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Micro Lab Quiz 1 > micro lab quiz 2 > Flashcards

micro lab quiz 2 Flashcards

1
Q

what is the problem w mixed culture

A

can’t effectively study

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2
Q

in order to study and characterize a single bacterial species, what do you need to do

A

obtain a pure culture (contains a single species and cells orig’d from a single parent cell)

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3
Q

list 3 methods to isolates bact

A

streak plate, agar dilution, liquid dilution

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4
Q

what is the streak plate method

A

sample taken from culture and streaked across the surface of an agar plate

use loop tool to take from liq or sol and then drag across surface of plate

cell density decreases across plate and single units left behind and get well isolated colonies

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5
Q

what is another word for colony

A

colony forming unit

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6
Q

what affects the pattern and method of preparing a streak plate

A

medium used, source of inocculation, ind pref based on experience

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7
Q

what is an advantage to streak plate method for isolation

A

1) no initial dilutions of starting cult is req’d

2)if contam is present in starting cult or happens via bad sterile tech, contaminants will be readily observed in medium

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8
Q

what must be assessed not assumed during an isolation method

A

purity

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9
Q

how do you test the purity of the culture

A

obs colony morph on solid media (want all same) or obs g stain from broth cult (want consistent)

could also do biochem testing or PCR to confirm ID

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10
Q

req’s of aseptic tech

A

near bunsen burner (creates updrafts to keep contams from air from falling in)

one thing open at a time

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11
Q

streak plate protocol from broth cult

A
  1. flame ster loop (cool)
  2. vortex tube (make sure to flame rim)
  3. dip loop; pick up agar side of plate
  4. streak 1/4 (don’t press too hard, don’t breathe over, hair fall)
  5. flame ster loop (cool 10-15s)
  6. rotate and drag 2 par lines from Q1-Q2
  7. zig zag from 2 pull lines in Q2 (don’t cross back and don’t overlap zig zags)
  8. rotate, pull, streak
  9. rotate, pull, streak in last Q
  10. return lid and flame ster loop
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11
Q

if cant see streaks

A

turn agar on angle w light

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12
Q

what is the main objective of streaking

A

to consistently have many well isolated colonies through dilution across plate

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13
Q

if you forget to cool the loop

A

damages and/or kill cells

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14
Q

forget to flame loop

A

before streaking out Q2, cells wont dilute well t/f probs wont get isolated colonies

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15
Q

streaking wet plate

A

causes colonies to fuse and run together thus distorting their true morph

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16
Q

not homogenizing a broth sample before streaking

A

may miss cells that are present in low numbers or heavier than other cells in the mix

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17
Q

insufficient zig zags in each Q

A

get insufficient dilution of cells/isolated colonies

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18
Q

breathing on agar

A

contam

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19
Q

incubating petri dish lid side up

A

condensation drips on plates causing colonies to spread out or run together

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20
Q

streak plate method from plate to plate

A
  1. hold loop in dom hand
  2. flame ster loop
  3. light bun burn and remove agar from lid
  4. use loop to pick up (avoid nearby colonies)
  5. return lid to plate and remove lid from new plate and streak in Q1
  6. flame ster and cool (10-15s)
  7. if col small, only flame after Q1; if col big also flame after Q2
  8. follow other steps of streaking
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21
Q

g stain procedure

A

cryst vi (60s)
water
gram’s iodine (60s)
water
acetone alcohol (5s)
water
safranin (60s)
water

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22
Q

smear prep from colony using a stab tool

A
  1. flame ster loop and cool
  2. transfer 1 loop of tap water to clean glass slide
  3. flame ster stab tool and cool
  4. gently touch isolated colony w stab tool
  5. resuspend cells on stab tool in water and smooth
  6. flam ster stab tool and cool
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23
Q

mic procedure

A

refer to lab summary

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24
Q

how does prepping a smear from colony differ from broth

A

cell density; loop taken from colony has too many cells

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25
Q

what is a viable cell

A

can grow and divide to produce daughter cells

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26
Q

how do we det viable cell counts

A

plate count method (cells deposited on top of an agar medium (spread) or throughout agar medium (pour plate)

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27
Q

what do the colony numbers on the plate provide

A

reasonable approx of the number of cells in the plated sample (each viable cell gives rise to one colony)

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28
Q

what is the spread plate method (general)

A

small volume of a diluted culture is spread onto the surface of a solid agar medium using a sterile spreader

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29
Q

what is the valid range

A

30-300 colonies (t/f usu need serial dilution)

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30
Q

if you spread plate directly from an overnight culture

A

get uncountable lawn of bact colonies

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31
Q

assume one cell gives rise to

A

one colony

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32
Q

what can affect cell viability

A

culture medium and incubation conditions (esp time)

