Microbiology: Exam 1 Essay

Essay Question Preparation

Part III. An essay – Dynamics and control of prokaryotic growth. (Up to 20 points available). Each part answered completely and correctly counts as 4 points. Each part must be clearly marked and separated from other part of the essay.

a) Principles and Phases of prokaryotic growth – Describe: • Exponential growth of bacteria: explain specifics of bacterial cell division and growth. Describe equation used to calculate the bacterial population – Lecture 2 – slides 2, 3 and notes • Phases of bacterial growth curve: Describe what occurs at each phase of growth – Lecture 2 – slide 7 and notes

Bacteria grow by means of binary fission:

1.     Chromosome attaches to plasma membrane.

2.     Chromosome duplicates and cell enlarges.

3.     DNA is moved into each future daughter cell, and cell wall is formed.

4.     Cell is divides into two and separates.

Bacteria grow exponentially. Formula: N_n = N₀ * 2ⁿ, where N₀ = initial # of cells and n = # of generations.

Bacteria growth phases:

1.     Lag: Bacteria still adjusting to environment and preparing to grow.

2.     Log - Early (exponential growth): Production of primary metabolites

3.     Log – Late (exponential growth): Production of secondary metabolites

4.     Stationary: Rate of death = rate of growth, using dead cells’ nutrients. Stage of endospore formation for some bacteria.

5.     Death: Rapid decrease in pop. By more than 99%, As cell death > cell growth

6.     Prolonged decline: Remaining live cells undergo gradual decrease on pop.

b) Environmental factors influencing microbial growth – Describe and give species examples of bacterial groups by their requirements of: • Temperature: Describe 5 bacterial groups and their importance to humans – Lecture 2 – slide 8 and notes • Oxygen: Describe 5 bacterial groups and give example for each – Lecture 3 – slide 9 and notes • pH: name 3 bacterial groups and their pH range – Lecture 2 – slide 10 • Water availability: describe two bacterial groups based on their salt tolerance – Lecture 2 – slide 10 and notes

Psychophiles (Optimal 5ºC – 10ºC): Inhibit artic regions, NO significant importance

Psychotrophs (Optimal 20º - 28ºC): Responsible for food spoilage in refrigerator

Mesophiles (Optimal 30º - 37ºC): Majority of pathogens

Thermophile (Optimal 60º - 80ºC): Hotsprings, source of useful enzyme (PCR rxns)

Extreme thermophile (Optimal 85º - 95ºC): Hydrothermal vents.

Obligate aerobes – Need oxygen Mycobacterium tuberculosis, Bordetella pertussis

Facultative anaerobe – Grow better in oxygen, but can live w/o Escherichia coli, S. aureus

Obligate anaerobe – Grows in absence of oxygen, O₂ toxic Clostridium tetani, C. perfringens

Microaerophiles – Grows in small O₂ concentrations (2-10%) Helicobacter pylori

Aerotolerant microbes – Grows w/ or w/o O₂, obligate fermenters Streptococcous pyogenes

pH acidophile – Listeria monocytogenes, Acetobacter aceti

pH neutrophile – Escherichia coli

pH alkaliphiles – Bacillus halodurans, Microbacteriaceae spp.

Vast majority of bacteria prefer isotonic solution

Bacteria that grow in high salt concentration: facultative halophiles, halophiles

Facultative halophiles: Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus

Bacteria sensitive to salt: Escherichia coli, Streptococcus pyogenes

c) Nutritional factors influencing microbial growth & Cultivating prokaryotes in the laboratory – Describe: • Essential elements, Growth factors and fastidious bacteria: give a definition for each and give examples – Lecture 2 – slide 11 • Energy sources used by bacteria – name 3 bacterial groups and source of energy they use – Lecture 2, slide 11 • Define selective media and use a particular example to describe how they work – Lecture 2 – slides 13, 14 and notes • Define differential media and use a particular example to describe how they work – Lecture 2 – slides 14 and notes

Essential elements = all chemical elements required to grow bacteria (carbon, nitrogen, hydrogen

Trace elements only needed in small amounts: zinc, iron, manganese

Growth factors = Complex organic molecules, needed for fastidious bacteria to grow. (ex: beef extract, blood)

Fastidious bacteria = Bacteria with very specific requirements for growth (ex: Neisseria gonorrhoeae)

Photoautotroph – sunlight, CO₂ carbon source (ex: cyanobacteria)
Photoheterotroph – sunlight, Organic C source (ex: purple non-sulfur bacteria, green non-sulfur bacteria, and heliobacteria)

Chemolithoautotroph – inorganic energy, CO₂ C source (ex: sulfur bacteria, nitrogen fixing bacteria)

Chemoorganoheterotroph – organic molecules for both energy and C source (human pathogens)

Selective media:

MacConkey agar → bile salts selects for only gram negative bacteria (in particular enteric bacteria)

EMB agar → dyes select for gram negative bacteria

Thayer-Martin → Selects for N. gonorrhea, but preventing growth of other bacteria and fungi (vancomycin, colistin, treimethoprim, nystatin).

