practical 1: enumerating bacteria in probiotic drinks Flashcards
give 3 examples of total cell counts
direct microscopic count
turbidity
dry weight analysis
what are the characteristics of direct microscopic count?
most common total cell count method
hard to do due to the size of the bacterial cells
what is turbidity also known as and what does it measure?
aka optical density
measures cell mass in bacterial culture
measure of cloudiness/haziness of a solution
culture absorbance proportional to cell numbers/weight
what is used to measure turbidity?
colorimeter or spectrophotometer
by dry weight analysis or chemical estimates
what does dry weight analysis entail?
drying cell mass after centrifuging
at what temperature is the cell mass dried at?
100-105 degrees C
what percentage is the dry weight of the wet weight?
dry weight = 20-30% of wet weight
what is the dry weight of prokaryotic cells?
10^-11g - 10^-15g
what does chemical estimates of cell numbers involve?
amount of a chemical component that a cell produces eg ATP or protein
name the most common method of viable cell counting?
plat or colony count
what is the assumption of the plate/colony count?
1 bacterial cell produces 1 colony forming unit (CFU)
what is the spread plate method?
culture diluted several times and spread onto agar plates
what is the pour plate method?
culture incorporated into molten agar and poured onto the plate
what are the CFUs used to calculate?
the number of CFUs on an agar plate used to calculate the number of bacteria per ml in the original culture
why would you use the miles and misra technique as an alternative?
several samples are needed for plate counts meaning number of plates can be cumbersome therefore use miles and misra method
what is the miles and misra technique?
divide plate into 12 squares
label the squares and plate the appropriate bacterial culture into each square
what are some of the advantages and disadvantages of the miles and misra technique?
smaller volumes needed for each dilution
more than on dilution can be plated therefore less plates are used
harder to count bacteria for CFU
must make sure the labelling is clear
why do you centrifuge?
to separate solids, liquids and gases based on density
how would you use centrifuge to count the wet weight of bacterial biomass?
pellets fall to bottom (dry mass)
remove superficial liquid (supernatant) = left with pellets = dry mass
why do you need to make sure the centrifuge is balanced?
to prevent the spin from breaking
what steps do you take to measure the wet weight of bacterial biomass?
measure weight of empty eppendorf
measure total weight (eppendorf + bacterial culture) before centrifugation
centrifuge
measure pellet weight after centrifugation
what is the equation used to calculate wet bacterial biomass?
(total weight - pellet weight) / total weight
x100 (expressed as a %
why do we add buffer solutions?
to maintain pH and therefore osmolarity
why do we use a blank cuvette?
used for reference
acts as a standard to measure against
why do we mix the probiotic solution well?
to ensure its an even solution throughout
what does a high OD mean?
high OD = less light passes through = more bacteria present in culture (larger size of bacteria)
is measuring biomass and turbidity a good quantitative method of enumerating bacteria in the probiotic sample? why?
no
measures alive and dead cells, we are only interested in the alive cells
for what other purpose would you make sequential measurements of OD of a cell suspension at 600 nm?
concentration of bacteria
colour in bacteria eg beetroot
600nm preferred as cells wont be killed (optimum wavelength)
what would happen to the bacterial cells if they were measured at higher than 600nm?
cells would be killed as too much UV present
why must you incubate cells when calculating density of viable cells in a sample?
allows cells to grow and divide
why is an undiluted bacterial sample plated directly onto agar plate inconvenient?
number of CFUs would be too numerous to count
why do we dilute the bacterial culture before plating it?
less concentrated culture means a more manageable amount of CFUs to count
what is the convention to follow when counting CFUs?
count the CFUs if it is between 30-300
any more or less dont count them
what is a serial dilution?
original solution is diluted several times
what is the most common serial dilutions?
10 fold dilution
what should you record dilutions as?