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32
Q

what is the most sig source of error during plate count method

A

student technique

esp pipetting/vol errors
not even spread
spreader is too hot

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32
Q

cfu/mL formual

A

col/vol x 1/dil

vol = vol pipetted onto medium and spread
dil = final dil of sample that was plated

33
Q

what is a serial dilution

A

stepwise series of transerfs from OG sam[le through series of dilution blanks (tubes that have known amnt of sterile water, saline, media, or buffer)

34
Q

what do serial dilutions do

A

minimize effects of tech errors and help achieve high accuracy and precision

35
Q

range of p1000 pip

A

200-1000

36
Q

colour tips of p1000

A

blue

37
Q

pip range of p200

A

20-200

38
Q

colour tips for p200

A

yellow

39
Q

how do you grab a glass pippette

A

flame rim, shake, grab one pip w out touching others, flame rim, put on lid, put canister horiz

40
Q

prepare serial dilution of broth cult procedure

A
  1. add 9 ml of saline to 6 sterile tubes
  2. set vol of p1000 to 1 mL
  3. vortex broth
  4. transfer 1 mL to first tube
  5. discard pip tip and vortex that tube
  6. transfer 1 mL of that to new tube
  7. repeat ill 1 x 10^-6
41
Q

spread plate procedure

A
  1. put tsa lid up on turntable
  2. vortex tube, put 100uL on agar from 1 x 10^-6
  3. discard pip tip
  4. flame ster spreader
  5. spread and rotate
  6. put spreader back in alcohol
42
Q

what could cause contam

A

forgetting to flame mouth of tubes or dil bottles w transfers

more than one tube open at a time

contam pip

working too close to open agar

forget to ster spreader

43
Q

why can you s/t get false high spread counts

A

forget to ster spreader bw rep plates (get carry on//high counts)

forget to vortex tube

forget to change pip tip bw transfers

pressing pip plunger to second stop before aspirating

44
Q

why can you s/t get false low spread counts

A

hot spreader

insuff spreading (wet plate) = run on colonies

spreading sample to quick/vigorous to edge of plate

bubbles in pip tip (get less vol in tip)

45
Q

a) calc cfu/mL for dil 1 x 10^-6; 0.1 mL, 199+123 cfu

b) calc cfu for tubes 1,2,3

A

a) 1.6 x 10^9

b)1.6E8, 1.6E7, 1.6E6

46
Q

if 2 dif dilutions for calc, what do you do

A

times the cfu’s that are more diluted by 10 (its ok if over 300 after multiplication)

47
Q

if dif vol plated

A

mult cfu by two if have 0.05 mL to 0.1 mL

48
Q

why is a plate w less than 30 col unreliable

A

inconsistencies in tech have large effect on final cfu count

49
Q

why is a plate w more than 300 unreliable

A

crowding and run on

50
Q

how are run on colonies counted

A

as one

51
Q

if way greater than 300 col

A

TNTC

(if above 300 but still countable, divide into sections then mult)

52
Q

calc % dif formual

A

plate A cfu-plate B cfu/av cfu for A+B x 100

usu vary by 20%

only used for plates prep’d at “ vol and dil (replicate spread plates)

53
Q

% error formula

A

% error = exp value - theo value/theo value x 100

54
Q

calc % error from pg 5-14

A

27%

55
Q

the dilution factor is

A

the reciprocal of the dilution

dil fact = 1/dil

never have units!

56
Q

formula for dil

A

[]f/[]i

57
Q

calc ex 1 and 2 from 5-15

A

see manual

58
Q

if dont know i/f []’s, can calc dilution w this formula

A

dil = vol A / vol A + vol B

59
Q

calc dil from ex 3 from 5-15

A

see manual

60
Q

calc dils from ex 4, 5 and 6 from 5-16

A

see manual

61
Q

draw out a basic serial dil procedure

A

manual

62
Q

do serial dil calc from ex 1 and 2 5-19/5-20

A

manual

63
Q

continue the instructions beyond Koehler Illumination and through sample viewing by correctly ordering the steps below.

A

__4__ Use the revolving turret to move the 40X lens out of the way such that you are between the 40X and 100X objective lenses.
__7__ Open the aperture diaphragm to 100X
__2__ Use the revolving turret to switch the objective lens to 40X then open the aperture diaphragm to 40X.
__5__ Place a drop or two of oil on the slide.
__3__ Use X-Y movement to center on a high density of cells and make fine focus adjustments.
__6__ Click the 100X oil immersion lens into place such that the lens contacts the oil.
__8__ While using the oculars, slowly find the sample using the fine focus knob.
__1__ While looking through the 10X objective lens use the X and Y knobs to find the sample and center on an area of high cell density then make fine focus adjustments.

64
Q

Troubleshooting: You have made a streak plate from a broth culture of Staphylococcus epidermidis. The streak plate is free of contamination, however the growth is unexpectedly dense. Even in the fourth quadrant, there is only a single well isolated colony. A gram stain of this colony showed both pink and purple cells.

