Transcript METHODS USED TO STUDY MICROORGANISMS

METHODS USED TO STUDY MICROORGANISMS
 Pure Culture Methods
 pure culture
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population of identical bacteria
 asexual reproduction of single cell
 microbial growth = cell reproduction
 sterile
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free of all living organisms
 sterilizing
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filtration; elevated temps; toxic chemicals; radiation
Aseptic Technique
 substances and equipment are
sterile
 minimize time vessels are open
 work in a clean area
 ensures that a pure sample is
transferred
 protects scientist
Microscopy
 Microscope
 produces enlarged images
 morphology not physiology
 Magnification
 Refraction: Bending light rays
 Resolution: ability to distinguish detail
Contrast & Staining
 Stain
 most microorganisms are colorless
 increase contrast between specimen and background
 Simple stains
 positive staining - cells are dark against light background
 Shapes
 Negative staining - clear microorganisms against light
background
 Differential staining
 multiple stains
 distinguish cell structures or cell types
 Gram stain – most widely used
 Acid-fast staining
 Mycobacterium - TB & leprosy
 Endospore staining
 reveals the presence of endospores
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heat resistant
produced by few bacteria
Clostridium and Bacillus
SPORE STAINING PROCEDURE
 Prepare a smear of bacterial cell
 Place a filter paper on top of the slide
 Place the slide on the staining rack in the sink and flood the smear
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with malachite green stain.
Heat the stain to steaming by passing a lit bunsen burner over the
smear. Don't overheat the stain! Once the steaming stops, pass the
bunsen burner over the slide again. As the stain evaporates add
more stain. Continue this procedure for 5-10 minutes.
Wash the smear gently and thoroughly with running water.
Counterstain with aqueous safranin for 1 minute.
Wash the slide with water, blot gently and allow the smear to air
dry.
Observe under oil immersion.
Spores stain green, while the rest of the cell stains pink.
Light Microscopes
 Bright-field Microscope
 field of view is brightly illuminated
 compound microscope
 Dark-field Microscope
 enhance contrast without staining
 most staining kills microorganisms
 can view live specimens
 special condenser
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focuses light at an angle
reflected off specimen
 Fluorescence Microscope
 use with fluorescent stains
 emit light at a different wavelength
 dyes can be chemically linked to anti-bodies
 specific chemical targets
 target can be visualized
 quickly and positively identified
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Phase Contrast Microscope
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contrast without staining
difference in density produces difference in light
Interference Microscope
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two light waves combined
beam is split - one through specimen; one around
recombined - 3-D effect
 Electron Microscopy
 electron beam not light
 higher magnification
 beam must be transmitted in vacuum
 A. Transmission Electron Microscope (TEM)
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500,000x
specimen must be killed
special preparation process
problem with artifacts
 B. Scanning Electron Microscope (SEM)
 knocks electrons out of specimen surface
 collected for image production (3-D)
VIROLOGY
Viral Replication
 Virus
Acellular, non-living; incapable of metabolism, growth,
reproduction; parasite or genetic extension.
 Specificity - physical attachment; viral nucleic acid to direct
replication.
 Host cell is either permissive or compatible.
 Viral nucleic acid is either RNA or DNA.
 Capsid - wall-like structure; protein; encloses nucleic acid
nucleocapsid.
Virus Replication
“making a copy of the nucleic acid and capsid
protein to construct a new nucleocapsid.”
 Nucleic acid directs formation:-
- Makes new viral protein and nucleic acid
- Shuts down host cell reproduction
Stages
of
Replication
1. Adsorption
 attaches to outer surface of host at specific binding sites
2. Penetration
 virus or nucleic acid genome crosses plasma membrane
3. Uncoating
 viral nucleic acid released from capsid. In bacteria,
penetration
and uncoating happen simultaneously; in plant and animal,
penetration, then uncoating.
4. Early protein synthesis
 polymerases needed to make copy of viral nucleic acid.
5. Nucleic acid synthesis
 Formation of multiple copies of viral nucleic
acid
genome.
6. Late protein synthesis
 new proteins for new capsids.