CFU ml -1
how do you make the dilutions comparable?
times the count you see on the plate by the degree of dilution
why would you use a new sterile tip each time?
to prevent cross contamination
why do you leave plates upside down until they dry?
to prevent condensation forming
why shouldnt you know the plate when doing miles and misra technique?
the drops will run into each other
what wavelength of light should be used to measure protein/DNA?
approx 280nm
absorbs max UV at this wavelength without damaging the protein
what does enumerating mean?
counting
what is characteristic about lactobacillus bacteria?
produce lactic acid through fermentation
what must yoghurt contain for it to be considered a probiotic?
live and active cultures which are usually added after sterilisation
what is the most common bacteria added to yoghurt?
L. caseii
what happens to those yoghurts that dont undergo sterilisation?
lactobacilli cultures added when bacteria have remained after fermentation
why would you take averages of colony counts?
to give a more accurate representation of original sample
why would there be a difference between samples CFU counts?
different bacteria grow best in different environments (pH, temperature, humidity)
if the average volume of a probiotic drink is 100ml, do probiotics fill the claim tht they deliver 1 billion live bacteria to the gut?
- work out at which sample at 100 ml would give 1 billion bacteria
eg. 1.2x10^7 x 100 = 1.2 x 10^9 (1.2 billion)
- work out at which sample at 100 ml would give 1 billion bacteria
- times the sample concentration by the volume of the drink
what is a colony?
generated from a single cell that has divided multiple times to form a mass of cells on the plate where the original single cell was deposited
what is the genetic characteristic of an isolated colony?
genetically identical
represent a pure culture
what is the morphological appearance of a colony influenced by?
unique characteristics of the bacterial strain
what is step 1 in identifying different bacterial strains in a mixed population?
identifying differences in colony morphology
if bacteria have the same morphology what can we assume about their colony?
same morphology = same colony
what property do bacteria with flagella have?
able to swarm across agar surface
filamentous appearance
what is characteristic about bacteria that are highly motile?
produce larger colonies due to bacterial spread
what do bacteria produce to give them a glossy/mucoid appearance?
bacteria that produce capsules or exopolysaccharides
what bacteria are classed as having a ground glass appearance?
bacterial colonies with a dry/rough appearance
what are the stages in gram-staining?
create uniform suspension with water (even distribution of bacteria)
let the smear dry (so bacteria dont move off the slide)
fix by passing through a bunsen flame
stain with crystal violet (1min)
pour off and rinse with water
cover with iodine (1min)
rinse with tap water
rinse with alcohol until colour no longer washes away
rinse with tap water
counterstain with safranin (1min)
rinse with water
leave by bunsen to dry
observe under immersion oil under microscope
what colour are gram positive bacteria after gram staining?
purple
what colour are gram negative bacteria after gram staining?
red
why do gram positives give a positive result for gram staining?
thicker peptidoglycan cell wall than gram negative therefore can absorb the dye
what is the purpose of the alcohol rinse during gram staining?
to destroy the little amount of peptidoglycan in gram negative bacteria so they give a negative result
where does the purple colour come from in gram postitive gram staining?
comes from the crystal violet-iodine complex
forms because gram positive cells can retain the primary stain
why is safranin used as a counterstain?
produces a contrasting background showing more of a difference between tissues to make it easier to view under a microscope
why are immersion oils used?
increase resolving power of the microscope to give a clearer image
are lactic acid bacteria gram positive or negative?
gram positive
what morphology do bacilli have?
long filaments and rods
give an example of bacilli with long filaments
Lactobacillus delbrueckii subsp. bulgaricus
what morphology do ovoid cocci have?
chains
give an example of ovoid cocci with chains
Leuconostoc
what morphology do cocci have?
chains
pairs
single
give an example of cocci with chains
streptococcus (lactococcus)
give an example of cocci with pairs
Pediococcus
Aerococcus
(lactococcus)
give an example of cocci with single morphology
lactococcus
what is the most common microbial biochemical activity tested in probiotics?
fermentation of sugars (carbohydrates)
how would you test for the fermentation of sugars from probiotic bacteria?
fermentation of sugars produces lactic acid
lowers pH
add indicator (bromocresol purple)
colour change
what are the colour changes observed when using bromocresol and what do they mean?
