working with microbes Flashcards
What does aseptic technique mean?
Without microorganisms
Why use aseptic techniques?
- Prevention of contamination of the laboratory by the organisms being handled
- prevention of contamination of the work with organisms from the environment
What does aseptic techniques do?
- remove or kill microorganisms from hands, equipment and objects
- employ sterile instruments and other items
- remove risk of contamination of experiments, enviro and people by microorganisms
what is autoclaving
pressurised steam steriliser
-autoclave conditions-121 degrees, 15psi, 15 mins
what is autoclaving used to sterilise
-bacteriological culture media
•Glassware
•Equipment e.g. pipette tips
name 3 physical ways of sterilisation
- heat sterilisation (dry or moist)
- filtration (asbestoses, membrane, sintered glass)
- radiation sterilisation (ionising or non ionising)
dry and moist sterilisation in heat sterilisation
- moist heat sterilisation e.g. autoclave, boiling water, water heating, pasteurisation, pressurised steam
- dry heat sterilisation e.g. dry oven, incineration
ionising and non ionising in radiation sterilisation
-ionising- x rays, U.V rays, cathode
non ionising- ultra violet
name two chemical ways of sterililsation
gas and liquid
name 2 gas ways of sterilisation
- formaldehyde
- ethylene oxide
Name 4 liquid ways of sterilisation
alcohol
Halogens
Phenols
Aldehydes
How do we know that what we have autoclaved is sterile
1) autoclave tape has strips which change colour when exposed to 121 degrees
2) the change in colour is permanent so in the lab you will know which equipment is sterilised
3) indicator valves also can be used to assess successful autoclaving
What is pasteurization
A carefully controlled heating to reduce microbial load and it is associated with milk
It is developed to kill pathogenic microorganisms but it’s not sterilisation coz it doesnf kill all organisms.
The milk is passed through a head exchanger at 71c for 15s and cooled rapidly
Types of radiation
Microwaves, ultraviolet, xrays, gamma Ray’s and electrons as well as heat is a form of energy
ionizing radiation used for which pre packed plastic equip
Petri dishes
Spreaders
Syringes
(Use one and dispose)
What is ionizing radiation
Energy to produce ions from other molecules
It can generate hydroxyl group (OH•) and hydride(H•)
What does ionizing radiation do
It causes damage to the DNA and other macro molecules-call death
And uses electron beams, x-ray machines and radioactive elements Co OR Cs to do so
What can depth filters be made of and what are they
They are a mesh of material overlapping in a random network and can be made up of paper or glass
What can filtration be used for
Liquid or airflow
Liquids- resist clogging so can remove large particulates
Airflow- HEPA filters
What is membrane filtration and what is it made off
It is a type of liquid sterilisation and is made up of hugh strength polymers such as nitro cellulose
What does membrane filtration act like so what do they do
Act like a sieve so trap microorganisms on the filter
What is chemical sterilisation used for
To sterilise surfaces e.g. 2% Trigene
Where are microbes grown ( bacteria/fungi)
Grown in either liquid or solid media
Solid media- called agar
Liquid media- called broth
And can be grown in petri dishes or plastic/ glass bottles
What is culture media
Mixture of nutrients in solution
Contains nutrients by microbes grown in lab
Contain buffer to maintain pH value
Liquid(broth) or solid(agar)
What is agar and what is it used to make
It is a red algae polysacharide and is used to make solid media
How to use agar
Ads 1.5-2% agar to broth and boil to dissolve agar
Remains solid over incubation conditions 0-80 degrees and then compare with gelatin
It liquifies at approx 100 degrees and gels at approx 42 degrees
How does plate culture work
Spread sample over agar surface
Bacteria grows on surface
Sample places in plate and agar poured over it
Bacteria grow within the agar
Plates inverted before incubation and prevents condensation falling into plate
What is defined media
It is the precise amount of nutrients I.e exact chemical composition
And Highly purified inorganic salts and organic chemicals e.g. glucose and amino acids
What is complex media
Rich in nutrients
Chemically undefined (milk protein ,beef,yeast, soybeans)
Support growth of microbes
What is media formulated to be
General
Selective
Differential
Enrichment
What is general formulation and what materials does it use
Grow wide variety of bacteria
It used nutrient agar, yeast extract, meat extract, blood agar
What does selective media include
Salt
Bile
Dyes e.g. crystal violet, malachite green
Antibiotics e.g. penicillin, streptomycin
Other chemicals
What does staphylovoccus use (media)
Nutrient agar +7.5% NaCl
Sodium deoxycholate which is used to inhibit gram postive skin bacteria in hand washing trials e.g. allows E coli k12 to grow
What is mannitol salt agar used for
Media for the isolation of pathogenic staphylococci
What is the ph indicator in mannitol salt agar
Phenol red
What does coagulase postive and negative staphylococci produce
C positive s - produce yellow colonies due to mannitol fermentation
C negative s - produce small pink or red colonies
Differential media
Used to distinguish between one group and another
Contains a chemical that is altered or utilised by certain microbes and not by others
It is a form of biochemical test
Chromagenic media
Contains substrates linked to chromogen (flurogen) Enzyme substrates (ONPG, CPRG, MUG) are organisms specific Results in colour change or flureosense under UV light
Oxide UTI media
Two chromogens cleared by the B galactosidase and B glucosidase
B gala… activity of E coli results in pink red colonies
B glu.. activity of enterococci produces blue colonies
Tryptophan deaminase activity produces a brown halo around colonies such as proteus sp
What is enrichment media
Used to increase growth of particular microbe and inbit growth of others
Used when organism is slow growing
What does enrichment media rely on
Selective agent
Specific nutrient
Environmental conditions e.g. light and temp
Example of enrichment media
Campylobacter
C jejuni present in food in low number and causes food poisoning
Need to enrich bacteria first can take several days
Enrich using Bolton broth at 37 degrees, 4-6 hours, 41.5 degrees for 44 hours
Incubate microaerobic at 41.5 degrees for 44 hours
What are the incubation temp generally for pathogens
30 to 45 degrees
Atmospheres for incubation
Aerobic
Anaerobic
Microaerophilic
Microaerophilic/ aerobic incubation
Optimal growth conditions: 3 to 5% oxygen, 2 to 10% carbon dioxide
Anaerobic need free oxygen enviro
Ways of counting bacteria
Direct count
Indirect methods
Viable count
Direct count
Number of cells are counted directly using microscope and chamber
Numbers in known volume of culture is counted and then multiplied
This is better for large cells e.g. yeast, fungal spores
Viable count
Way of assessing growth of single celled organisms such as bacteria is the viable count
Bacteria are grown on agar plate
Plate count
Each colony formed from single bacteria
Count number of colonies you can determine number of bacteria present
What is Dilutions used for
Sample with too many colonies can be diluted to ensure plates are countable. We need a plate between 30 and 300 colonies
How does membrane filtration methods work
Membrane filter on a filter support
Water sample filtered through membrane filter 0.45 um
Membrane filter removed and places in plate containing the appropriate medium
Incubation for 24 hours
Typical colonies count etc
What is membrane filter method useful for
Analysing aquatic samples or low number of organisms
And it can be combined with direct counting via fluorescent dyes
What are Contact plates used for and how does it work
Used to fount number of bacteria present on surface
Good for low numbers
Plate places on surface and incubated then counted
