Lab Exam 2

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animal viruses

-a multitude that can infect humans and cause disease

Work with animal viruses cannot be done in this lab for several reasons:

-Obvious safety concerns
-proper facilities and equipment not available
-working with viruses requires eukaryotic cells for virus replication
-working with viruses & eukaryotic cells requires a high level of sterility
-work must be done in biosafety cabinets that have a filtered air supply

BACTERIOPHAGE

-viruses that only infect bacteria
-most complex in structure than most animal viruses

Bacteriophage vs Animal virus

1.Bacteriophage:
a.requires a host (bacteria) in which to replicate
b.does not have enzymes, ribosomes etc... it uses the cellular machinery of its host
c.contains DNA
d.complex structure
e.lyse bacteria

2.Animal Virus
a.requires a host (eukaryotic cell) in which to replicate
b.does not have enzymes, ribosomes etc... it uses the cellular machinery of its host
c.contains RNA or DNA but never both
d.simple structure
e.lyse eukaryotic cell

Lyse cycle:

1.Attachment
2.Penetration
3.Synthesis
4.Assembly
5.Release

The Plaque Assay:

-A technique that can be used to determine:
1.Which species of bacteria the bacteriophage can infect
2.The # of bacteriophage particles in a sample

How a plaque assay works:

1)Step 1: A dilution series of the bacteriophage sample is prepared
-1 in 10 dilution series of the bacteriophage sample made in sterile saline

2)Step 2: Portion of each diluted phage sample added to a constant volume of the host bacterium to be infected
-Step #2 results in bacteriophage/bacteria mixtures

3)Step 3: Bacteria and bacteriophage mixed together and the bacteriophage allowed to attach to the bacteria (pre-adsorption)
-More phage than bacteria.
All bacteria have phage attached
-More bacteria than phage.
Few bacteria have phage attached

Step 4: Each bacteria + bacteriophage mixture added to a tube of soft agar that has been autoclaved and cooled to 45


Step 5: Each soft agar + bacteria + bacteriophage tube is mixed and poured onto a regular agar plate so that the soft agar spreads evenly across the plate (agar overlay).


-The phage that infected a bacterial cell will replicate and destroy the cell releasing new phage that can go on to infect cells that are in direct contact with the cells that have lysed. All bacterial cells that have been destroyed produce an area of clearing on the plate.
-Different phage can produce different looking plaques

Plaque

the area of clearing on the bacterial lawn, is the result of a bacteriophage infecting and destroying bacterial cells

Why is soft agar used?

it's consistency is firm enough that bacteria can't move through it but not firm enough to let the bacteriophage move short distances so that the bacteriophage can infect neighboring bacterial cells

Tube with bacteria only

-Only bacteria so the plate is covered with a lawn of bacteria
-No phage so no bacteria destroyed

Tube with more phage than bacteria

-More phage than bacteria so all bacteria are destroyed
-No lawn since all bacteria destroyed

Tube with more bacteria than phage

-More bacteria than phage so not all bacteria are destroyed
-Plaques are formed

PFU/ml

-plaque-forming units per ml
-In a plaque assay individual phage cannot be counted so plaques are counted instead.
-Plate for PFU/ml determination should contain ~20-300 plaques

No phage

A lawn of bacteria

"stippled" effect

-More phage than bacteria
-lysis of all bacteria

AEROTOLERANCE

-the ability or inability to live in the presence of O2
-Different bacterial species have different O2 requirements

OBLIGATE AEROBES

MUST have O2 to survive and grow

FACULTATIVE ANAEROBES

can grow in the presence OR absence of O2

AEROTOLERANT ANAEROBES

do NOT need O2 BUT can survive in the presence of O2

AEROTOLERANCE

-the ability or inability to live in the presence of O2
-Different bacterial species have different O2 requirements
-autoclaving removes most free O2 from media; however, as the media cools oxygen starts to diffuse back into the media

MICROAEROPHILES

survive ONLY when O2 levels are low

CAPNOLPHILES

survive ONLY when CO2 levels are high

OBLIGATE ANAEROBES

-ANY O2 will KILL the bacteria

AEROTOLERANCE

-autoclaving removes most free O2 from media; however, as the media cools oxygen starts to diffuse back into the media
-when media is in a tube the diffusion of O2 back into the broth or agar results in a CONCENTRATION GRADIENT
-surface of the media in a tube will have a high O2 concentration since it is exposed to the air while media at the bottom of the tube will have no O2
-bacteria will grow where the O2 concentration in the tube is optimal for that organism
-bacteria will grow where the O2 concentration in the tube is optimal for that organism

