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microbiology > practical 1: enumerating bacteria in probiotic drinks > Flashcards

practical 1: enumerating bacteria in probiotic drinks Flashcards

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Biology 101 flashcards
1
Q

give 3 examples of total cell counts

A

direct microscopic count
turbidity
dry weight analysis

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2
Q

what are the characteristics of direct microscopic count?

A

most common total cell count method

hard to do due to the size of the bacterial cells

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3
Q

what is turbidity also known as and what does it measure?

A

aka optical density
measures cell mass in bacterial culture
measure of cloudiness/haziness of a solution
culture absorbance proportional to cell numbers/weight

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4
Q

what is used to measure turbidity?

A

colorimeter or spectrophotometer

by dry weight analysis or chemical estimates

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5
Q

what does dry weight analysis entail?

A

drying cell mass after centrifuging

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6
Q

at what temperature is the cell mass dried at?

A

100-105 degrees C

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7
Q

what percentage is the dry weight of the wet weight?

A

dry weight = 20-30% of wet weight

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8
Q

what is the dry weight of prokaryotic cells?

A

10^-11g - 10^-15g

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9
Q

what does chemical estimates of cell numbers involve?

A

amount of a chemical component that a cell produces eg ATP or protein

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10
Q

name the most common method of viable cell counting?

A

plat or colony count

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11
Q

what is the assumption of the plate/colony count?

A

1 bacterial cell produces 1 colony forming unit (CFU)

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12
Q

what is the spread plate method?

A

culture diluted several times and spread onto agar plates

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13
Q

what is the pour plate method?

A

culture incorporated into molten agar and poured onto the plate

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14
Q

what are the CFUs used to calculate?

A

the number of CFUs on an agar plate used to calculate the number of bacteria per ml in the original culture

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15
Q

why would you use the miles and misra technique as an alternative?

A

several samples are needed for plate counts meaning number of plates can be cumbersome therefore use miles and misra method

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16
Q

what is the miles and misra technique?

A

divide plate into 12 squares

label the squares and plate the appropriate bacterial culture into each square

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17
Q

what are some of the advantages and disadvantages of the miles and misra technique?

A

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

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18
Q

why do you centrifuge?

A

to separate solids, liquids and gases based on density

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19
Q

how would you use centrifuge to count the wet weight of bacterial biomass?

A

pellets fall to bottom (dry mass)

remove superficial liquid (supernatant) = left with pellets = dry mass

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20
Q

why do you need to make sure the centrifuge is balanced?

A

to prevent the spin from breaking

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21
Q

what steps do you take to measure the wet weight of bacterial biomass?

A

measure weight of empty eppendorf
measure total weight (eppendorf + bacterial culture) before centrifugation
centrifuge
measure pellet weight after centrifugation

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22
Q

what is the equation used to calculate wet bacterial biomass?

A

(total weight - pellet weight) / total weight

x100 (expressed as a %

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23
Q

why do we add buffer solutions?

A

to maintain pH and therefore osmolarity

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24
Q

why do we use a blank cuvette?

A

used for reference

acts as a standard to measure against

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25
Q

why do we mix the probiotic solution well?

A

to ensure its an even solution throughout

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26
Q

what does a high OD mean?

A

high OD = less light passes through = more bacteria present in culture (larger size of bacteria)

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27
Q

is measuring biomass and turbidity a good quantitative method of enumerating bacteria in the probiotic sample? why?

A

no

measures alive and dead cells, we are only interested in the alive cells

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28
Q

for what other purpose would you make sequential measurements of OD of a cell suspension at 600 nm?

A

concentration of bacteria
colour in bacteria eg beetroot
600nm preferred as cells wont be killed (optimum wavelength)

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29
Q

what would happen to the bacterial cells if they were measured at higher than 600nm?

A

cells would be killed as too much UV present

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30
Q

why must you incubate cells when calculating density of viable cells in a sample?

A

allows cells to grow and divide

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31
Q

why is an undiluted bacterial sample plated directly onto agar plate inconvenient?

A

number of CFUs would be too numerous to count

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32
Q

why do we dilute the bacterial culture before plating it?

A

less concentrated culture means a more manageable amount of CFUs to count

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33
Q

what is the convention to follow when counting CFUs?