Differential media: Separate growing bacteria into groups. Maybe overlap with selective.

MacConkey agar → Differentiates between fermenting (pink) and non-fermenting bacteria (no change).

EMB agar → Differentiates between non-lactose fermenters (colorless) and lactose fermenters (produce lactic acid making colony stained dark, esp. E. coli = green metallic sheen).

Blood agar → alpha-hemolytic (green ring = limited pathogenicity) , beta-hemolytic (white halo = extreme pathogenicity), gamma-hemolytic (No effect - Less likely to be pathogenic).

d) Selection of antimicrobial procedure – Describe how selection of antimicrobial

procedure is affected by:

• Type of microbes present (use the graphs reviewed in lecture): give

examples of the most and the least resistant microbes. Define decimal reduction

time and explain how it affects duration of antimicrobial treatment – Lecture 2 –

slide 20 and notes • Number of microbes (use the graphs reviewed in lecture): explain how the

level of microbial contamination affects duration of treatment – Lecture 2 – slide

20 and notes • Risk of infection & medical instruments: describe how medical instruments

are treated depending on its use – Lecture 2, Slide 21 –

• Composition of the item: describe how the composition of an item to be

treated affects the selection of the antimicrobial treatment – Lecture 2, Slide 21

Most resistant = Prions

Endospores

Mycobacteria → acid-fast, hydrophobic waxy cell wall

Protozoan cysts → disinfectant resistant, but heat sensitive

Vegetative protozoan

Gram negative bacteria

Fungi

Naked viruses

Gram positive bacteria

Least resistant = enveloped viruses

Decimal reduction time (D) – Time required to reduce microbial population by 90%.

t = D * (lgN+1)

Longer decimal reduction time = longer treatment

If sample with 10⁴ microbes D = 1min, sample is sterilized in 4 mins → D = 2mins, sample is sterilized in 8 mins.

More microbes present in sample = longer treatment (Direct relationship)

If sample with 10⁴ microbes D = 1min, sample sterilized in 4mins → sample with 10² microbes, sample sterilized in 2mins.

Risk of infection:

Critical instruments - contacts with open wound (sterilized)

Semi-critical – mucous contact (disinfected)

Non-critical instruments – skin contact (sanitized)

                                                                             

Composition of item affects choice of treatment:

- Moisture sensitive → NO water/steam → Dry heat/(g) steriliants

- Heat sensitive → NO heat → (g) steriliants, irridation, chemical solutions

e) Use of Heat, Alcohol and Ethylene oxide to destroy microbes – Describe: • Moist heat: compare conditions (time, temp, pressure etc) and outcomes (what is killed, what survives) of pasteurization, boiling and autoclaving – Lecture 2 – slide 22 and notes • Dry heat: compare conditions and outcomes of flaming and hot air sterilization – Lecture 2 – slide 23 and notes • Alcohol: describe conditions of treatment. Compare the outcomes of alcohol treatment to outcomes of autoclaving – Lecture 2 – slide 27 • Ethylene oxide: describe conditions of treatment. Compare the outcomes of ethylene oxide treatment to that of autoclaving – Lecture 2 – slide 27

Boiling – 100ºC for at least 5mins

Pasteurization (3 settings):

Classical 63ºC for 30mins

High temp. Short Time 72ºC for 15sec

Ultra high 140ºC for 2 sec

Autoclaving 121ºC at 15psi for 20mins → Kills all except prions

Dry heat 170ºC for 2hrs / 200ºC for 1.5hrs

Flaming – burns microbes instantly 600 – 1600ºC

Alcohol requires at least 60% alcohol for 10 sec → kills bacteria and enveloped viruses, but NOT non-enveloped viruses and endospores

Ethylene oxide – Very strong oxidant that damages proteins, nucleic acids, lipids → Kills everything (except maybe prions). Treatment 3-5hrs → 8-12 heated forced air to remove possible carcinogenic chemicals.

