lowest risk of infectivity

Fewest precautions required

Don’t cause infections in healthy individuals

BSL-2

Moderate risk to humans

Microbes commonly found in the area

Precautions include PPE and safety cabinets

S. aureus

BSL-3

Potential to be lethal via inhalation

Indigenous or exotic

Mycobacterium tuberculosis, Bacillus anthracis

Respirator, safety cabinet

West Nile virus, HIV

BSL-4

Most dangerous

Often fatal

Transmitted by inhalation

Agents have no treatment or vaccination

Highest level of precautions

What are the factors that influence the level of microbial control needed?

What is the item going to be used for?

Identify situation

What’s being eliminated?

What's the potential risk of infection?

Environmental conditions (pH, temperature, presence of organic material)

What is the number of microbes to eliminate?

What is the D-value?

What situations require specific levels of control?

Daily life

Hospitals

Microbiology labs

Various industries - food production, water treatment, other industries

Fomite

Inanimate object used by humans that can transmit microbes

What are the most difficult microbes to eliminate?

Bacterial endospores - C. boutlinum

Protozoan cysts

Mycobacterium species

Pseudomonas species

Naked viruses

Why are endospores hard to eliminate?

Resistant to heat, drying, and numerous chemicals

Why are protozoan cysts hard to eliminate?

Endospore-like state for protozoan

Generally excreted in feces and cause diarrheal disease

Why are Mycobacterium spp. hard to eliminate?

Cell wall structure initiates resistance

Why are Pseudomonas spp. hard to eliminate?

Can grow in presence of many chemical disinfectants

Why are naked viruses hard to eliminate?

Lack envelope and are more resistant to chemical killing

Enveloped viruses are easier to kill with disinfectants because phospholipid bilayer is destroyed by disinfectant

Critical items

Used inside the body and penetrate body tissue

Must be sterile

Surgical instruments, catheters, IV fluids

Semi-critical items

Contacts mucous membrane and nonintact skin

Doesn’t penetrate body tissues

GI endoscopes, some respiratory therapies

Non-critical items

Unbroke skin

Stethoscope

What are the various methods of control?

Sterilization

Aseptic technique

Commercial sterilization

Disinfection

Pasteurization

Decontamination

Sanitation

Degermining

Antiseptics

What is sterilization?

Eliminates all forms of life (except prions)

Lab, medical, manufacturing, and food industry settings

Achieved thru physical means - high heat, pressure, or filtrations

Achieved thru chemical means - sterilant

What is aseptic technique and why is it important in lab and clinical settings?

Prevents contamination of sterile surfaces

Protocols that maintain sterility (asepsis)

Prevent contamination of patients (avoiding sepsis)

Sterile field - necessary for medical procedures that carry contamination risk

What is commercial sterilization?

Uses heat at a temperature low enough to preserve food quality but high enough to destroy common pathogens

Main target are C. botulinum spores

What is disinfection?

Inactivates most microbes on fomite surface using antimicrobial chemicals or heat

Not sterilization

What is pasteurization?

Reduces number of spoilage organisms and pathogens while maintaining food quality

Not sterile and product will still spoil

High-temperature short-time (HTST) pasteurization

Ultra-high temperature (UHT) pasteurization

What is HTST pasteurization?

72C for 15 seconds

Lowers bacterial numbers while preserving the quality of the milk

What is UHT pasteruization?

138C for 2 or more seconds

Can be stored for a long time in sealed containers without being refrigerated

Helpful method when refrigeration is limited

What is decontamination?

Reduces pathogens to a level considered safe

What is sanitation?

Reduces microbial populations to levels acceptable for public health

What is degerming?

Significantly reduces microbial numbers

Handwashing

What are antiseptics?

Antimicrobial chemicals safe for use on skin surfaces

Hydrogen peroxide, isopropyl alcohol

What are the conditions in an autoclave?

121 C and 15 psi for 15 minutes

Sterilizes and kills endospores

What is moist heat?

Denatures proteins

Relatively fast, reliable, safe, and inexpensive

Widely used

What is boiling?