A

count the CFUs if it is between 30-300

any more or less dont count them

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34
Q

what is a serial dilution?

A

original solution is diluted several times

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35
Q

what is the most common serial dilutions?

A

10 fold dilution

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36
Q

what should you record dilutions as?

A

CFU ml -1

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37
Q

how do you make the dilutions comparable?

A

times the count you see on the plate by the degree of dilution

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38
Q

why would you use a new sterile tip each time?

A

to prevent cross contamination

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39
Q

why do you leave plates upside down until they dry?

A

to prevent condensation forming

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40
Q

why shouldnt you know the plate when doing miles and misra technique?

A

the drops will run into each other

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41
Q

what wavelength of light should be used to measure protein/DNA?

A

approx 280nm

absorbs max UV at this wavelength without damaging the protein

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42
Q

what does enumerating mean?

A

counting

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43
Q

what is characteristic about lactobacillus bacteria?

A

produce lactic acid through fermentation

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44
Q

what must yoghurt contain for it to be considered a probiotic?

A

live and active cultures which are usually added after sterilisation

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45
Q

what is the most common bacteria added to yoghurt?

A

L. caseii

46
Q

what happens to those yoghurts that dont undergo sterilisation?

A

lactobacilli cultures added when bacteria have remained after fermentation

47
Q

why would you take averages of colony counts?

A

to give a more accurate representation of original sample

48
Q

why would there be a difference between samples CFU counts?

A

different bacteria grow best in different environments (pH, temperature, humidity)

49
Q

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?

A
    • 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)
    • times the sample concentration by the volume of the drink
50
Q

what is a colony?

A

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

51
Q

what is the genetic characteristic of an isolated colony?

A

genetically identical

represent a pure culture

52
Q

what is the morphological appearance of a colony influenced by?

A

unique characteristics of the bacterial strain

53
Q

what is step 1 in identifying different bacterial strains in a mixed population?

A

identifying differences in colony morphology

54
Q

if bacteria have the same morphology what can we assume about their colony?

A

same morphology = same colony

55
Q

what property do bacteria with flagella have?

A

able to swarm across agar surface

filamentous appearance

56
Q

what is characteristic about bacteria that are highly motile?

A

produce larger colonies due to bacterial spread

57
Q

what do bacteria produce to give them a glossy/mucoid appearance?

A

bacteria that produce capsules or exopolysaccharides

58
Q

what bacteria are classed as having a ground glass appearance?

A

bacterial colonies with a dry/rough appearance

59
Q

what are the stages in gram-staining?

A

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

60
Q

what colour are gram positive bacteria after gram staining?

A

purple

61
Q

what colour are gram negative bacteria after gram staining?

A

red

62
Q

why do gram positives give a positive result for gram staining?

A

thicker peptidoglycan cell wall than gram negative therefore can absorb the dye

63
Q

what is the purpose of the alcohol rinse during gram staining?

A

to destroy the little amount of peptidoglycan in gram negative bacteria so they give a negative result

64
Q

where does the purple colour come from in gram postitive gram staining?

A

comes from the crystal violet-iodine complex

forms because gram positive cells can retain the primary stain

65
Q

why is safranin used as a counterstain?

A

produces a contrasting background showing more of a difference between tissues to make it easier to view under a microscope

66
Q

why are immersion oils used?

A

increase resolving power of the microscope to give a clearer image

67
Q

are lactic acid bacteria gram positive or negative?

A

gram positive

68
Q

what morphology do bacilli have?

A

long filaments and rods

69
Q

give an example of bacilli with long filaments

A

Lactobacillus delbrueckii subsp. bulgaricus

70
Q

what morphology do ovoid cocci have?

A

chains

71
Q

give an example of ovoid cocci with chains

A

Leuconostoc

72
Q

what morphology do cocci have?

A

chains
pairs
single

73
Q

give an example of cocci with chains

A

streptococcus (lactococcus)

74
Q

give an example of cocci with pairs

A

Pediococcus
Aerococcus
(lactococcus)

75
Q

give an example of cocci with single morphology

A

lactococcus

76
Q

what is the most common microbial biochemical activity tested in probiotics?

