Lecture 4C - Culture Media, Biosafety, and Aseptic Technique Flashcards
Pathogen Risk Group Classification
- RG 1
- RG 2
- RG 3
- RG 4
- no or low individual and community risk
- unlikely to cause human or animal disease
RG 1
examples of microorganisms in RG 1
- E. coli
- S. cerevisiae
- Lactobacillus
- B. subtilis
- moderate individual risk, low community risk
- can cause disease but unlikely to ba a serious hazard
- lab exposure may cause serious infection, but effective treatment and preventive measures are available and risk of spread of infection is limited
RG 2
examples of microorganisms in RG 2
- Streptococcus
- Herpes virus
- most mammalian cell lines
- high individual risk, low community risk
- usually causes serious human or animal disease but does not ordinarily spread
- effective treatment and preventive measures are available
RG 3
examples of microorganisms in RG 3
- Yersinia pestis
- HIV
- SARS virus
- MERS-CoV
- high individual and community risk
- usually causes serious human or animal disease and can be readily transmitted
- effective treatment and preventative measures are not usually available
RG 4
examples of microorganisms in RG 4
- Ebola virus
- Marburg virus
- Lassa virus
Biosafety levels
- BSL-1
- BSL-2
- BSL-3
- BSL-4
- not known to consistently cause disease in humans
- minimal potential risk to lab personnel and envt.
BSL-1
example in BSL-1
non-infectious E.coli
- moderate potential hazard to humans
- mild disease or difficult to contract via aerosol in a lab setting
BSL-2
example in BSL-2
- HepaA virus
- S. pyogenes
- B. burgdorferi (lyme)
- Salmonella sp.
- microbes can either be indigenous or exotic
- can cause serious or potentially lethal disease through respiratory transmission
BSL-3
example in BSL-3
- Yersinia pestis
- M. tuberculosis
- SARS
- rabies virus
- hantavirus
- West Nile virus
- dangerous and exotic
- pose ah igh risk of aerosol-transmitted infections
- fatal and without treatment or vaccines
BSL-4
example in BSL-4
- ebola virus
- small pox virus
the risk group classification of a pathogen is associated but…
not always correlated with biosafety level
open bench
BSL-1
- biosafety cabinet
- laminar flowhood
- autoclave
- mandatory biohazard sign
BSL-2
- laboratory is separated from general traffic flow and accessed through anteroom or airlock
- autoclave
- sink with hands-free operation
- inward direcional airfow
- all work with infectious material is conducted within biological safety cabinet
BSL-3
- airflow systems
- multiple containment rooms
- sealed containers
- positive pressure personnel suits
- established protocols for all procedures
- extensive personnel training
- high levels of security to control access to facility
BSL-4
clean air enters, dirty air exists and not recycled
positive pressure personnel suits
- set of steps designed to prevent contamination form microorganisms
- involves applying strictest rules and utilizing what is known about infection prevention to minimize the risk that you’ll ecperience getting containated/infected
aseptic technique
when is aseptic technique used
- transferring cultures
- inoculating media
- isolating pure cultures
- performing microbiological test
General aseptic technique steps
- disinfetion and sterilization
- always wipe hands and work area (10% bleach/70% alcohol)
- always cap bottles and flasks
- never uncover sterile materials unless needed. always work near the flame if not working in laminar flowhood
- ensure absence of air movement
- be careful not to talk, sing, or whistle when performing sterile procedures
population of cells arising from a single cells developed by who
Robert Koch
techniques used to isolate pure cultures
- spread plate
- streak plate
- pour plate
population of cells arising from single cell
colony
involves techniques of spreading a mixture of cells on an agar surface so that individual cells are well separated from each other
streak plate
what is involved in streak plating
- bacteriological loop
- Bunsen burner
- solid agar medium
Different way of streaking
- three-way streak
- four-way streak
why do we need to sterilize in between streaking
prevent carry over
reduce number of original population
serial dilution
how is the viable cell determined in serial dilution
Most Probable Number (MPN)
statistically acceptable no. of colonies
30-300 colonies (or 25-250)
number of alive and active microorganisms in one serving
Colony Forming Units (CFU)
- small volume of diluted mixture containing approx. 30-300 cells is transferred
- spread evenly over surface with a sterile bent rod
spread plate
- sample is serially diluted
- diluted samples are mixed with liquid agar
- mixture of cells and agar are poured into sterile culture dishes
pour plate
portion of the whole, especially a sample taken for biological analysis or other treatment
aliquot
where can colonies grow in pour plating
- surface
- subsurface
- bottom
where can colonies grow in spread plating
surface only
colony forming units per milliliter or original sample
(plate count) (dilution factor)
RDD
researcher directed decision
colony characteristicts that develop in agar surfaces or biofilms aid in what
identification of desired colony
differences in growth rate from edges to center is due to what
- oxygen, nutrients, and toxic products
- cells may be dead in some areas
different types of form
- punctiform
- circular
- filamentous
- irregular
- rhizoid
- spindle
different types of elevation
- flat
- raised
- convex
- pulvinate
- umbonate
different types of margin
- entire
- undulate
- lobate
- erose
- filamentous
- curled
different types of surface
- glistening
- smooth
- dull/dry/powdery
- rough
- wrinkled