Destroys most microbes and viruses but not endospores

Not a form of sterilization

What is inceration?

Burns cell components to ashes

Flaming inoculating loops

Destroy medical wastes and contaminated animal carcasses

What are dry heat ovens?

Destroys cell components and denatures proteins

Less efficient than moist heat and require longer times and higher temperatures

What are physical methods of microbial control?

Refrigeration/freezing

Radiation

Filtration

What is refrigeration/freezing?

Refrigeration - temperatures between 0-7C

Inhibits microbial metabolism, slowing the growth of microbes

Preserves refrigerated products such as foods or medical supplies

Freezing - below -2C stops microbial growth and may kill susceptible organisms

Cultures and medical specimens can be stored for long periods at ultra-low temperatures

Ultra-low freezer maintains temperatures at or below -70C

What is radiation?

Causes damage to DNA and potentially to plasma membrane

Used to sterilize heat sensitive materials - medical equipment, surgical supplies, medications

Some endospores can be resistant

3 sources - gamma radiation, x-rays, electron accelerators

What is filtration?

Air filtration - HEPA filter removes nearly all microbes from air (.3 micron pore size filter)

Membrane filtration - liquid filtration, for heat sensitive fluids, traps microbes on filter

What are the different types of germicidal chemicals?

Sterilants

High-level disinfectants

Intermediate-level disinfectants

Low-level disinfectants

What are sterilants?

Treat heat sensitive critical instruments

Kills endospores, Mycobacterium spp., fungi, and all viruses

What are high-level disinfectants?

Treat semi-critical instruments

Kills some endospores, Mycobacterium spp., all fungi, and all viruses

What are intermediate-level disinfectants?

Disinfects non-critical instruments

Doesn’t kill endospores, kills some Mycobacterium spp., all fungi, some non-enveloped viruses, and all enveloped viruses

What are low-level disinfectants?

General purpose

Disinfect furniture, floors, and walls

Doesn’t kill endospores and Mycobacterium spp., kills some fungi and enveloped viruses but no non-enveloped viruses

How is the appropriate chemical chosen?

Toxicity

Presence of organic matter

Compatibility with material being treated

Residue

Cost and availability

Ease of use

Storage/stability

Environmental risk

Alcohols

Kill bacteria and fungi but not endospores or non-enveloped viruses

Denature proteins (inhibit cell metabolism)

Disrupt membranes (cell lysis)

Common use - antiseptic on skin, swabbing skin for degerming before needle injection, alcohols also are the active ingredient in instant hand sanitizers

SARS-CoV-2 is an enveloped virus and may be inactivated by alcohol-containing products

How do you properly use hand sanitizer?

CDC recommends covering hands with sanitizer

Rub hands together until dry - ~20 seconds

Hand sanitizer should be between 60-90% alcohol to be effective

Works both by denaturing proteins and by disrupting microbial cell membrane but will not work effectively in presence of visible dirt

Aldehydes

Inactivates enzymes and nucleic acids

Strong, broad-spectrum disinfectant

Kills bacteria, fungi, viruses, and endospores - sterilization at low temperatures (alternative for autoclaving/heat sterilization)

Formaldehyde - preserves biological specimens by crosslinking proteins

Formalin - used in vaccines to kill bacteria and inactivate viruses

Irritating to skin - not a good antiseptic

Ethylene oxide

Gaseous sterilizing agent - fabric, pillows, artificial hips, pacemakers, petri dishes

Highly penetrating and can sterilize items within plastic bags such as catheters, disposable items in labs and clinical settings

Items are sterilized in a special chamber

Cold sterilization - good for heat sensitive items

Bisbiguanides

Antiseptics before surgery - surgeon hand scrub

Chlorhexidine - common ingredient in skin creams, disinfectants, mouthwashes/oral rinses, catheters, and surgical mesh

Broad-spectrum activity against yeasts, gram-positive/negative bacteria, and enveloped viruses - exception is Pseudomonas aeruginosa which may develop resistance on repeated exposure; not effective for Mycobacterium, non-enveloped viruses, or spores