A

fermentation of sugars (carbohydrates)

77
Q

how would you test for the fermentation of sugars from probiotic bacteria?

A

fermentation of sugars produces lactic acid
lowers pH
add indicator (bromocresol purple)
colour change

78
Q

what are the colour changes observed when using bromocresol and what do they mean?

A 
yellow = pH < 5.2 = more acidic = more sugar fermented
purple = pH > 6.8 = more alkaline/neutral = less sugar fermented
79
Q

to get a clear result using bromocresol purple, why must you have a pure culture?

A

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

80
Q

why are Mann Rogosa Sharpe agar plates used?

A

contain the most suitable growth media for lactobacilli

81
Q

would a culture of the same bacterial species have a varied genotype?

A

yes

wide variation in genotype = wide variation in phenotype

82
Q

what is a HAI?

A

hospital acquired infection

aka nosocomial

83
Q

what does a varied genotype mean in relation to antibiotic resistance?

A

different genotypes mean that the phenotype (antibiotic resistance) will be different between the isolates too

84
Q

what type of antibiotic is ampicillin?

A

B lactam

85
Q

what type of antibiotic is kanamycin?

A

aminoglycoside

86
Q

Neomycin phosphotransferase II gene (NPT II/Neo) encodes for resistance to which antibiotic?

A

kanamycin

87
Q

B lactamase gene (bla) encodes for what enzyme that confers resistance to B-lactam antibiotics and which reaction does it catalyse?

A

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

88
Q

what does MIC stand for and what does it mean?

A

minimum inhibitory concentration

the lowest concentration of a drug needed to inhibit visible growth of a microorganism in overnight incubation

89
Q

what experiment would be carried out to measure minimum inhibitory concentration?

A

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

90
Q

where might the gene coding for antibiotic resistance be located?

A

bacterial chromosome

encoded by plasmids

91
Q

why is it important to obtain a plasmid ‘profile’ of the bacterial strain when carrying out plasmid DNA extraction?

A

plasmids are easy to transfer and many antimicrobial genes can be encoded on one plasmid

92
Q

what are the 2 steps involved in plasmid genotypic profiling?

A

extract plasmid DNA

purify plasmid DNA

93
Q

what is the function of the Qiagen kit?

A

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

94
Q

what is a cosmid?

A

hybrid plasmid

95
Q

what does the P1 buffer do?

A

resuspend cells
make sure there are no cell clumps
used in step 1 of plasmid profiling

96
Q

what does the P2 buffer do?

A

detergent for lysis
used in step 2 of plasmid profiling
mix thoroughly by inverting until solution becomes clear = cells have lysed

97
Q

what does the N3 buffer do?

A

precipitation
used in step 3 of plasmid profiling
add and mix thoroughly
should contain white clumps

98
Q

what does the PE buffer do?

A

washes

used in step 4 of plasmid profiling

99
Q

what does the EB buffer do?

A

elute DNA
used in step 5 (final) of plasmid profiling
low salt used to dissolve DNA

100
Q

what do restriction endonucleases do?

A

cut DNA at specific target sequences

101
Q

what is the mode of action of DNA dependent on?

A

DNA sequence

102
Q

give 2 examples that use restriction endonucleases for identifying differences in DNA sequences

A

pulsed-field gel electerophoresis (PFGE)

single nucleotide polymorphisms (SNP)

103
Q

what are the 2 principles assumed when cutting plasmid DNA at a single location?

A

DNA will be linearised

will be cut into 2 fragments if cut twice

104
Q

what is meant by length polymorphism?

A

difference in length

105
Q

when are length polymorphisms seen and what do they indicate?

A

after gel electerophoresis

indicate which plasmid has been isolated

106
Q

if all species fully metabolise glucose, why do only some partially ferment lactose which is a milk sugar?

A

different enzymes are needed to metabolise lactose

some bacterial strains wont have this enzyme therefore cant metabolise it fully

107
Q

where would the sugar ribose be found?

A

RNA

108
Q

where would the sugar deoxyribose be found?

A

DNA

109
Q

what is heterofermentative metabolism?

A

produce lactic acid and a sugar

110
Q

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?

A

mannitol could be toxic to some bacteria
different enzyme needed
salty environment more suited to gram positive

microbiology (17 decks)

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