Agar Deep Stabs

-Agar deep stabs = 10 ml of enriched TSA (allows more types of bacterial species to grow)
-more media is used in an agar deep stab to ensure that the bottom of the tube is anaerobic
-bacteria are stabbed ONCE all the way to the bottom on the tube with an inoculating needle

Obligate anaerobe (Agar deep stabs)

No growth near the top of the stab line

Facultative anaerobe (Agar deep stabs)

Growth all along the stab line

Obligate aerobe (Obligate aerobe)

Growth only at the very top of the stab line

Important ingredients in fluid thioglycollate medium:

1.Sodium thioglycollate
2.L-cystine
3.Resazurin

Sodium thioglycollate

-reducing agent
-reduces O2 to H2O

L-cystine

-reducing agent
-reduces O2 to H2O

Resazurin

-oxidation-reduction indicator
-red/pink in the presence of O2
-straw color in the absence of O2

Anaerobic Jar

-Aerotolerance can also be examined on agar plates; however, this requires that plates be incubated under both aerobic and anaerobic conditions
-Anaerobic conditions can be produced using a commercially available anaerobic GasPak system (i.e., an anaerobic jar)

To produce an anaerobic system requires:

chemical gas generating packet

chemical gas generating packet

-gas packet contains inorganic carbonate, activated carbon, ascorbic acid and water
-packet becomes "activated" by exposure to air and CO2 gas is produced
-Within 2.5 hr anaerobic conditions (≥ 15% CO2) are achieved

To confirm anaerobic conditions:

METHYLENE BLUE INDICATOR STRIP

METHYLENE BLUE INDICATOR STRIP

Methylene Blue:
oxidation-reduction indicator
blue in the presence of O2
white in the absence of O2

Most common reason why anaerobic conditions were NOT achieved (i.e., methylene blue indicator strip remains blue

incomplete gasket seal

A little bit more on anaerobic bacteria:

-anaerobic bacteria can cause infections in humans some of which can be life threatening
-anaerobic bacteria associated with deep wound infections, intestinal and dental abscesses

How can an anaerobic bacterial species that is the cause of an infection be identified in a clinical sample?

If a clinical sample is processed & cultured incorrectly (i.e., in the presence of O2) an anaerobic pathogen could be overlooked

SYRINGE ASPIRATION

-to obtain a clinical sample from the site of an infection believed to be caused by an anaerobic bacteria
-after aspiration contents of the syringe is expelled into a bottle of media that has had all O2 purged from it

in a clinical lab the culture and maintenance of anaerobic bacteria is typically performed in an ___________ that is completely devoid of all O2

anaerobic chamber

The Effect of Different Environmental Conditions on Microbial Growth

-Since bacteria are ubiquitous bacterial species can be found in many different habitats and therefore can survive and grow under many different kinds of environmental conditions
-all bacteria have a limited temperature range at which they can grow

The environmental conditions that a bacteria may encounter include:

1.Different temperatures
2.Different pH levels
3.Different osmotic pressures

CARDINAL temperatures

-minimal, optimal & maximal temperatures at which a bacterium can GROW
-based on their cardinal temperatures bacteria can be placed in different thermal categories

PSYCHROPHILES

-can ONLY grow at temperatures less than 20°C
-typical range -5°C to 20°C (32-68°F)

PSYCHROTROPHS

-typical range for growth 0°C to 30°C (23-86°F)
-psychrotrophic bacteria can be found in soils, surface water and in foods
-psychrotrophs can be human pathogens

PSYCHROPHILES

-80% of all bacteria can grow at ≤ 10°C
-psychrophilic bacteria can be found in alpine soils, icefields and oceans (marine water < 5°C)
-psychrophiles are NOT human pathogens

MESOPHILES

-typical range for growth ~15°C to ~40°C (59-113°F)
-optimal temperature for growth is approximately normal human body temperature
-mesophilic bacteria can be human pathogens that cause disease

THERMOPHILES

-typical range for growth ~40°C to ~75°C (104-167°F)
-thermophiles can be found growing in hot springs

HYPERTHERMOPHILES or EXTREME THERMOPHILES

-typical range for growth ~65°C to ~110°C (149-230°F)
-hyperthermophiles can be isolated from ocean floor thermal vents/ridges

pH

-the concentration of hydrogen ions (H+) in a solution
-is measured on a logarithmic scale from 0-14 based on the H+ concentration
-pH = -log [H+]

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