Microbiology week 3

Question 1

Eukaryote or prokaryote?

No nuclear membrane

Answer 1

Prokaryote

Question 2

Eukaryote or prokaryote?

Linear, diploid DNA

Answer 2

Eukaryote

Question 3

Eukaryote or Prokaryote?

80S Ribosome; (60S+40S)

Answer 3

Eukaryote

Question 4

Eukaryote or Prokaryote?

70S Ribosome; (50S+30S)

Answer 4

Prokaryote

Question 5

List the properties of prokaryotes that are distinct from those of eukaryotes.

Answer 5

• No true nucleus/no nuclear membrane
  
  • Translation (protein synthesis) is usually coupled to transcription in the cytoplasm
  • The rate of translation regulates the rate of transcription
• No internal membrane-bound organelles
• Usually haploid: single chromosome and extrachromosomal plasmids
  
  • Hypercoiled
  • Do not carry out endocytosis
• Most membranes lack sterols
• Ribosomes are different
  
  • Prokaryote: 70S (50S subunit and 30S subunit)
  • Eukaryote: 80S (60S subunit and 40S subunit)
• Respiration happens via the cell membrane (vs the mitochondria in eukaryotes)

Question 6

Diagram a bacterial cell.

Question 7

Descibe the function of the cell wall.

Answer 7

• Unique to bacteria
• Dictates bacterial shape
  
  • spherical: coccus (cocci)
  • rod: bacillus (bacilli)
  • spiral
  • No cell wall: mycoplasma
  • Coccobacilli: in between shape (rods that look like spheres, comma shaped)
• Murein/peptidoglycan: major constituent of wall that is unique to bacteria

Question 8

Describe murein/peptidoglycan and explain its synthesis.

Answer 8

• critical component in maintaining the shape and rigidity
• Synthesis
  
  • Building block: un-acetyl muramic acid (MurNac)
    
    • Decorated with different D-amino acids for Gram positive and negative
  • Building block: n-acetyl glucosamine (GlucNac)
  • Transglycosidation: a peptide chain attached to assembly of disaccharide subunits
  • Peptide chain attached to MurNac
  • Transpeptidation: additional cross-linking between opposing MurNac and GlucNac strands occurs via the pentapeptides
  • Third aa of one strand establishes peptide bond with 4th aa of another strand
    
    • energy is derived from the cleavage of D-alas

​

Question 9

Describe bacteria’s internal cell structures.

Answer 9

Cytoplasmic membrane (inner membrane)

• Where respiration takes place
• Lipid bilayer (no sterols like cholesterol)
• Components for
  
  • electron transport system
  • motility machinery
  • ion transport
  • metabolite uptake and release

Chromosome:

• Usually singular
• Haploid (single copy of double-stranded DNA)
• No histone proteins encasing it
• No nucleus (no nuclear membrane)
• Hypercoiled

Plasmids

• Self-replicate in synchrony with host chromosome
• Easily transmitted between bacteria
• Carry genes that encode resistance to antibiotics

Ribosomes

• different from eukaryotic ribosomes (70s=S v 80S)
• No ER or golgi
• protein synthesis happens right after transcription in cytoplasm

Spores

• Aberrant daughter cells with thick protective cell wall

Question 10

Describe bacteria’s external cell structures.

Answer 10

Peptidoglycan

• Disaccharide backbone

Outer membrane

• Gram (-) only

Flagella (legs)

• locomotory organelle
• usually too small to be seen in microscope
• provides motiliity to allow bacteria to swim towards nutriets and away from poisons
• anchored in cell wall
• gets energy from inner cell membrane
• bacteria can have one or many
• can be used to separate types of species (stereodeterminatnts)

Pili (fimbriae) (the fur)

• hairlike structures
• thinner than flagella
• make bacteria stick to other cells
• aggregation
• attachment
• sex

Capsule

• usually composed of glycolyx (except for bacillus anthracis)
• Found on some but not all bacteria, gram (-) or (+)
• not essential for growth in vitro, protects in vivo
  
  • provides a physical barrier between the environment and the bacterial cell
  • Resists opsonization and phagocytosis by interfering with complement deposition
  • evades immune system through molecular mimicry to host glycans

Question 11

Compare the cell envelop of Gram positive and Gram negative bacteria

Question 12

Explain how the different structures of the cell envelope of Gram positive and Gram negative bacteriaconfer distinct Gram stain phenotypes.