Which of the options below could reasonably explain the unexpected gram variable result?

A

acetone alcohol was left on slide for too long

cells were damaged after excessive heat fixation

65
Q

Which of the following technique errors would result in a streak plate from a broth culture with unexpectedly dense growth (and a lack of isolated colonies)?

A

Broad side of the loop was used for streaking secondary and tertiary sections

Forgot to flame after primary quadrant

Zig zags in secondary and tertiary crossed back into primary section

Primary section was too large

66
Q

In order to calculate the concentration of an overnight broth culture of E. coli you spread plate two different dilutions, in triplicate, using a volume of 100 µl for each plate. Use the tabulated data to answer the questions below and to calculate the cfu/ml of the original culture.

colonies (1E-6* dilution); # colonies (1E-7 dilution)
285, 290, 307; 28, 31, 32

A

3.0E9

67
Q

Calculate the concentration of an overnight broth culture of Staphylococcus epidermidis based on the tabulated data below. Each dilution was spread, in triplicate, using a volume of 100 µl for each plate. Ensure you are using the correct format, with the appropriate number of significant figures.

colonies (5E-5* dilution) # colonies (5E-6* dilution) # colonies (5E-7 dilution)
TNTC 240, 253, 301 24, 29, 31

A

_5.4E8

68
Q

Calculate the dilution of E. coli cells required if the initial overnight concentration of the broth culture is 6.5x108 cells/ml and the desired final concentration is 3.5x104 cells/ml.

calc dil factor

A

5.4E-5_

1.9E4

69
Q

Determine the dilution of Staphylococcus required if the initial concentration of the broth culture is 4.0x108 cells/mL and your goal is to have a spread plate with 100 colonies when 0.1 mL is plated. Hint: rearrange the cfu/ml equation to solve for dilution.

A

2.5E-6

70
Q

Which of the following would result in a 1x10-1 (1E-1) dilution?

0.5 ml of culture added to 5.0 ml of diluent

0.5 ml of culture added to 4.5 ml of diluent

100 ul culture added to 900ul of diluent

1 ml of culture added to 10 ml of diluent

1 ml of culture added to 9ml of diluent

A

b c e

71
Q

Which of the following would result in a 1x10-2 (1E-2) dilution?

0.5 ml of culture added to 49.5 ml of diluent

100 ul culture added to 900ul of diluent

1 ml of culture added to 99ml of diluent

0.1 ml of culture added to 10 ml of diluent

0.1 ml into 9.9 ml of diluent

A

a c e

72
Q

Answer the following question on paper showing all work and a complete diagram with a depiction of spread plates included. Then insert this answer as an attachment or image. See page 5-20 in the manual for an example.

A culture of Pseudomonas putida was grown in TSB for 22 hours with shaking. The concentration of this culture is known to be 2.0x109. How would the culture be serially diluted to allow a volume of 100 uL to result in 50 colonies on a spread plate?

See attachment for in depth calculations and serial dilution diagram.

A

see manual

73
Q

When preparing a streak plate from a broth culture, when is it necessary for you to heat-sterilize the inoculating loop?

A

Before culture transfer; after quadrant one; after quadrant four.

74
Q

When preparing a streak plate, what are the functions of the Bunsen burner in relation to aseptic technique?

A

sterilizing the inoculating loop tool

flaming the open mouth of culture tubes

generating a heated updraft that prevents air contaminants from settling onto the work surface

75
Q

What colony morphology characteristic cannot be determined until an organism is present in pure culture on an agar plate?

A

odor

76
Q

How does the preparation of a streak plate differ between taking a loopful of cells from a broth vs taking a loopful of cells taken from a colony?

A

The density of cells is higher from the colony. Need to flame after the primary streak (quadrant 1) and secondary streak (quadrant 2).

77
Q

After streaking from the mixed broth culture, how many different colony morphologies should appear on your plates after they have been incubated overnight?

A

2

78
Q

What two assumptions are made when we enumerate bacteria using the spread plate methods?

A

a colony is derived from a single cell; the # of colonies is equivalent to the # of viable cells in the dilution plated.

79
Q

A “1 in 10 dilution” means what?

A

1 part solution + 9 parts diluent (dilution = 0.1)

80
Q

What is the expected cellular morphology, using correct terminology, for a pure culture of Staphylococcus epidermidis?

A

gram-positive, 1 μm cocci, clusters (some single)

81
Q

In Lab 4, you will be performing a Gram Stain from a colony instead of a broth culture. What is different in preparing the smear on the slide?

A

You will put a loopful of tap water on the slide and use the inoculating needle to resuspend the colony.

82
Q

e coli spelling

A

escherichia coli

Micro Lab Quiz 1 (3 decks)

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