Halogens

Chlorine - swimming pools, disinfect waste liquid, surfaces, drinking water

Sodium hypochlorite (bleach) - kills bacteria and viruses, less reliable when organic material is present

Drinking water contains .5ppm chlorine

Chloramine - disinfection of drinking water (tablets used by military)

Chlorine dioxide - gaseous agent used for fumigation and sterilization of enclosed areas; also commonly used for disinfection of water

Chlorine compounds can irritate skin, nose, and eyes - some protozoan cysts are resistant to chlorine (Cryptosporidium)

Iodine - oxidizes cellular components, destabilized macromolecules

Heavy metals

Kill microbes by binding to proteins, which inhibits enzymatic activity

Small concentrations of metal ions accumulate in cells - cause proteins to denature

Not selectively toxic to microbial cells

Mercury - not used in the US due to toxicity concerns

Silver - used as an antiseptic, some catheters and bandages contain silver

Silvadene cream - topical for burn wounds

Silver nitrate drops - were used for ophtalmia neonatorum (eye infection in newborn) and replaced by antibiotic creams

Copper nickel, zinc

Copper - algacide

Zinc chloride - mouthwashes

Phenolics

1800s - scientists started using chemicials for disinfection

1860s - Joseph Lister began using carbolic acid as a disinfectant for the treatment of surgical wounds

Phenolics - compound in mouthwashes and throat lozenges

Less toxic than phenol - disrupt membrane, denature proteins to inhibit microbial growth

Listerine, Lysol

pHisoHex - handwashing in hospital settings, effective for Staph and Strep that cause skin infections

Triclosan - in many antibacterial products until 2016, FDA banned products containing it and 18 other chemicals

Why was triclosan banned?

Researchers examined data from many different studies and found that people who washed with plain soap had the same chances of getting sick as people who used antibacterial soap containing triclosan

What are the targets in bacterial cells that are affected by germicidal chemicals?

Cytoplasmic membrane

Proteins

DNA

How does the assay for testing the effectiveness of germicidal chemicals work?

Disk-diffusion assay is used to determine effectiveness of chemical agents against a particular microbe

Plate is inoculated with various antimicrobial discs

Zone of inhibition around each disc indicates how effective the antimicrobial agent is

No zone of inhibition = doesn’t work

Characteristics of viruses

Infectious acellular particles

DNA/RNA contained in protein coat

Inert - no metabolism, replication, motility

Need a host cell to replicate

Can’t be grown in a pure culture

Specific for cell type or host

Viral structure

Nucleic acid surround by a protein coat (capsid)

How are viruses are visualized?

Can’t be seen with light microscopy

Electron microscope is used instead

Comparative size of viruses

~10 nanometers

Smaller than bacteria and human cells

Smallest to largest - viruses → bacteria → human cells

Capsid

Outer protein coat of viruses

Surrounds genome - protect and transport the genome to other cells

Capsomeres

Subunits that comprise the virus’s capsid

Nucelocapsid

Capsid plus nucleic acids

Why are viruses considered to be nonliving?

Acellular

Don’t exhibit characteristics of living things - inert

No metabolism, replication, motility

Needs a host cell to do things

What are viruses composed of?

DNA/RNA in a protein coat

Viral genome strucutre

Genomes are small

DNA or RNA

Single- or double-stranded

Segmented or single molecule

Can’t be DNA + RNA

What are the viral shapes?

Polyhedral/icosahedral - 20 flat triangles

Helical - capsomeres arranged in helix

Complex - phage (sheath, tail pins, and fibers), icosahedral nucleocapsid (head) and helical protein (tail)

Protein spikes

Attach to receptor sites on host cells

Phages attach by tail fibers

Protein structures

Enveloped viruses

Surrounded by lipid bilayer obtained from host cell

Envelope is small portion of phospholipid membrane obtained as the virion buds from a host cell

Matrix between nucleocapsid and envelope

Contain viral glycoproteins required for infecting cells

Non-enveloped (naked) viruses

More resistant to disinfectnats

Classifications of viruses

International Committee on Taxonomy of Viruses - viral species names are italicized using a genus and species name

Baltimore system - genome structure, virus shape, mechanisms of replication, presence or absence of envelope, disease symptoms the virus causes

Enteric transmission

Intestines

Polio, rotavirus

Respiratory transmission

Respiratory system

Influenza, measles, rhinovirus

Zoonotic transmission (zoonoses)

Animals → humans

Rabies, West Nile virus

Arbovirus transmission

Arthropods

Sexual transmission

Sex

Herpes, hepatitis, HIV

What are some components that viral genes code for?

