Micro 1 Flashcards
Eukaryotes size, metabolism, cell organisation, replication, maintenance of shape, nucleic acid handling, ribosomal structure, genome
Size - vary Metabolism - generally aerobic Cell organisation - compartmentalise, enclosed nucleus Replication - sexual repro Maintenance of shape - intracellular skeleton Nucleic acid - all within the nucleus Ribosomal - same origins Genome - divided into chromosomes
Prokaryotes size, metabolism, cell organisation, replication, maintenance of shape, nucleic acid handling, ribosomal structure, genome
Size - vary
Metabolism - variety
Cell organisation - all metabolic activity in same space
replication - binary fission
maintenance of shape - extracellular skeletal structure cell wall
nucleic acid - occurs at the same time, transcription and translation
ribosomal - same origins
genome - single circular chromosome within cytoplasm
taxonomy when useful and what used for
- Can be useful in parasites and most viruses
- Organisms in the same group tend to share habitats and life cycles
- Similar epidemiologies and pathogeneses
- Not helpful with bacteria as they have been evolving since the beginning and they are very diverse
Light microscopy what occurs, when sensitive and what are the 2 mechanisms
1) Unstained wet preparations - generally fungi and some bacteria
2) Oil immersion - fixed, stained specimens - allows us to have high magnification - bacteria
○ Refracted light around the organism so not so sensitive and generally only detected when in large numbers
Culture how does it work, how are most cultured
- Success depends on the quality of the sample as well as how it is stored and transported
- Some pathogens cannot be cultured
Most are cultured on cell free media - range of nutrients in a liquid (broth generally in 5ml bottles) or agar form
○ Agar allow single colonies from a single cell - pure so can identify based on colour, shape, consistency, smell - Additives, temperature and atmosphere
○ Generally 37 for mammals, fish 15-10 degrees, fungi about 25 degrees
What only grows in animals cells and what type of cultured cells are there
- Only grow in animal cells - viruses
- Cultured cells
○ Primary cell culture - cells directly from the animal, foetal cells used generally as generally unlikely that it has other viruses within
○ Established lines - transformed cells that replicate and grow on different media, stop natural cell death, limited in species that can get them from
○ Grown in complex media, often containing serum - cell factors that cells need to grow in vitro
What are the 5 ways to identify microbes
1) Microscopy
2) Culture
3) Organ culture
4) chick embryo
5) Animal inoculation
Chick embryo features
- Next step if won’t grow in an organ culture, many vaccinations such as the influenza and Q fever has to be grown within a chick embryo
- Specific ages
- Specific sites
○ Amniotic cavity
○ Allantoic cavity
○ Yolk sac
○ Chorioallantoic membrane
List 6 tests that detect microbial proteins or carbohydrates and describe
1) Toxin-Antitoxin Tests - animal is inoculated with tissue from clinical case and second animal is inoculated with sample and specific antitoxin. If animal inoculated with sample along is affected and animal with antitoxin is not than positive test
2) Labelled Antibody Tests - Antibodies specific for proteins are labelled and applied to sample, the sample then can have a fluorescein stain applied and examined microscopically.
3) ELISAs - antibody bound to plate is used to capture pathogen proteins from solution and a second labelled antibody then detect whole organism or bound protein
4) Neutralisation or Inhibition Assays - the growth of some bacteria or viruses can be blocked by specific antibodies
5) Hemagglutination - some microorganisms agglutinate certain RBCs such as parvo virus
6) Latex Agglutination - within latex beads mix has an antibody that is specific to a carbohydrate that will agglutinate (clump beads together) if present
List the 4 methods of detection of nucleic acids
1) genome itself
2) hybridisation - single stranded DNA will bind to complementary DNA
3) PCR - polymerase chain reaction
4) High throughput sequencing
Polymerase chain reaction what occurs and advantages
○ Segments of DNA are separated and compared to reference range to identify
○ Can determine different strains where the lines are just slightly off where the reference range is, if same strain than identical banding will be shown
○ Real time PCR - every cycle measure how much DNA product is produced, once amount of DNA reaches threshold then say positive and compare to other dilutions to determine amount of DNA in the first place - determine amount of disease present
§ If RNA need to add a reverse transcriptase
○ Can overestimate the likelihood of the animal being infectious as DNA can still be present
○ A lot quicker turnaround time for result than culture and more sensitive
High throughput sequencing how does it work and compare to PCR
○ Collecting nucleic acid from sample and putting into DNA sequencing team
○ Search through nucleic acid to find certain pathogens
