practical microbiology Flashcards
why is direct microscopic count hard
due to the size of bacterial cells
turbidity
Optical density- absorbance is proportional to cell number and weight. Colorimeter or spectrophometer.
what temp is used to achieve dry weight
100-108 degrees
dry weight is …….. of wet weight
20-30%
what does chemical estimate of cell number use
amount of ATP or protein
what is the number of CFUs used to estimate
the number of bacteria in the original sample
miles and msra
splits plate into grids- smaller volumes used. Means more than 1 dilution can be plated per petri dish. however harder to count bacteria- has to be well labeled.
what helps achieve dry weight
centrifuge
why are dilutions used
so you can count number of bacteria on plate
why do filamentous bacteria look the way they do
they can swarm
highly motile bacteria may produce
larger colonies due to larger bacterial spread
capsule ot rxopolysacchardies look
mucosal or glossy and are circular and raised
dry and and rough
“ground glass”
why are counter stains used
producesczntrasting background and also makes differentiation clear
example of a counter stain
Safranin
immersion oil used to
increase resolving power
difference in biochemical activity can identify
diff species
most common biochemical test
fermentation of sugars
why is it important to have a pure culture
no contamination from other strains that have the same biochemical processes
MRS
growth medium good for lactobacilli- mannitol
why is fermentation of sugar a good process of differentiation
production of lactic acid will change the pH and this can be measured.
although two e.coli may appear identical by gram stain and colony morphology
they may have a diff genotype and antimicrobial susceptibility phenotype
Ampicillin
B-lactam
Kanamycin
aminoglycoside
genes for resistance can be coded on by either the
chromosome or the plasmid
QIA-prep
isolation of u to 20ug of high purity plasmid or cosmic DNA- can be used in the process of sequencing
heterofermentative
produces lactic acid and sugar
how to calculate % wet bacterial biomass
total- pellet/ total x100
turbidity
cloudiness
why do we use buffers
to maintain pH and osmolarity. when used alone- standard
accepted range of CFU on plate
30-300 CFU’s
is measuring biomass and turbidity a good quantitative method of enumerating bacteria in the probiotic same?
no- measures dead and alive cells- we are only interested in alive cells
why do you multiply by the degree of the dilution
so they are comparable
why is it important to change the tip between each serial dilution?
to prevent cross-contaminantion
why do we measure cell number at 600nm?
won’t kill the cells
what wavelength of light would we use to measure protein or DNA?
280 nm- absorb max UV light at this wavelength
enumerating
counting
TMTC
too many to count
based on the ops obtained from the biomass and turbidity experiments compared with the viable count (CFs), was there a diff between samples? Why would this be?
yes, because diff bacteria grow best in diff environments
GRAM +VE
purple- larger peptidoglycan level
GRAM -VE
red
what is used in gram staining
crystal violet iodide comple
Mann Rugosa Sharpe (MRS) agar is
designed to favour growth of lacobacilli
at <5.2 pH bromocresol is
yellow
at >6.8 pH bromocresol is
purple
MIC
minimum inhibitory concentration
how MIC can be measured
using a micro broth dilution method. identical amount of bacteria is put in each well, but trying conc of antibiotic- higher turbidity indicates more resistance- more bacteria
location of DNA in gel electrophoresis indicates
BP length
endonuclease cut at a
specific DNA sequence
if all species metabolise glucose, why do some only partially ferment lactose, which is a milk sugar?
diff enzymes need to metabolise lactose
where would the sugar ribose be found?
RNA
mannitol s a product of heterofermentative metabolism carried out by a class of lactic acid bacteria. Why do only small groups of bacteria from yoghurt ferment mannitol?
mannitol can inhibit the growth of some bacteria, instead of encouraging it
MRS stands for
mann rogues sharpe
MRS Agar and Broth were designed to encourage the growth of the `lactic acid bacteria’ which includes species of the following genera:
Lactobacillus, Streptococcus, Pediococcus and Leuconostoc.
