Transcript Exp 2A, Isolation of Pure Cultures Streak Plate Dilution Technique

MICROBIOLOGY LAB, 156
Day 3
Exp 1: Culture Transfer
Techniques , with organisms
Exp 2A, Isolation of Pure
Cultures
•Streak Plate Dilution
Technique , SPS
Microorganism: a living
organism too small to be seen
with the naked eye, include
bacteria, fungi,protozoa,
microscopic algae, and viruses
Bacteria: simple, single celled
organisms, prokaryotes ( no
nuclear membrane) measured in
um (10-6m), 1-10 um
•Spread Plate Technique
5/9/2005
MICROBIOLOGY LAB, 156
Day 3
Exp 1: Culture Transfer
Techniques , w/ organisms
• Cultures: one BS, SM culture per
table
•Media: 2 broth, 2 slant, 2 deep, 2
plate per person ( min)
•properly label each medium,
• aseptically transfer, inoculate, to
each medium.
• Prep for incubation at 37C/24hrs.
Exp 2A, Isolation of Pure
Cultures
Streak Plate Dilution
Technique , SPS
•Spread Plate Technique
Terms: pure culture,
sterilization, sub culturing,
aseptic technique, media,
streak plate dilution
technique (and theory),
spread plate technique
5/9/05
Flaming- prevents contamination of
culture
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Hold Inoculating loop
Insert in flame until
loop glows red
Allow to cool
Broth to slant
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1. Wrap fingers of non dominant hand around the culture
tube containing broth for transfer
2. Using the pinkie finger of your dominant hand twist
the red cap from the tube. Hold in your pinkie and do not
place it on the counter
3.
Pass the mouth of the culture tube across the flame
4.
Direct the inoculating needle into the broth.
5.
Flame the mouth of your broth culture tube and
replace the cap. Place it in your rack
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Pick up the slant in your non dominant hand
Transfer of broth to broth
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Steps for Transfer of Broth to Broth
Hold loop or needle with dominant hand( right )
Flame the loop
Hold culture tube in left hand
Remove red cap with pinkie of right hand
Flame mouth of culture tube
Place loop into broth( water)
Flame mouth of culture tube and close
Open culture tube with broth( should be labeled)
Dip loop into new broth and mix
Flame mouth of tube and close
Flame loop
Place to the side of your rack
Part 2
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Twist off the red cap
8.
Flame the mouth of the slant tube
9.
Direct the inoculating needle into the tube and “
stab” the agar in the base( butt)
10. Withdraw on the entry line and when you reach the
surface make a simple streak along the face.
11. Flame the mouth of the tube and replace the cap.
12. Flame your inoculating needle and replace in your rack.
Broth to streak plate
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Procedure for Streaking a Plate for Isolation:
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Procedure:
1. Flame the loop and wire and streak a loopful of
broth as at A in the diagram.
2. Reflame the loop and cool it.
3. Streak as at B to spread the original inoculum
over more of the agar.
4. Reflame the loop and cool it.
5. Streak as at C.
6. Reflame the loop and cool it.
7. Streak as at D.
8. Label the plate and incubate it inverted.
Streak plate
Growth of organism over plate
Journal Entry
Exp 2. Exp 1 Culture Transfer Tech.
date
Pg #
Purpose: In lab book
Materials: Cultures: BS, SM. Media: .. Equipment: …
Procedures: bullet procedures,
and ref page # in lab book ( citation: Cappucino & Sherman,
7th ed. Pages : ---)
Data: next period
Conclusion:
signed
Journal Entry
Exp 3. Exp 2A. Isolation of Pure Cultures date
Pg #
Purpose: In lab book
Materials: Cultures: Mixed BS, SM. & SM, ML, Media: ..
Equipment: …
Procedures: bullet procedures,
and ref page # in lab book ( citation: Cappucino & Sherman,
7th ed. Pages : ---)
Data: next period
Conclusion:
signed
Organisms
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SM, Serratia marcescens
ML, Micrococcus luteus
BS, Bacillus subtilis
Exp 1: Culture Transfer Techniques , w/
organisms
BC
Materials:
•per table: cultures: one BS broth, SM broth,
ML broth and slants
•per person : media: 3 broth, 3slant,3 deeps, 3
plate ( min)
RF,BC, 1/27
SM
Procedure:
•properly label each medium
• aseptically transfer, inoculate each organism
to the three different media.
• Prep for incubation at 25C, /24hrs
RF,SM, 1/27
Exp 2A, Isolation of Pure Cultures
Materials: mixed cultures: SM/ML & BS/SM , one per table, 4 TSA Plates per person
•Streak Plate
Dilution
Technique ,
SPD
SM, Serratia marcescens
ML, Micrococcus luteus
BS, Bacillus subtlus
cultures
cultures
SM/ ML
•Spread Plate
Technique
SM/ ML
BS/SM
BS/SM
SP
SPD
SPD,SM/ML RF
SPD,bs/sm RF
SM/ML RF
Incubation, 25C, 24 hrs
Bs/sm RF
Exp 2A, Isolation of Pure Cultures
•Streak Plate Dilution
Technique , SPD
•Spread Plate Technique
cultures
cultures
SM/ ML
22C/24hr
SPD
SPD,SM/ML RF
BS/SM
SM/ ML
BS/SM
SPD,BS/SM RF
SM/ML RF
SP
BS/SM RF
Materials: mixed cultures: SM/ML & BS/SM , one per table, 4 TSA Plates per person
Culture characteristics
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Use supplement and handout to observe
the growth of the four organisms in the
slant, deep, broth, and on the plate.
Do the organisms look like one of the
examples on your sheet?
Try to record their appearance on your
templates
Culture observations on the agar
plate
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Color production( chromogenesis). An
example of this is the pink color of
Serratia
Growth pattern and characteristics
Amount of growth( scant or heavy)
Comparison of E. coli and Micrococcus
luteus
Colony morphology
Colony morphology
Margin of the colonies
Elevation
Broth culture( refer to supplement)
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Cloudy
Turbid( Flocculent)
Sediment formation
Pellicle formation
Slants
Is there growth in the
bottom ?
 Is there growth on the
slant itself
 What are the growth
characteristics on the
slant?
 Key words
Aerobic
Anaerobic
Facultative
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Isolation of Pure culture
Observe your dilution streak of your mixed culture
 On the bottom of your Petri dish circle colonies of
two organisms
 Example
ML/SM mixture – circle yellow and pink cultures
 With your inoculating loop lift cells from circled
colonies and streak on new plate or inoculate a
slant per detailed instructions in class
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New work( supply table)
Eight Organisms for Study/Table
 8 Plates
 8 Deeps( if available)
 8 Slants
 8 Broths
Preparation
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Label all tubes and plates carefully
Assign each member of the group 2
organisms
Transfer the organisms to the culture
media using aseptic techniques used in
weeks one and two
Organisms for study
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Gram negative organisms
PA - Pseudomonas aeruginosa
PV- Proteus vulgaris
EC- Escherichia coli
EA- Enterobacter aerogenes
Gram Positive Organisms
BS - Bacillus subtilis
SA - Staphylococcus aureus
SE - Staphylococcus epidermidis
SS- Streptococcus salivarius