BIOL 251 - Final Exam

How is total magnification of compound microscopes calculated?

Objective x ocular

Why are microbial cells stained?

Most microbes are transparent/translucent so staining…

Improves contrast

Provides information about the bacteria

How are bacteria stained?

Fixation occurs first - preserves and fixes structures; firmly attaches them to slide

Heat or chemical fixation is used

Smear is then flooded with stain of choice

What are basic dyes?

Have positive charge; attracted to negative charge

Stains the cell itself

What are acidic dyes?

Have negative charge; attracted to positive charge

Stains the background

What are the steps to Gram staining?

Crystal violet

Iodine

Alcohol

Safranin

What is crystal violet?

Primary stain that adheres to Gram-positive bacteria

What is the iodine solution used for?

A mordant which makes dye less soluble so it adheres to cell walls

What is alcohol used for?

A decolorizer that washes away stain from Gram-negative cell walls

What is safranin?

A counterstain that allows dye to adhere to Gram-negative cells

What is the most critical step in the Gram stain and why?

The 3rd or decolorizing stage is the most critical

Timing is very important and if done incorrectly, will lead to wrong conclusions

Left on too long - Gram-positive will look Gram-negative

Left on too short - Gram-negative will look Gram-positive

What is the purpose of the Gram stain?

To differentiate Gram-positive from Gram-negative bacteria

How does the Gram stain work?

The primary stain will stay and adhere to Gram-positive bacteria for the whole process

Gram-negative bacteria will get decolorized and counterstained in the last two steps

Dyes show differences in cell wall structure

Why does the Gram stain technique not work on acid-fast bacteria?

Has a waxy layer of mycolic acid that repels the dyes

Why does staining not work for bacteria with capsules?

Dye doesn’t penetrate the capsule

What is resolution?

Degree of detail visible

Why is oil used to improve resolution?

Has the same refractive index as glass

Prevents light refraction which prevents loss of light rays

What is the limit of resolution for light microscopy?

~.2 microns

Can’t visualize viruses

Gram-positive wall structure

Thick layer of peptidoglycan

Alternating layers of NAM and NAG sugars connected with peptide interbridges (pentapeptides) and tetrapeptide chains

Teichoic acid - enhance rigidity and binding

Porous, fully permeable - many small molecules can enter

Gram-negative wall structure

Thin layer of peptidoglycan

Periplasmic space

Inner and outer membrane

Bilayer composed of phospholipids and lipopolysaccharides (LPS)

Barrier to some antibiotics

Relative sizes from smallest to largest

Lipid → protein → virus → bacteria, mitochondria → RBC → animal/plant cell → pollen/human egg

What are the types of prokaryotic cells?

Bacteria and Archaea

What are some descriptions of Bacteria?

Found nearly everywhere

Some are pathogenic

Cell walls have peptidoglycan

What are some descriptions of Archaea?

Can live in very extreme environments (extremophiles)

None have shown to be pathogenic

What are some classes of eukaryotic cells?

Protists, Fungi, Helminths

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)

Capsid

Outer protein coat of viruses

Surrounds genome - protect and transport the genome to other cells

Capsomeres

Subunits that comprise the virus’s capsid

Nucleocapsid

Capsid plus nucleic acids

How are viruses are visualized?

Can’t be seen with light microscopy

Electron microscope is used instead

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

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

What are the domains of life?

Bacteria, Archaea, Eukarya

What is the science of taxonomy?

Classification, description, identification, and naming of living organisms based on common characteristics

System to name and categorize organism, format standardized

Provided consistent terminology for related organisms

Based on genetic relatedness

What are the rules of nomenclature for living things?

Binomial system of Genus species naming

Genus is Capitalized

Species is lowercased and italicized

How is genetic relatedness determined?

Relatedness is based on comparisons of DNA sequences of rRNA

rRNA genes slowly mutate at constant, predictable rates

Closely related species have similar rRNA sequences and vice versa

How do species and strains differ?

Species - basic unit of classification that contains specific strains/isolates

Strains - genetic variant of a species; different subtype/culture

Different strains have different levels of pathogenicity

How are prokaryotic and eukaryotic cells similar?

Chemically similar - nucleic acids, proteins, lipids, carbs

Have ribosomes

How are prokaryotic and eukaryotic cells different?

Prokaryotes - NO membrane-bound nucleus; 70S ribosomes

Eukaryotes - membrane-bound nucleus; more complex; contain membrane-bound organelles; 80S ribosomes

Why is it important to understand the differences/similarities between the cell types?

Effective antibiotics target unique features of prokaryotic cells

What are flagella?

Most common mechanism of motility for prokaryotes

Composed of flagellin

Different arrangements

What are pili?

