micro lab quiz 2 Flashcards
what is the problem w mixed culture
can’t effectively study
in order to study and characterize a single bacterial species, what do you need to do
obtain a pure culture (contains a single species and cells orig’d from a single parent cell)
list 3 methods to isolates bact
streak plate, agar dilution, liquid dilution
what is the streak plate method
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
what is another word for colony
colony forming unit
what affects the pattern and method of preparing a streak plate
medium used, source of inocculation, ind pref based on experience
what is an advantage to streak plate method for isolation
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
what must be assessed not assumed during an isolation method
purity
how do you test the purity of the culture
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
req’s of aseptic tech
near bunsen burner (creates updrafts to keep contams from air from falling in)
one thing open at a time
streak plate protocol from broth cult
- flame ster loop (cool)
- vortex tube (make sure to flame rim)
- dip loop; pick up agar side of plate
- streak 1/4 (don’t press too hard, don’t breathe over, hair fall)
- flame ster loop (cool 10-15s)
- rotate and drag 2 par lines from Q1-Q2
- zig zag from 2 pull lines in Q2 (don’t cross back and don’t overlap zig zags)
- rotate, pull, streak
- rotate, pull, streak in last Q
- return lid and flame ster loop
if cant see streaks
turn agar on angle w light
what is the main objective of streaking
to consistently have many well isolated colonies through dilution across plate
if you forget to cool the loop
damages and/or kill cells
forget to flame loop
before streaking out Q2, cells wont dilute well t/f probs wont get isolated colonies
streaking wet plate
causes colonies to fuse and run together thus distorting their true morph
not homogenizing a broth sample before streaking
may miss cells that are present in low numbers or heavier than other cells in the mix
insufficient zig zags in each Q
get insufficient dilution of cells/isolated colonies
breathing on agar
contam
incubating petri dish lid side up
condensation drips on plates causing colonies to spread out or run together
streak plate method from plate to plate
- hold loop in dom hand
- flame ster loop
- light bun burn and remove agar from lid
- use loop to pick up (avoid nearby colonies)
- return lid to plate and remove lid from new plate and streak in Q1
- flame ster and cool (10-15s)
- if col small, only flame after Q1; if col big also flame after Q2
- follow other steps of streaking
g stain procedure
cryst vi (60s)
water
gram’s iodine (60s)
water
acetone alcohol (5s)
water
safranin (60s)
water
smear prep from colony using a stab tool
- flame ster loop and cool
- transfer 1 loop of tap water to clean glass slide
- flame ster stab tool and cool
- gently touch isolated colony w stab tool
- resuspend cells on stab tool in water and smooth
- flam ster stab tool and cool
mic procedure
refer to lab summary
how does prepping a smear from colony differ from broth
cell density; loop taken from colony has too many cells
what is a viable cell
can grow and divide to produce daughter cells
how do we det viable cell counts
plate count method (cells deposited on top of an agar medium (spread) or throughout agar medium (pour plate)
what do the colony numbers on the plate provide
reasonable approx of the number of cells in the plated sample (each viable cell gives rise to one colony)
what is the spread plate method (general)
small volume of a diluted culture is spread onto the surface of a solid agar medium using a sterile spreader
what is the valid range
30-300 colonies (t/f usu need serial dilution)
if you spread plate directly from an overnight culture
get uncountable lawn of bact colonies
assume one cell gives rise to
one colony
what can affect cell viability
culture medium and incubation conditions (esp time)
what is the most sig source of error during plate count method
student technique
esp pipetting/vol errors
not even spread
spreader is too hot
cfu/mL formual
col/vol x 1/dil
vol = vol pipetted onto medium and spread
dil = final dil of sample that was plated
what is a serial dilution
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)
what do serial dilutions do
minimize effects of tech errors and help achieve high accuracy and precision
range of p1000 pip
200-1000
colour tips of p1000
blue
pip range of p200
20-200
colour tips for p200
yellow
how do you grab a glass pippette
flame rim, shake, grab one pip w out touching others, flame rim, put on lid, put canister horiz
prepare serial dilution of broth cult procedure
- add 9 ml of saline to 6 sterile tubes
- set vol of p1000 to 1 mL
- vortex broth
- transfer 1 mL to first tube
- discard pip tip and vortex that tube
- transfer 1 mL of that to new tube
- repeat ill 1 x 10^-6
spread plate procedure
- put tsa lid up on turntable
- vortex tube, put 100uL on agar from 1 x 10^-6
- discard pip tip
- flame ster spreader
- spread and rotate
- put spreader back in alcohol
what could cause contam
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
why can you s/t get false high spread counts
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
why can you s/t get false low spread counts
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)
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) 1.6 x 10^9
b)1.6E8, 1.6E7, 1.6E6
if 2 dif dilutions for calc, what do you do
times the cfu’s that are more diluted by 10 (its ok if over 300 after multiplication)
if dif vol plated
mult cfu by two if have 0.05 mL to 0.1 mL
why is a plate w less than 30 col unreliable
inconsistencies in tech have large effect on final cfu count
why is a plate w more than 300 unreliable
crowding and run on
how are run on colonies counted
as one
if way greater than 300 col
TNTC
(if above 300 but still countable, divide into sections then mult)
calc % dif formual
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)
% error formula
% error = exp value - theo value/theo value x 100
calc % error from pg 5-14
27%
the dilution factor is
the reciprocal of the dilution
dil fact = 1/dil
never have units!
formula for dil
[]f/[]i
calc ex 1 and 2 from 5-15
see manual
if dont know i/f []’s, can calc dilution w this formula
dil = vol A / vol A + vol B
calc dil from ex 3 from 5-15
see manual
calc dils from ex 4, 5 and 6 from 5-16
see manual
draw out a basic serial dil procedure
manual
do serial dil calc from ex 1 and 2 5-19/5-20
manual
continue the instructions beyond Koehler Illumination and through sample viewing by correctly ordering the steps below.
__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.
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?
acetone alcohol was left on slide for too long
cells were damaged after excessive heat fixation
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)?
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
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
3.0E9
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
_5.4E8
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
5.4E-5_
1.9E4
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.
2.5E-6
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
b c e
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 c e
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.
see manual
When preparing a streak plate from a broth culture, when is it necessary for you to heat-sterilize the inoculating loop?
Before culture transfer; after quadrant one; after quadrant four.
When preparing a streak plate, what are the functions of the Bunsen burner in relation to aseptic technique?
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
What colony morphology characteristic cannot be determined until an organism is present in pure culture on an agar plate?
odor
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?
The density of cells is higher from the colony. Need to flame after the primary streak (quadrant 1) and secondary streak (quadrant 2).
After streaking from the mixed broth culture, how many different colony morphologies should appear on your plates after they have been incubated overnight?
2
What two assumptions are made when we enumerate bacteria using the spread plate methods?
a colony is derived from a single cell; the # of colonies is equivalent to the # of viable cells in the dilution plated.
A “1 in 10 dilution” means what?
1 part solution + 9 parts diluent (dilution = 0.1)
What is the expected cellular morphology, using correct terminology, for a pure culture of Staphylococcus epidermidis?
gram-positive, 1 μm cocci, clusters (some single)
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?
You will put a loopful of tap water on the slide and use the inoculating needle to resuspend the colony.
e coli spelling
escherichia coli