Answer 12

Gram staining depends on the ability of Gram-positive bacteria to retain a complex of purple dye and iodine when challenged with a brief alcohol wash; gram-negatives do not retain the the purple dye and are rinsed with safranin (pink)

• Gram-positive: cell wall has many layers of murein; the sugars and charged amino acids of murine make the highly polar structure surrounding the cell a dense hydrophilic layer which gives the cell the ability to retain the dye-iodine complex
  
  1. contains teichoic acid which also evokes non-specific cytokine-induced inflammation
• Gram-negative: has a very thick cell wall

Question 13

What type of cell has lipopolysaccharide?

Answer 13

Gram negative bacteria

Question 14

Define lipopolysaccharide

Answer 14

• Potent activator of immune cells and stimulates release of major pyrogenic substances (inflammatory cytokines) such as IL-1, IL-6, and TNF
• Binds Toll Like Receptor on many cell types
• Results in fever, hypotension, potential shock and death (Septic Shock)

Question 15

Describe lipopolysaccharide’s structure

Question 16

What type of antibiotics interfere with cell wall cross-linking through interaction with penicillin-binding proteins (transpeptidases) and cause autolysis?

Answer 16

B-lactams:

• penicillins
• cephalosporins
• carbapenems
• monobactams

Question 17

What antibiotics interfere with incorporation of new subunits into growing murein chains?

Answer 17

Glycopeptides

Vancomycin

Question 18

What antibiotic interferes with DNA replication by inhibiting the action of DNA gyrase or topoisomerase?

Answer 18

Fluoroquinolones:

• ciprofloxacin
• levofloxacin
• gatifloxacin
• moxifloxacin
• gemifloxacin

Question 19

Compare and contrast the mycobacterial and mycoplasmal cell envelopes with those of Gram-positive and negative organisms.

Question 20

Describe the growth curve of bacteria grown in culture and label the stages of growth.

Answer 20

1. ) During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.
2. )The log phase (sometimes called the logarithmic phase or the exponential phase) is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population. If growth is not limited, doubling will continue at a constant rate so both the number of cells and the rate of population increase doubles with each consecutive time period. For this type of exponential growth, plotting the natural logarithm of cell number against time produces a straight line. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with wastes.
3. ) The stationary phase is often due to a growth-limiting factor such as the depletion of an essential nutrient, and/or the formation of an inhibitory product such as an organic acid. Stationary phase results from a situation in which growth rate and death rate are equal. The number of new cells created is limited by the growth factor and as a result the rate of cell growth matches the rate of cell death. The result is a “smooth,” horizontal linear part of the curve during the stationary phase.
4. ) At death phase,(Decline phase) bacteria die. This could be due to lack of nutrients, a temperature which is too high or low, or the wrong living conditions.

Question 21

Define the terms aerobe, anaerobe, microaerophile, facultative anaerobe, aerotolerant, strict (or obligate):

Answer 21

Obligate (strict) Aerobe: (obligate = requires)

● Requires O2 for growth, do not ferment substrates, use aerobic respiration, oxidative pathways.

● examples: Mycobacterium tuberculosis, Pseudomonas aeruginosa

Obligate (strict) Anaerobe: (obligate = requires)

● O2 is toxic to them

● Use fermentative metabolism

● ex. Clostridium tetani, Clostridium botulinum

Microaerophile: aerobes (5% O2)

●Need O2 - BUT TOO MUCH CAN KILL

● use aerobic respiration, not fermentation

●Example: Campylobacter jejuni

Facultative anaerobes:

“The Swiss Army Knife of Bacteria”

● Grow in the presence or absence of O2

● Use aerobic respiration and fermentation

● Grow FASTER aerobically than anaerobically.

●Example: Escherichia coli

Aerotolerant:

● Tolerate a small amount of O2

● Use fermentation

● Example: Bacteroides fragilis

Question 22

State four reasons why anaerobes are sensitive to oxygen.

Answer 22

Molecular oxygen contains two unpaired electrons in its outer orbital, it is readily reduced to superoxide (O−2) and hydrogen peroxide (H2O2) within cells.[1] Aerobic organisms produce superoxide dismutase and catalase to detoxify these products, but obligate anaerobes produce these enzymes in very small quantities (or not at all).

Dissolved oxygen increases the redox potential of a solution, and high redox potential inhibits the growth of some obligate anaerobes.[3][5][6] For example, methanogens grow at a redox potential lower than -0.3 V.[6]

Sulfide is an essential component of some enzymes, and molecular oxygen oxidizes this to form disulfide, thus inactivating certain enzymes (e.g. nitrogenase). Organisms may not be able to grow with these essential enzymes deactivated.

Growth may be inhibited due to a lack of reducing equivalents for biosynthesis, because electrons are exhausted in reducing oxygen.