Microbiology Lecture 4 Review: Chapter 11,13

Lecture Review - By: Remy Voloshchak

Chapter 11 Diversity of Bacteria

1.     Compare properties of bacterial phyla Proteobacteria, Firmicutes and Actinobacteria – Lecture 4, Slide 2 –
Properties of Proteobacteria, Firmicutes, and Actinobacteria (phylum of bacteria)

a.     Proteobacteria is gram-negative, firmicutes is gram-positive, and actinobacteria is gram-positive or acid-fast

b.     GC content: Actino – 69-73%, proteo – 40-50%, firmi – 23-53%

c.     They can all be aerobic; firmi and action can also be obligate anaerobes

d.     Morphology:

                                               i.     Proteo – bacilli, cocci, coccobacilli

                                             ii.     Firmi – bacilli, cocci, pleoimorphic

                                            iii.     Actino – bacilli, cocci, filamentous, branching

e.     All have small colonies, but action can have large colonies too

f.      Only proteo produces endotoxins, since it’s GRAM-NEGATIVE; they ALL produce exotoxins

g.     Proteo – not antibiotic producer; firmi and action – yes antibiotic producers

h.     They are ALL human pathogens

2.     Compare the properties of bacterial genera Haemophilus and Vibrio. Name principal species that are human pathogens – Lecture 4, Slides 3, 5-7 –
Ptoreobacteria à haemophilus, vibrio, pseudomonas, legionella

a.     Haemophilus – gram-negative gamma-proteobacteria, coccobacilli, not motile, and no flagella

                                               i.     Aerobic

                                             ii.     Tolerant to stomach acid

                                            iii.     Has a capsule and forms biofilm

                                            iv.     Produces endotoxin but not exotoxin

                                              v.     Natural reservoir – humans and other animals

b.     Vibrio – gram-negative gamma-proteobacteria, curved rod, motile, has flagellum

                                               i.     Facultative anaerobe

                                             ii.     Not tolerant to stomach acid

                                            iii.     Has capsule and forms biofilm

                                            iv.     Produces exotoxin and endotoxin

                                              v.     Natural reservoir – coastal and brackish waters

3.     Describe the properties of enteric bacteria. Name principal species that are human pathogens and diseases they cause – Lecture 4, Slides 3, 8, 9 –
Properties of enteric bacteria:

a.     Gram-negative

b.     Bacilli

c.     Facultative anaerobes

d.     Associated with human and animal guts

e.     Most are flagellated

f.      Produce acid in fermentation of sugars

g.     Examples:

                                               i.     Shigella causes shigellosis

                                             ii.     E.coli can cause UTI when it enters the urinary tract

                                            iii.     Salmonella causes intestinal infections in human guts, but is part of normal flora of birds

                                            iv.     Klebsiella causes pneumonia

4.     Compare the properties of bacterial genera Clostridium and Mycoplasma. Name principal species that are human pathogens and diseases they cause – Lecture 4, Slides 10-14 –
Comparison of firmicutes important species bacteria

Clostridium · Firmicutes: low GC content · Doesn’t form endotoxins · Motile · Bacillus · Has a cell wall (gram-positive) · Forms endospores · Is sensitive to penicillin · Forms exotoxins Ex: C. perfringes: food poisoning, gangrene

Mycoplasma · Firmicutes: low GC content · Doesn’t form endotoxins · Motile · Pleiomorphic · No cell wall · Doesn’t form endospores · Not sensitive to penicillin · Doesn’t form exotoxins Ex: M. hominis: pelvic inflammation, bacterial vaginosis, male infertility

5.     Compare the properties of bacterial genera Staphylococcus and Streptococcus Name principal species that are human pathogens and diseases they cause – Lecture 4, Slides 10, 11, 15, 16 –
Further comparison. Firmicutes: low GC content, gram-positive

Streptococcus · Firmicutes: gram-positive, low GC content · Forms exotoxins · Not motile · Bacillus · Has a cell wall (gram-positive) · Doesn’t form endospores · Is sensitive to penicillin · Forms exotoxins · Cocci grow in clusters · Facultative anaerobe Ex: S pyogenes: cellulitis, impertigo, necrotizing fasciitis, toxic shock syndrome, scarlet fever, streptococcal pharyngitis

Staphylococcus · Firmicutes: gram-positive, low GC content · Forms exotoxins · Not motile · Bacillus · Has a cell wall (gram-positive) · Doesn’t form endospores · Is sensitive to penicillin · Forms exotoxins · Cocci grow in chains · Aerotolerant anaerobe Ex: S. aureus: opportunistic pathogen, normally vital in limiting skin pathogens; pneumonia, scalded skin syndrome, toxic shock syndrome, food poisoning, impertigo

