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Micro Lab Quiz 1 > Micro Lab Final > Flashcards

Micro Lab Final Flashcards

1
Q

Set up Kholer illumination

A

refer to lab manual 2-9

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2
Q

Smear prep from broth culture

A

manual 3-6

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3
Q

gram stain

A

3-7

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4
Q

streak plate from broth

A

4-3

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5
Q

cell morph (terms and table prep for g stain)

A

4-13
colony colour
g rxn (-/+)
cell shapes
cell size (um)
cell grping

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6
Q

mic clean up

A

discard slides in beaker
remove oil from 100x lens w paper
10x lens and aperature
turn off light

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7
Q

set up serial dilution

A

manual 5-5

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8
Q

spread plate

A

manual 5-6

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9
Q

why is a sample turbid

A

cloudiness bc scattered light (more cells = more scatter = greater turbitity)

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10
Q

how do we meas turbidity/scattered light

A

spectrophotometer can meas dif bw amnt of light that enters sample and amnt of unscattered light that leaves sample

unit = optical density (OD)

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11
Q

what is optical density

A

directly proportional to number of cells in sample

as turb increase, so does OD

allows us to see total number of cells

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12
Q

why are OD meas’s better than plate count method

A

same broth can be checked many times and can track rate cells numbers increase

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13
Q

what is a standard curve

A

scatter plot that relates OD (y axis) to cells per mL (x axis)

to est standard curve, need to count # of cells in sample you’re taking OD reading from

get cells #’s by plate count method

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14
Q

what are the lim’s of OD to cell #’s relationship

A
  1. direct rel bw OD and # of cells/mL breaks down at high turb (rel strongest bw 10^7-9)
  2. a/t that can affect cell size/shape/grpings will change standard curve (make new curve is change org, growth med, incub condition)
  3. some bact form clumps/long chains as rep/divide and some grow in films (vortex before sampling to avoid)
  4. dif media have dif baseline optical densities dep on colour/pigmentation
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15
Q

what is imp to know ab standard curve

A

stand curve does not provide any info ab how quickly a pop is growing

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16
Q

what is the cell density meter set to

A

600 nm for bact and yeast (not for mammalian cells)

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17
Q

why should the red set be redone every 15 min in density meter

A

avoid drift (use NB and press R)

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18
Q

when looking at series of OD readings, how do you check if they’re valid

A

should drop by half

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19
Q

what are coliforms

A

indicator organisms

from fam enterobacteriaceae that serves as index for potential contam by entero pathogens

safer, cheaper, easier faster

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20
Q

what do bacteriological evaluations of water meas

A

specific subpops of bact rather than testing for every potential pathogens individually

quality of water det’d by kinds of bact present not how many

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21
Q

what do water quality analytical procedures and purification/treatment methods rely on

A

vol, source, initial quality of water

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22
Q

what is the total coliform (TC)

A

grp of bact includes many dif genus and species

coliforms found in many env including intestinal flora (imp habitat bc many coliforms are thermotolerant and passed through fecal wastes)

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23
Q

why are coliforms used as indicators for potential contam

A
  1. natural habitats are imp sources of water contam
  2. capacity to survive fresh and marine water is similar to that of entero-pathogens
  3. density usu correlates w degree of water contam
  4. do not reprod in water (as long as water is not nutrient laden)
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24
Q

how do we distinguish coliforms

A

facultative aerobic
g neg
rod
non-spore forming
non motile or motile by peritrichous flagella
ferment lactose w prod of gas w in 48h at 35C