Viral protein coat

Proteins required for replication of viral nucleic acid

Proteins required to get the virus into and out of the cell

How do viruses get into and out of cells?

Enter

Endocytosis - enveloped or naked

Fusion with host cell - enveloped virus

Leave

Budding - virus leaves with phospholipid bilayer from host cell

Apoptosis - non-enveloped viruses released when host cell dies; initiated by virus or host

Steps of viral replication

Attachment

Penetration and uncoating

Synthesis

Assembly

Release

Attachment (adsorption)

First step in viral replication

Virus binds specifically to one or more receptor sites on host cell

Penetration/uncoating of virus

Second stage in viral replication

Entire virus enters

Endocytosis (non-enveloped) or fusion with host cell membrane (enveloped)

Bacteriophage - puncture and inject their DNA

Virus uncoated and genome released

Synthesis

Third step in viral replication

Genome released

Synthesis of viral nucleic acids and proteins

Eclipse phase

Interval between penetration and production of virions

Assembly

Fourth step in viral replication

New viruses are fully made

Release

Fifth step in viral replication

Lysis and budding are both common

Budding - most enveloped viruses; covered with matrix protein and lipid envelope

Apoptosis - non-enveloped viruses released when host cell dies; initiated by virus or host

Viral host specificity

Host range is very small and specific

Virion must bind to protein receptor found in the host cell’s membrane

Host cell receptor carries out normal functions for cell

Non-enveloped viruses attach via arrangement of shape of capsids

Bacteriophage replication

Good model for understanding how viruses replicate

Virulent - attachment → penetration → biosynthesis → maturation → lysis

Temperate - infects cell → phage DNA incorporated into host genome → cell divides and DNA is passed on → under stressful condition, prophage DNA is excised from chromosome and enters lytic cycle → DNA and proteins replicate → new phage particles made → lysis

Temperate phage

Phage DNA integrate into host’s DNA - phage DNA → prophage

Prophage is replicated along with host chromosomes

Most often occurs in slower growing, nutrient-deprived cells

Lysogenic cycle - phage DNA is incorporated into host genome, forming prophage, which is passed onto subsequent generations of cells

Lytic cycle - usually actively growing cells, environmental stressors cause prophage to enter lytic cycle, cell lysis and phages released

Lytic phage replication

Phage bind specific receptors on bacterial cell

Phage DNA is injected into cell

Transcription

Replication of phage DNA

Assembly of phage components

Cell lysis and phage release - cell is destroyed

Virulent phage

Lyses cells

Leads to death of host cell

Only does lytic cycle

Lysogen

Bacterium that carries a prophage integrated into its chromosome

Lysogenic conversion

Properties of a bacterial cell are changed due to the bacterium carrying a phage

Prophage

Form of a phage whose DNA has been integrated into the host’s DNA

What are some properties encoded by a prophage?

Toxins and antibiotic resistance

Why is studying DNA important?

Understand how genetic diversity occurs in bacterial cells

Proteins involved in bacterial DNA replication make great targets for antibiotics

What is the flow genetic info?

Central dogma

DNA →Transcription → RNA → Translation → Proteins

Phenotype

Physical expression of genomic properties

May be influenced by the environment

Genotype

DNA content, genetic makeup

Determined by genetic content of an organism

What are the two genotypic changes that can occur in bacteria?

Mutations

Gene transfer

Mutations

Change in DNA sequence

Mutations are relatively stable

Genetic changes are inherited

Gene transfer

Process by which new donor genes are introduced into a recipient cell

Vertical gene transfer

From generation to generation

Mutations passed to progeny cells