6.     Compare cellular properties of Staphylococcus aureus and Streptococcus pyogenes. Name diseases they cause – Lecture 4, Slides 15, 17, 18 –
Staphylococcus aureus belongs to phylum firmicutes and is a gram-positive coccus, growing in clusters; it has golden colonies, and is alpha, beta, and gamma hemolytic on blood agar. It grows well and produces acid on mannitol salt agar; it is a facultative anaerobe, catalase-positive, and coagulase-positive.
Streptococcus pyogenes also belongs to phylum firmicutes and is a gram-positive coccus, but it grown in chains. It is beta-hemolytic, and grows in white colonies on blood agar. It does not grow on mannitol salt agar, because it is salt-sensitive. It is both coagulase-negative, and catalase-negative.

7.     Besides capsules, what are other virulence factors of S. aureus and S. pyogenes preventing killing of bacterial cells by phagocytes? – Lecture 4, Slide 18 –
Virulence factors are molecules produced by microbes that help them colonize a niche in the host, avoid being killed by the host’s immune system.

a.     Staphylococcus aureus is typically not pathogenic – part of normal flora in humans, but it is an opportunistic pathogen, which has the following virulence factors:

                                               i.     Capsule/biofilm (interferes with ingestion by phagocytes

                                             ii.     Adhesins – clumping factor A binds to fibrin, for example

                                            iii.     Protein A – binds to IgG inverted, thus making the cell invisible to phagocytes.

                                            iv.     Enzymes –

1.     penicillases that digest penicillin

2.     beta-lactamases, that digest beta-antibiotics

3.     catalase – inactivates hydrogen peroxide

4.     staphylokinase – dissolves blood clots

                                              v.     Penicillin-binding protein A – does NOT bind penicillin, making resistant

                                            vi.     Staphyloxantin – inactivated hydrogen peroxide

b.     Streptococcus pyogenes – NOT a part of normal human flora, is always pathogen

                                               i.     Calsule/biofilm – helps avoid being phagocyted

                                             ii.     Adhesins – M protein has two functions:

1.     Being and adhesin

2.     Inhibiting the activity of C3 catalase, which prevents activation of the complement system, interfering with attraction of phagocytes

                                            iii.     Protein G – binds IgG inverted (like protein A, just produced by a different bacteria)

                                            iv.     Enzymes:

1.     Hyaluronidase – breaks connections between human cells

2.     Streprokinase – dissolves blood clots

3.     C5e peptidase – digest C5a, preventing inflammation reaction and hence attraction of phagocytes to site of infection

                                              v.     Exotoxins – superantigens (non-specifically activate T-helper cells leading to cytokine storm), lipid-digesting enzymes, Streptolysin O and Streptolysin S, which form pores in cholesterol containing membranes, leading to cell lysis

8.     Describe the functions of virulence factors involved in covering the cells of S. aureus and S. pyogenes by human proteins – Lecture 4, Slides 15, 16, 18 –
Mention some virulence factors of Staphylococcus aureus (capsule/biofilm, Protein A – binds IgG inverted, staphylokinase enzyme, which dissolves blood clots, adhesin (protein A again), exotoxins. OK, now mention some for Streptococcus pyogones – capsule/iofilm, protein G (protein M, which also inhibits C3 convertase, preventing the activation of complement system, which is designed to attract phagocytes to the cell), Streptokinase enzyme (does what staphylokinase does – dissolves blood clots), superantigens, which activate T-helper cells.

9.     Compare the properties of bacterial genera Corynebacterium and Mycobacterium. Name principal species that are human pathogens and diseases they cause – Lecture 4, Slides 19-22 –
Corynebacterium and mycobacterium are general of the phylum actinobacteria (high GC content – 69-73%).

a.     Similatiries: neither produce endospores, antibiotics, or endotoxins (since they are not gram-negative).