Hollow, short, thick protein filaments

For conjugation (Gram-negative)

Useful for adhesion to surfaces

What are F pilus (sex pilus)?

Facilitates transfer of DNA between cells

What is the structure of peptidoglycan?

Alternating NAM and NAG sugars

What is the function of peptidoglycan?

Protects the cell from lysing

Why is peptidoglycan unique?

Only found in bacteria

What is a capsule?

Distinct and gelatinous

Firmly attached to the cell wall, encloses one or two cells

Allows some organisms to evade defense systems and cause disease

What is a slime layer?

Diffuse and irregular

Allows bacteria to adhere to specific surfaces

Loosely attached to the cell wall, often many cells are embedded

How is prokaryotic DNA arranged?

Single, circular, double-stranded

What are plasmids?

Extra-chromosomal DNA

Small, contain only a few genes

Self-replicating

Not essential for the cell’s survival

May be gained, lost, or transferred

Why are plasmids important?

Code for resistance to antibiotics, disinfectants, ability to metabolize unusual nutrients, or ability to produce certain toxins

Biotech/genetic engineering tool

What is an endospore?

Type of dormant cell

Formed in the cytoplasm

Thick walled, dehydrated structures

Resistant to heat, harsh chemicals

Found basically anywhere

Protects bacterial genome until environment is favorable

Organism can exist in the spore state for 100s of years

Why is gene transfer significant?

New genetic material can code for resistance or virulence

What are three mechanisms of gene transfer in bacteria?

Transformation

Transduction

Conjugation

What is transformation?

Naked DNA is taken up from the environment

What is transduction?

Genes are transferred between cells in a phage

What is conjugation?

Use of a hollow tube called a conjugation pilus to transfer genes between cells

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

How does lysogenic conversion contribute to strain variation?

One strain produces toxin and one doesn’t - the toxic strain went thru lysogenic conversion

What are some toxins that were transferred to bacteria via transduction?

E. coli - shiga toxin

V. cholerae - cholera toxin

C. diptheriae - diphtheria toxin

C. botulinum - botulinum toxin

What are the steps of a growth curve?

Lag phase

Log phase

Stationary phase

Death/decline phase

What is the lag phase?

No increase in number of living bacterial cells

Cells are preparing to multiply

What is the log phase?

Exponential increase in number of living bacterial cells

What is the stationary phase?

Plateau in number of living bacterial cells

Rate of cell division and death are roughly equal

What is the death/decline phase?

Exponential decrease in number of living bacterial cells

What kind of information can be derived from a growth curve?

Can deduce the rate of growth of bacteria

What are obligate aerobes?

Microbes that require oxygen

Produce SOD and catalase

What are obligate anaerobes?

Microbes that grow in the absence of oxygen

Oxygen intolerant

Doesn’t produce SOD or catalase

What are facultative anaerobes?

Microbes that grow in aerobic or anaerobic conditions

Produces SOD and catalase

What are microaerophiles?

Microbes that require between 5-15% of atmospheric oxygen

Produces some SOD and catalase

What are aerotolerant anaerobes?

Microbes that are indifferent to the presence of oxygen

Anaerobic respiration

Produces SOD but NOT catalase

What is pH and its ranges?

Measure of H+ ion concentrations

Acidic - 0 - <7

Neutral - 7

Basic - 7< - 14

What are neutrophiles?

Microbes that survive in a pH range of 5-8

What are acidophiles?

Microbes that survive in a pH below 5.5

What are alkaliphiles?

Microbes that survive in a pH above 8.5

What are some practical applications of pH in relationship with microbes?

Using highly acidic or alkaline environments to inhibit bacterial growth

Ex) lemon juice, vinegar, pickled foods

What are psychrophiles?

Microbes that grow in cold environments

What are mesophiles?

Microbes that grow in room temperature environments

~37C

Most common human pathogens

What are thermophiles?

Microbes that grow in hot environments

What hyperthermophiles?

Microbes that grow in extremely hot environments

How does salt concentration contribute to food preservation?

High salt concentrations lead to a hypertonic solution

The hypertonic solution causes microbes to go through plasmolysis

What are halotolerant bacteria?

Bacteria that can tolerate high concentration of salt (10% +)

Source of foodborne infections

What are halophilic bacteria?

Bacteria that require a high concentration of salt in their environment (salt water)

3%-9%

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 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

What is sterilization?

Eliminates all forms of life (except prions)

Autoclave

Membrane filtration

Ethylene oxide

Commercial sterilization

2% alkaline gluteraldehyde

Autoclave

Sterilization

121C, 15psi, 15 mins

Membrane filtration

Sterilization

Used for heat-sensitive liquids