○ Costs about $1000 and can just plug into the laptop - easily accessible
○ Not as sensitive as PCR as looking at everything that is there unlike PCR that zones in on exact sequence you are after, this could also be a positive as it may identify novel pathogens that weren’t expected to be identified
Mechanism of detection of specific immune responses
Acute and convalescent test
- Produces an antibody response
- If antibody is present than get clumping/agglutination
- At some time in this patient it was exposed to the pathogen - not necessarily recent
- Generally take initially (Acute) and 2 weeks later and look for difference in antibody level - if recent infection than the antibody levels would have risen in this time
List 8 ways specific immune responses or antibodies can be detected
- Agglutination
- ELISAs
- Neutralisation
- Western blots
- Immunoprecipitation
- Complement fixation
- Immunofluorescence
- Haemagglutination inhibition
Cell mediates responses how determined
- Lymphocyte stimulation test and detection of cytokine release (especially interferon gamma and/or interleukin 2)
- Will generate mild inflammatory response - slight lump and Redding
○ Used with the Q fever vaccination pre-test
Again doesn’t tell you what is happening now just a few weeks ago and beyond
what is the difference between infection and disease
Infections (entry into the body) occur frequently
• Disease (loss of function) does not
Normal flora features and roles
• Always present
• Variation
• Role in health
○ Digestive process and Vitamins (K and B)
• Protective role - prevent growth
○ Detoxify toxic substances, competition for other microbes
• Source of opportunists
○ If in other parts of the body like deeper tissues
• Significance in diagnosis
Complicates the diagnosis - identify strains and characteristics
Koch’s postulates what does it do and list them
- Determinants that need to be passes in order to determine between normal flora and infectious agents
• regularly found in lesions of the disease
• Isolated in pure culture on artificial media
• Experimental reproduction
• Recovered from experimental disease
List 4 problems of Koch’s postulates
- Most pathogens cannot be grown in culture
- Synergistic infection - needs another disease - sometimes normal flora opportunistic
- Environment may play a role in disease
- Some organisms may lose virulence when cultured on artificial media
Questions asked when making a microbiological diagnosis if detect an organism, if don’t detect an organism
If I detect an organism:
• Is it from the animal? - need to take the sample carefully, if it is the surface of the skin
• Is it the one causing disease?
• Is it the only one causing disease? - synergistic is common
If I don’t detect an organism:
• Is it because there wasn’t one there?
• Is it because it didn’t survive the trip?
• Did I look for the right one?
List the 5 important features when collecting samples for diagnosis
1) History - everything you know
2) Site of sampling - an active or fresh lesion and preferable not one that is open to the surface of the body
○ try to avoid sampling a dead animal due to bacterial overgrowth and the agent generally has declines in numbers
○ Best site is the medulla of the long bone (bone marrow) if dead sample
3) Method of sampling and transport - generally cool (chilled but not frozen) it and send it off to the laboratory as soon as possible
4) Consultation - Consult laboratory if necessary and maintain a relationship
5) Handle with Caution - zoonotic disease potential, can be dangerous to the health of other animals seen that day
What are the 4 classes of containment facilities
Class I
• Class II - what we are working in - 80% laboratory infections from exposure to aerosols
• Class III - completely change clothing before and after you go in
• Class IV - positive pressure suits - protects the person within or negative pressure within the chamber so if any break the organisms can’t get out because sucked back in
sterilisation and disinfection definition
- Sterilisation - absolute - no infectious risk, no infectious spores or organisms
- Disinfection - selective process - taking out the riskiest organisms, may be better at killing some organisms than others
Antisepsis, bacteriostatic agents, bacteriocidal agents define
Antisepsis - lower level, removal of majority of surface organisms - used when cleaning skin surface before surgery
- Application of chemicals to a body surface to kill or inhibit pathogens
• Bacteriostatic agents - used as treatments, stop the growth of the organism but rely on animals immune system to destroy the agent
• Bacteriocidal agents - kill the organisms - quite toxic for animals
How is heat used to destroy microbes
most efficient and generally the quickest and most available - Most would be inactivated 50-70degrees spores at 100 degrees
- Pasteurisation - doesn’t completely sterilise but important in milk in terms of public health and zoonosis spread
- Moist Heat - not as good on metal as may rust but better than just heat as the water makes bonds with separated molecules so lower temperatures are necessary to sterilise wet rather than dry material