b.     Corynebacterium – gram-positive, straight or slightly curved bacilli

                                               i.     Obligate aerobe OR facultative anaerobe

                                             ii.     Produces exotoxins

                                            iii.     C diphtheriae causes diphtheria

c.     Mycobacterium – acid-fast, slender bacilli grow in chains

                                               i.     All obligate aerobes

                                             ii.     Does not produce exotoxins

                                            iii.     M tuberculosis causes tuberculosis and M leprae causes leprosy

10.  Compare the properties of bacterial genera Mycoplasma and Mycobacterium. Name human pathogens from these genera. – Lecture 4, Slides 10, 11, 13, 14, 20, 22 –
Mycobacterium vs mycoplasma:

a.     Similarities: both are prokaryotes and source of human pathogens

                                               i.     Obligate aerobes

                                             ii.     Don’t produce endotoxins, since neither is gram-negative

                                            iii.     Don’t produce exotoxins either

                                            iv.     Don’t produce endospores

b.     Mycoplasma – firmicutes, so low GC content

                                               i.     No cell wall

                                             ii.     Pleiomorphic

                                            iii.     Exhibits gliding motility

                                            iv.     M pneumoniae is a pathogen that causes pneumonia

c.     Mycobacterium – actinobacteria, so high GC content

                                               i.     Acid-fast cells wall

                                             ii.     Slender bacilli growing in chains

                                            iii.     Usually nonmotile

M. tuberculosis causes tuberculosis

Chapter 13: Viruses

1.     Describe the universal characteristics of viruses and their architecture – Lecture 4, Slide 23 –.
Universal characteristics of viruses: no cellular organization, obligate parasites because they can’t synthesize their own DNA, so they have to hijack the host’s DNA replication machinery, very small, very host-specific, because they need to a very specific protein in the cell membrane to bind to.
Viral architecture:

a.     Genetic material – can be single- or double-stranded DRA or RNA

b.     Capsule – made of proteins, houses genetic information

c.     Envelope – optional; has a lipid membrane and hosts viral adhesins

2.     What defines the virus’ host specificity? – Lecture, Slide 24 –
The virus’ host specificity means that the virus is unique, because it can only infect the cells which it can adhere to, which are very specific.

a.     Defined by viral adhesins – proteins the virus uses to bind to the host

b.     Restriction-modification system, consisting of two proteins – restrictase and DNA modifying enzyme – this allow the virus to actually replicate. This is because typically a cell has defenses against viruses, like the restriction enzyme might recognize foreign DNA and destroy it, but restrictase protects from that. Modification enzyme can attach a methyl group to the restriction site of viral DNA, thus making it look like it’s not foreign.

3.     What is lysogenic conversion? Give two examples naming bacterial species and virus involved, toxin produced, and disease caused in humans – Lecture 4, Slide 28 –
Lysogenic conversion is when the phenotype of normal bacteria differs from the phenotype of bacteria infected by some virus. This is important because some bacteria, such as E.coli that comprise part of normal flora of humans may become pathogenic once infected with the virus.

a.     Ex: C. diphtheriae gets infected by beta-virus, which makes it produce diphtheria toxin, which causes diphtheria in humans

b.     Ex: v cholera gets infected with CTX alpha virus, which codes for cholera toxin, which the cell then expresses. This causes cholera in humans

4.     Describe replication of reverse transcribing viruses (retroviruses) in eukaryotic cell. – Lecture 4, Slide 31 –
Each retrovirus will have:

a.     dsDNA: goes to nucleus to make mRNA and DNA copies

b.     ssDNA: goes to nucleus and gets converted into dsDNA, which is used to synthesize ss (+) DNA copies used for new viral particles

c.     it replicates like this:

                                               i.     it attaches to host cell

                                             ii.     it enters the cell

                                            iii.     uncoats and releases viral genetic material

                                            iv.     if it is DNA, it goes to the nucleus for replication, if it is RNA, it replicates in the cytoplasm

                                              v.     the virus assembles

                                            vi.     the virus gets released

5.     Compare three types of persistent viral infections – Lecture 4, Slide 32 –
There are two major types of viral infections: acute and persistent.

a.     Acute viral infection usually lasts for a short time and the host likely develops long-lasting immunity. Large amounts of viral particles are made during a short amount of time and then they die

b.     Persistent viral infections occur when the virus stays insite the host for a long time, like for life. When the virus replicates, it likely buds out of the cell and the cell would usually die from the host immune system, rather than the viral infection itself there are three types of persistent viral infections:          

                                               i.     Chronic infection – virus present for a long time; symptoms develop in the beginning of the disease, but can disappear later (hepatitis B)

                                             ii.     Latent infection – the virus is present and then not detectable in the blood stream because it is dormant inside the host cells during the symptomless period. The symptoms may reappear later after reactivating the virus, and the symptoms may be different from the first time (herpes)

                                            iii.     Slow infection – the presence of infectious virus in the blood gradually increases over a prolonged period of time, and no apparent symptoms may be detectable during that period (HIV, retroviral infections)

A good website covering the various types of viruses: 
https://www.kumc.edu/AMA-MSS/Study/micro4.htm