Lab Four Flashcards
What is the calculation for colony-forming units?
Number of colonies x 1/volume plated x 1/dilution = CFU/ml
why do we use the units of colonies forming units?
Colony-forming units are used because we cannot be certain that each colony was the result of a single cell and so we record our count as CFU instead of the number of microorganisms
what is the cell range to count on a plate?
25-250
why do we count more than 25 cells?
less than 25 colonies are not statistically significant
why do we count less than 250 cells?
- more than 250 is hard to count and there is likely to be an error
- there is also more likely to be interactions between colonies which would affect the visible colonies
what is a class II biological safety cabinet?
- double filter unit
- HEPA filters
why is a class II biological safety cabinet used?
provides safety to the worker, sample, and environment
what is a HEPA filter?
High Efficiency Particulate Air filter
what is a HEPA filter used for?
human and fungal samples
How do you ensure you are not interrupting airflow on class II biological safety cabinet?
don’t want to interrupt airflow
- Do not lean on the vents, use armrests
- stay 1m back if observing
what is a eukaryotic microorganism?
consists of single cells or simple aggregates of cells that contain a membrane-bound nucleus and intracellular organelles
where are eukaryotic microorganisms found?
widely distributed in the environment in both free-living and various types of symbiotic relationship
what are the features of a virus?
- found in every ecosystem on earth
- are the most abundant type of biological entity
- require a host cell to multiply and are considered obligate, intracellular parasites
what do viruses infect?
- humans
- animals
- plants
- bacteria
- archaea
what are fungi?
eukaryotic multicellular organisms
where do fungi grow on media?
grow within or on the surface of the growth medium and extract nutrients for growth
what determines fungi taxonomy?
based on the morphology of the various spores and the specialized hypha that bear them
what are the three major phyla of fungi?
Zygomycota
Ascomycota
Basidiomycota
what are the features of Zygomucota?
- asexual sporangiospores in compact sacs called sporangia
- Sporangia are borne on the tips of reproductive hyphae (sporangiophores), extend aerially above the surface
- the sacs burst and each sporangiospores are capable of forming a new fungus
-sexual reproduction, the hyphae from one mycelium contacts and fuses with the hyphae from another mycelium and forms a zygospore
what are the features of Ascomycota?
- produce asexual conidiospores, now known as conidia
- conidia are borne externally on aerial hyphae called conidiophores
-in sexual reproducation they form ascospores in a sac called an ascus
what are basidiomycota?
-produce sexual spores on club-shaped basidia
what are examples of basidiomycota?
mushrooms
toadstools
puffballs
what bacteria causes athlete’s foot?
dermatophytes
most commonly caused by trichophyton rubrum or trichophyton mentagrophytes
how do dermatophytes cause athlete’s foot?
fungi that inhabit dead layers of skin and destroy keratin
how do antifungal drugs work?
most target sterols in the plasma membrane
what is lamsil?
a drug used to treat dermatophyte skin infections
what is the active ingredient is lamsil?
terbinafine
how does terbinafine work?
inhibits ergosterol synthesis by inhibiting an enzyme in the fungal membrane synthesis pathway
this changes the cell membrane permeability and causes fungal cell lysis
what is penicillin?
earliest discovered and very widely used antibiotic agent
what is penicillin derived from?
from the common soil fungus, Penicillium
what is the common morphology of yeast cells?
usually ellipsoidal, spherical or cylindrical cells. Several times larger than the average bacterial cell
what is Candida albicans?
a fungus present on the skin and mucus membranes. Overgrowth results in candidiasis, oral or vaginal thrush
what is some treatment for candidiasis?
Canesten
Diflucan
what is the active ingredient in Canesten?
clotrimazole
what is the active ingredient in Diflucan?
fluconazole
how do candidiasis treatments work?
they inhibit sterol synthesis
what does polymorphic mean?
many forms
what are the two forms that yeast cells can alternate between?
pseudo hyphae and true hyphae
what influences the morphological state?
depends on the environmental conditions
what are the features of true hyphae?
have parallel sides
what are the features of pseudo hyphae?
comprise of a series on conjoined, elongated yeast cells
how are eukaryotic cells grown?
in proper nutrients eventually cover the surface of a dish in a monolayer (confluent growth)
what is cell morphology in a monolayer?
cells are thin, transparent and elongated
what happens to cells in a monolayer when a virus infects them?
the infected cells produce ‘cytopathic effects’ (CPE)
- formations of holes (plaques) in the monolayer
- cells become rounded
- cells become enlarged
what genetic information does a bacteriophage contain?
either DNA or RNA but never both
what are the two types of bacteriophage infection?
can either be lytic or lysogenic
what is the lytic lifecycle?
the infected cell is destroyed, lysed
what is the lysogenic lifecycle?
bacteriophage genome is integrated into the host genome
what is the features of a tissue culture media?
- contains salts, nutrients, and a buffer to maintain the correct pH
- media is specific to each cell type
- most contain foetal-calf serum (FCS)
- cells can be grown in plastic flasks, tubes or multi-well plates
what is subculturing?
cells growing on the container surface are washed, detached from the surface by the enzyme of physical means, resuspended in a new media, and then incubated
-cells are incubated until confluent growth
what is a plaque assay used for?
to estimate the concentration or titre of a bacteriophage in a suspension
how is a plaque assay done?
a few virus particles and lots of host cells are spread across the surface of an agar plate
once the host bacteria create a lawn, there will be some plaques where the bacteria have been infected by the bacteriophage and so cannot grow
the plaques are counted and used to calculate the original number of bacteria in the suspension
what is plaque morphology used for?
useful in determining the differences between types of bacteriophage
what are some practical uses for bacteriophages?
- use in the fish, cheese, and kiwifruit industries to target pathogens
- in humans as a treatment for bacterial infections
what are the two bacteriophage strains used in our plaque assay?
T1 and T4
what is the plaque assay procedure?
- prepare a 10-fold dilution of bacteriophage mixture to 1x10-6
- label plates
- collect a bijou containing molten agar
- using the aseptic technique add 100 microlitres of diluted bacteriophage to the agar
- add 100 microlitres of the host bacteria
- invert the bijou without creating bubbles
- pour agar over a plate
- once agar has set. invert
- incubate at 35 degrees for 18-24 hours