yellow = pH < 5.2 = more acidic = more sugar fermented purple = pH > 6.8 = more alkaline/neutral = less sugar fermented
to get a clear result using bromocresol purple, why must you have a pure culture?
in an impure culture there will be more than one strain
each strains have different biochemical profile
results will be skewed as each strain will ferment sugar differently
why are Mann Rogosa Sharpe agar plates used?
contain the most suitable growth media for lactobacilli
would a culture of the same bacterial species have a varied genotype?
yes
wide variation in genotype = wide variation in phenotype
what is a HAI?
hospital acquired infection
aka nosocomial
what does a varied genotype mean in relation to antibiotic resistance?
different genotypes mean that the phenotype (antibiotic resistance) will be different between the isolates too
what type of antibiotic is ampicillin?
B lactam
what type of antibiotic is kanamycin?
aminoglycoside
Neomycin phosphotransferase II gene (NPT II/Neo) encodes for resistance to which antibiotic?
kanamycin
B lactamase gene (bla) encodes for what enzyme that confers resistance to B-lactam antibiotics and which reaction does it catalyse?
enzyme it encodes for is OXA B-lactamase
catalyses the hydrolysis of the amide bond in B-lactam ring of penicillins/B-lactams and cephalosporins
what does MIC stand for and what does it mean?
minimum inhibitory concentration
the lowest concentration of a drug needed to inhibit visible growth of a microorganism in overnight incubation
what experiment would be carried out to measure minimum inhibitory concentration?
pipette identical amount of bacteria into wells at progressively lower concentrations
cloudy well = bacterial growth
indistinguishable well = no bacterial growth
MIC lies between these two wells
where might the gene coding for antibiotic resistance be located?
bacterial chromosome
encoded by plasmids
why is it important to obtain a plasmid ‘profile’ of the bacterial strain when carrying out plasmid DNA extraction?
plasmids are easy to transfer and many antimicrobial genes can be encoded on one plasmid
what are the 2 steps involved in plasmid genotypic profiling?
extract plasmid DNA
purify plasmid DNA
what is the function of the Qiagen kit?
purifies plasmid DNA through a column containing DNA binding resin
designed for isolation of up to 20mg high purity plasmid or cosmid DNA for uses in routine molecular biology applications
what is a cosmid?
hybrid plasmid
what does the P1 buffer do?
resuspend cells
make sure there are no cell clumps
used in step 1 of plasmid profiling
what does the P2 buffer do?
detergent for lysis
used in step 2 of plasmid profiling
mix thoroughly by inverting until solution becomes clear = cells have lysed
what does the N3 buffer do?
precipitation
used in step 3 of plasmid profiling
add and mix thoroughly
should contain white clumps
what does the PE buffer do?
washes
used in step 4 of plasmid profiling
what does the EB buffer do?
elute DNA
used in step 5 (final) of plasmid profiling
low salt used to dissolve DNA
what do restriction endonucleases do?
cut DNA at specific target sequences
what is the mode of action of DNA dependent on?
DNA sequence
give 2 examples that use restriction endonucleases for identifying differences in DNA sequences
pulsed-field gel electerophoresis (PFGE)
single nucleotide polymorphisms (SNP)
what are the 2 principles assumed when cutting plasmid DNA at a single location?
DNA will be linearised
will be cut into 2 fragments if cut twice
what is meant by length polymorphism?
difference in length
when are length polymorphisms seen and what do they indicate?
after gel electerophoresis
indicate which plasmid has been isolated
if all species fully metabolise glucose, why do only some partially ferment lactose which is a milk sugar?
different enzymes are needed to metabolise lactose
some bacterial strains wont have this enzyme therefore cant metabolise it fully
where would the sugar ribose be found?
RNA
where would the sugar deoxyribose be found?
DNA
what is heterofermentative metabolism?
produce lactic acid and a sugar
mannitol is a product of heterofermentative metabolism carried out by a class of lactic acid bacteria. why do only a small amount of bacteria from yoghurt ferment mannitol?
mannitol could be toxic to some bacteria
different enzyme needed
salty environment more suited to gram positive