Question 23

List some predisposing factors that contribute to anaerobic infections:

Answer 23

● Exposure of a sterile body location to a high inoculum of indigenous bacteria of mucous membrane flora origin, inadequate blood supply and tissue necrosis which lower the oxidation and reduction potential which support the growth of anaerobes.

● Decreased blood supply and can predispose to anaerobic infection are: trauma, foreign body, malignancy, surgery, edema, shock, colitis and vascular disease.

● Other predisposing conditions: splenectomy, neutropenia, immunosuppression, hypogammaglobinemia, leukemia, collagen vascular disease and cytotoxic drugs and diabetes mellitus.

● A preexisting infection caused by aerobic or facultative organisms can alter the local tissue conditions and make them more favorable for the growth of anaerobes.

● Impairment in defense mechanisms due to anaerobic conditions can also favor anaerobic infection.

Question 24

Give an example of a spore-forming organism that causes severe hospital acquired infections:

Answer 24

Clostridium difficile (c-diff)

Question 25

Distinguish the features of these culture media: nutritive, enriched, selective, and differential:

Answer 25

Nutrient media: contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections.

Enriched media: contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen.

Selective media: used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing.

Differential media / indicator media: distinguish one microorganism type from another growing on the same media.[7] This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.

Question 26

Suggest some appropriate applications for the following methods of bacterial killing: Autoclaving, ionizing radiation, pasteurization, antisepsis, filtration, Clorox decontamination:

Answer 26

Sterilization = destroy all microbial forms (vegetative cells and spores)

Disinfection = destroy most bacterial cells and spores on surfaces and objects

Clorox decontamination:
Use of physical or chemical means to destroy most bacterial cells and spores on surfaces and objects.

Autoclaving: (sterilization)
•increased pressure raises boiling point and steam temperature

• *Ionizing radiation:** (sterilization)
• Bacteria, Viruses, Fungi
• Foods, Plastics
• Hazard for occupational users
• *Pasteurization:**
• Heat treatment between 62oC and 74oC
• Duration from seconds to minutes
• Kills vegetative bacterial cells without altering nature of food product.
• *Antisepsis:**
• Use of chemical agents on skin or other tissues to remove or inhibit bacterial agents.
• *Filtration:**
• High,Size exclusion
• Bacteria, Fungi, Some viruses
• Liquids, drug solutions
• Smallest virus particles may pass through pores. Pyrogens may be present

Question 27

Describe two methods with which we can determine the number of bacteria present in a liquid medium:

Answer 27

1. Dilute from broth culture and count resulting colonies. This is more accurate, but takes overnight and yields colony forming units
2. Measure turbidity (spectrophotometry) of broth culture over time.

• Inadequate for estimating very low or very high numbers of organisms
• Correlates well with viable counts (and is faster, easier)

Question 28

Name an important food pathogen that replicates at low temperatures:

Answer 28

Listeria monocytogenes

Question 29

What is a fomite?

Answer 29

Any object or substance capable of carrying infectious organisms, such as germs or parasites, and hence transferring them from one individual to another. Skin cells, hair, clothing, and bedding are common hospital sources of contamination.

Question 30

What is the purpose of a siderophore in bacteria?

Answer 30

•Some bacteria have mechanisms for scavenging iron with“siderophores”

Question 31

Describe the role of fermentation in bacteria metabolism?

Answer 31

Fermentation is metabolism in the absence of O2

• Lower energy yield than metabolism linked to electron transport
• Electron transport without O2 as e- acceptor is anaerobic respiration

Question 32

How do bacteria replicate?

Answer 32

Bacteria replicate by binary fission.

Question 33

The best method for decontamination of a colonoscope is to rinse thoroughly in clean water, then:

A.Autoclave

B.Dry heat for 45 minutes

C.Immerse in gluteraldehyde solution for 90 minutes, the rinse with sterile water.

D.Soak in alcohol for 30 minutes then rinse in sterile water.

Answer 33

C: Endoscopic instruments are fragile, have lenses and adhesives that can be distorted or destroyed by heat, but they should be sterile to prevent transfer of pathogens between patients. Therefore, “cold decontamination” with an appropriate cleaner such as gluteraldehyde for 90 minutes is recommended. Alcohol may not be as effective as gluteraldehyde.

Question 34

While in Haiti on a public health mission, you discover your cholera clinic has run out of IV rehydration solution. To prepare emergency IV solutions from tap water you decide to:

A.Autoclave it

B.Boil it

C.Filter then autoclave it

D.Put a few drops of iodine in it

E.Put a few drops of chlorine bleach in it.

Answer 34

C: To administer fluids by IV they must be sterile AND free of LPS. Filter first to remove any bacteria, then autoclave. Boiling is not as effective. Although iodine and chlorine may make the tap water potable, they are toxic by IV.