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25
what is dif ab thermotolerant coliforms
usu of fecal origin prod acid (alongside gas) w in 24 h at 44C if get thermotol fecal coliforms, indicates fecal contam
26
what is the most common fecal coliform
Escherichia coli
27
what is imp to remember ab testing for coliform contam
most coliforms are not pathogenic but presence in water indicates the likely presence of pathogens
28
two techniques to test for total coliforms and/or thermotolerant (fecal coliforms)
membrane filtration method most probable number of coliforms technique
29
what is selective media
permit or enhance growth of desired org while suppressing/inhibiting growth of another
30
what is differential media
support growth of dif orgs but allow orgs to be distinguished phenotypically based on their biochem
31
how are g neg selected for medium in lab 7
bile salts, detergents, amphipathic dye salts
32
what do bile salts do and how do enteric bact resist
bile salts destroy lipid bilayer of cell membrane enteric bact resistant to them bc have crosslinked matrix which acts as exclusion barrier to phobic mols and amphi mols like detergents and bile salts and neg charge mols
33
what also select for g neg
BG, basic fuschin, rosolic acid, aniline blue which are ph indicators cant penetrate LPS of g neg but can stop growth of g pos
34
how are coliforms differentiated from other g neg orgs
based on ability to ferment the sugar lactose w prod of gas
35
when does fermentation occur
when bact/yeast import and break down a carb (sugar) from surrounding medium/env to release E w production of alcohol and/or acid and CO2 ETC not involved and e A usu pyruvate
36
what is resp
uses ETC w O or a dif mol as final e A
37
how to detect non soluble gases that may be produced during fermentation
use broth medium w durham tube tube collects gas prod'd by bact during incubation
38
how to detect acids prod'd during fermentation
add ph indicator dye to growth medium in addition to specific carb
39
what is M-FC agar selective for
thermotolerant g neg bact based on ability to grow in presence of high incubation temps, bile salts, rosolic acid and aniline blue differential for lactose fermenting abilities on basis of ph of major end prods
40
what colour do mixed acid lactose fermentors (e coli) produce
dark blue colonies bc drop in ph
41
what colour do butadienol lactose fermentors (enterobacter) appear
blue to blue grey (butad raises ph)
42
what colour do lact fermentors appear
pink-reddish blue grey salmonella and shigella
43
issues of tsa plates
obs of colonies are often difficult due to crowding no info ab hazardous bact vs harmless bact max vol plated = 100 uL
44
how does the membrane filter (MF) work
water passes thru sterile mem filt w pore size 0.45 um and only things smaller pass filter then placed on sel and dif med for thermotolerant fecal coliforms (44.5C)
45
MF advantages
1. range of vols can be processed (can be large, up to 10L) 2.water sol impurities that might interfere w indicator org growth pass through small pores and removed from growth medium 3. provides precise quantitative enumeration 4. usu only need single incubation step (fast and simple) 5. colonies are sep'd on filter, makes downstream isolation easier
46
MF disadvantages
1. cant be used to test waters w high turbidity w out pre filter bc filter will clog 2. scoring of colonies (differentiation) can be difficult if sample contains large numbers of non coliform bact and excessive crowding occurs in filter 3. solid particles and chems may absorb to filter and interfere w growth coliforms
47
calc fecal coliforms/100mL
fecal coliform colonies x 1/dil
48
rules for M-FC
1. lim 20-60 fecal coliforms (blue) 2. <200 total col put est if more than 200 or if no col present
49
what is MPN of coliforms
indirect quant analysis pos test indicated by growth and gas
50
advantages of MPN
1. flexible vols can be processed (<10mL) 2. all kinds of water samples can be used 3. stressed and injured coliforms can be recovered 4. test is easy to set up and results easy to interpret 5. precision and sensitivity can be increased w larger vol, many diltuions or greater number of replicate tubes 6. media is cheap
51
MPN disadvantages
1. only semi quantitative 2. precision is lower and relies on number of tubes used in test 3. presumptive phase takes 48h + 24 (fec)/48h(tot) subculture confirmation 4. if isolation req'd, need more steps on solid media 5. coliforms can be suppressed or overgrown 6. water sol impurities can affect coliform growth 7. hard to process large vol
52
what is the presumptive coliform step
enrichment step medium contains peptones and lactose g pos and neg can grow but s/t get false pos (would need selective media to confirm) must produce gas and grow
53
what is the confirmed (total) coliform test
only use pos from presump test use brilliant green lactose broth (has peptones, sel dyes, lactose, bile salts) need growth and gas
54
what is the fecal (thermotolerant) coliform test
from presump tests that were pos medium selects for thermotol coliforms w 44.5C inc temp growth and gas aka EC broth tubes
55
what are the recreational water quality standards
health can - <500 TC/100mL <200 fecal coliforms/100mL <35 enterococci/100mL US env protection agency <200 fecal coliforms/100ml WHO <100 e coli/100 ml
56
what are drinking water quality standards
no coliform pres/detectable
57
what are enteric bact
oxidase-neg, catalase pos, prod acid from glucose, reduce nitrate to nitrite many = chemoorganotrophs hard to ID bc all g neg rods that are beige cols, round smooth
58
how do enterobact break down sugars
embden meyerhoff pathway/glycolysis happens during: aerobic resp (e A = O; etc), anaer resp (NO3, CO2 etc; etc), ferm (pyruvate, not etc)
59
what are the dif categories of ferm and prods generated useful for
ID process of orgs in ent fam
60
key pts when looking at microbial fermentations
NADH ox'd to nad+ O2 not req'd term e A usu pyruvate and no etc
61
what are mixed acid fermentors
prod ethanol and stable acids
62
what are butanediol fermentors
prod butanediol, ethanol, co2, some lactic acid and formic acid
63
what is the phenol red broth test
general medium, not sel have durham tube dye = yellow at low ph; bright pink at high ph if bact can ferment test carb, acidic end prods lower ph (yellow) and get growth if no ferm, no acids, still get growth but neg, high ph (orange/red)
64
what is MRVP broth test
to dif enterobact based on major ferm end prods when supplied w glucose MR - red at low ph, yellow at high mixed acid ferm's prod stable acid end prods and lower ph VP - detects for presence of acetoin (precur of but) brown-red = pos brown green-yellow if neg
65
citrate slant
sel for bact that can use citrate as sole c source bromothymol blue dye = green at low ph/blue at high ph (blue if pos)
66
urea broth
differential for bact that are rapid urease pos (hydrolyze urea fast) pos = high ph = neon pink neg = pale orange/pink
67
TTC motility
use triphenyl tetrazolium chloride semi solid agar medium red colour where cells have grown (all enterobact can grow) but will see red beyond stab line if mobile
68
SIM
1. tests for sulfur reduction (get black precipitate) 2. tests for tryptophan deamination (bact that prod tryptophanase can deaminate aa tryptophan and get indole); use kovac's reagent 3. motility (look for cream colour growth beyond stab line)
69
which media are dif and sel
an blue, bril gr, carmen red, bas fusch
70
which media is sel
bile salts
71
which media is dif
phenol red
72
Ingredient that allows the media to be differential for lactose fermentation
rosilci acid
73
Ingredients that inhibit gram positive bacteria:
aniline blue, bile salts
74
pos/neg phen red
yellow/red
75
pos/neg mr
red/yellow-orange
76
pos/neg vp
brown red/brown green
77
pos/neg citrate
blue/green
78
pos/neg urea
bright pink/pale orange
79
tryptophan deamination pos/neg
reagent layer bright pink/yellow-orange

Micro Lab Quiz 1 (5 decks)

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