○ Boiling - not efficient at destroying the spores
○ Autoclaving - steam sterilisation and under pressure - complete sterilisation besides mad cow disease
- Dry Heat - need to be used higher temp and longer as explained above
How is radiation used to destroy microbes
- Ultraviolet Light - cannot penetrate through glass or objects
- Ionising Radiation - gama rays, effective sterilisation and generally not much damage to the material itself
What are 7 features of chemicals agents that needs to be considered
- Activity spectrum - few disinfectants are effective against spores and viruses are generally more resistant than bacteria
- Inhibition vs Inactivation - need inactivation
- Rate of action - can take extended period of time to act
- Compatability
- Effect of organic material
- Side effects - on yourself, staff, animals and materials
- Price and stability - can have a short lifespan of efficacy
List some major groups of chemicals used as disinfectants
Soluble Alcohols - Ethanol, isopropylalcohol • Alkylating Agents - Ethylene Oxide, Formaldehyde • Halogens - Chlorines, Iodines • Phenolics (Creosol, semi-synthetic phenols) • Quarternary Ammonium Compounds (cationic detergents) • Anionic Detergents (soaps) • Biguanide Compounds (chlorhexidine) • Dialdehydes (gluteraldehyde) • Alkalis (sodium hydroxide, lime) and Acids
Preservation of microorganisms
- Chilling and freezing typically used to preserve microorganisms
- Killing effect of freezing due to crystallisation of the water together with salt solutions damages the cell or viral envelope
- Effect of multiple freeze-thaw cycles will kill most of the organisms
- Cryoprotectants
- Low temperature storage within liquid nitrogen, solid carbon dioxide or low temperatures refrigerators
- Freeze drying or lyophilisation organisms can be stored at room temperature
List the 4 features that virus taxonomy is based on
- Type of nucleic acid
- Strategy of viral replication
- Morphology of the virion
- Sequence analysis of the viral genome
how do you get chains and clumps of bacteria
Divide in same planes = chains
Divide in different planes = clumps
What are the steps in the gram stain and the result
1) gentiane violet -> rinse
3) iodine -> rinse
4) alcohol acetone -> rinse
5) dilute carbol fuschine
Gram positive is purple
Gram negative is pink
what are the characteristics of gram positive and gram negative bacteria
Gram positive
- Thicker capsule
- Large peptidoglycan layer
- Harder for stain to leave so retains the gentiane violet
Gram negative
- Instead of large peptidoglycan layer only small broken layer
- Also have lipopolysaccharides
What are some components of the bacterial capsule and function
- Extracellular material not always present
- Usually polysaccharide (sometimes polypeptide)
- Mucoid colonies (makes the colonies look this way)
- Capsular antigen associated with pathogenicity (E.coli)
Function - Protects from desiccation
- Facilitates adherence to surfaces
Interferes with phagocytosis
At what phase of bacterial growth do you want for control methods
Want bacteria in the exponential phase
- When they are metabolising and growing
- Actively replicating
Using biochemical pathways that are used as identification
List 3 types of special media
- Enrichment media
- Selective media
- Indicator or differential media
Virus structure what are the two common structures
Nucleocapsid: nucleic acid surrounded by protein coat (capsid)
- Icosahedral or helical shape of capsid
Envelope: acquired by budding through cellular membranes
- Glycoproteins on surface of envelope
- Susceptible to environment
Enveloped viruses components, properties, treatment and how released
Components: lipids, proteins, glycoproteins Properties/Treatment: • Labile in the environment • Sensitive to acid, detergent, • drying and heat • Modifies cell membrane Released by budding and cell lysis
Non-enveloped virus components, properties, treatment and how released
Components: proteins Properties/Treatment • Stable in the environment • Insensitive to acid, detergent, • drying and heat Released by cell lysis
Enveloped virus what conditions, how spread, environment, immunoprotection
• Must be in moist conditions
• Spread in large droplets, secretions, transfusions etc
• Do not survive adverse conditions (GIT)
• Do not need lysis to spread within host
Antibody alone may not provide immunoprotection (CMI)
Non-Enveloped virus what conditions, how spread, environment, immunoprotection
- Can be spread easily
- Infective after drying
- Survive adverse conditions (inside gastrointestinal tract)
- Resistant to detergents
- Antibody may provide immunoprotection
what are the bad components of embryonated egg inoculation
1) time consuming
2) labor intensive
3) technically difficult
4) need infectious virus
what are the bad components of cell culture
1) Time consuming
2) Labour intensive
3) Technically difficult
4) need Infectious virus
- Cytopathic effect (CPE)
PCR advantages, disadvantages and what need
- Rapid diagnosis
- Specialised equipment
- (May be) Quantitative
- Doesn’t need infectious virus
- Don’t need to have a viable organism arrive at the lab