7. Assembly
 viral maturation; nucleic acid packaged in
capsid.
8. Release
 assembled viruses leave host; host cell is killed
Plant Viruses
 Viruses that replicate within plant cells.
 Enter through abrasion and insect bite.
 Crosses over plasma membrane by endocytosis.
 Plant viruses are named for disease they cause
e.g. tobacco mosaic virus.
 Chloroplast becomes chlorotic; can’t carry out
photosynthesis;
plant cell dies and plant develops disease symptoms e.g.
mosaic pattern of chlorotic spots.
Animal Viruses
 Essential steps are the same – adsorption; penetration;
uncoating; early protein synthesis; replication; late
protein synthesis; assembly; release.
 Adsorption is chemical dependent
 Uncoating takes place outside of host and some inside.
 Most viruses enter by endocytosis (a process whereby
cells absorb material (molecules such as proteins)
from the outside by engulfing it with their cell
membrane).
 Plasma membrane surrounds adsorbed virus - form
membrane-bound vesicle; vesicle released in cell with
lysosome; lysosome degrade capsid; viral nucleic acid is
released.
 Assembly - takes place within cytoplasm or within
nucleus
 Release - lysis; (hours instead of minutes); gradual release
- budding
 Budding - plasma membrane engulfs assembled virus;
forms vesicle; transported out of cell; released viruses
enclosed in membrane; protracted infection; host cell is
debilitated.
Herpes Simplex Virus
Replication of herpes simplex virus
Retroviruses
 RNA viruses that use reverse transcriptase to produce
DNA within host cell (a reverse transcriptase, also
known as RNA-directed DNA polymerase, is a DNA
polymerase enzyme that transcribes single-stranded
RNA into double-stranded DNA).
 making DNA using an RNA template
 integrated into host genome by an integrase
 Released by budding
- slowly and continuously from an infected cell
- eventually causes cell death
HIV
 Infection is persistent due to budding
 Virus replicates in T lymphocytes, essential
components of immune system resulting in
great decrease in body’s ability to defend
against microbial infections.
AZT
Azidothymidine - zidovudine
 AZT is incorporated instead of thymidine nucleotides in
reverse transcription
 Can block replication of HIV
 Useful treatment, not a cure
Transformation of Animal Cells
 Animal virus infection does not always lead to viral disease
 DNA incorporated into host DNA
- Temperate phage (A phage that can enter into lysogeny with its host.
- A phage that can become a prophage (a virus that exists in a bacterial cell and
undergoes division with its host without destroying it) .
- Lysogeny- is the fusion of the nucleic acid of a bacteriophage with that of a host
bacterium so that the potential exists for the newly integrated genetic material to be
transmitted to daughter cells at each subsequent cell division.
- Provirus - a virus genome that has integrated itself into the DNA of a
host cell. One kind of virus that can become a provirus is a retrovirus.
- inherited viral genes
 Can transform animal cell into malignant cell (malignant is a clinical
term that is used to describe a clinical course that progresses rapidly
to death).
 Lysogeny, or the lysogenic cycle, is one of two methods
of viral reproduction.
 Lysogeny in prokaryotes is characterized by integration
of the bacteriophage nucleic acid into the host
bacterium's genome.
 The newly integrated genetic material, called a prophage
can be transmitted to daughter cells at each subsequent
cell division, causing proliferation of new phages.
 Lysogenic cycles can also occur in eukaryotes, although
the method of incorporation of DNA is not fully
understood.
 Transformed cells
- altered surface properties
- continue to grow even when contact is
established
 Tumors
 Oncogenic viruses
- transform cells and cause cancerous growth
 Oncogenes
- genes that induce cancerous
transformations only when expressed
 Activation of multiple oncogenes necessary to cause
cancer does not usually involve viruses.
 Exposure to mutagenic agent that alters gene regulation
could allow oncogenes to be expressed
 Carcinogens include – asbestos, smoke, benzene,
radiation, UV etc.
Some viral infections leading to specific forms of cancer
papillomaviruses – genital warts; cervical cancer