day 3 exp 1: culture transfer techniques, with organisms microbiology lab, 156 exp 2a, isolation of...
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Day 3 Day 3
Exp 1: Culture Transfer Techniques , with organisms
MICROBIOLOGY LAB, 156
Exp 2A, Isolation of Pure Cultures
•Streak Plate Dilution Technique , SPS
•Spread Plate Technique
5/9/2005
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
Day 3 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.
MICROBIOLOGY LAB, 156
Exp 2A, Isolation of Pure Cultures
Streak Plate Dilution Technique , SPS
•Spread Plate Technique
5/9/05
Terms: pure culture, sterilization, sub culturing, aseptic technique, media, streak plate dilution technique (and theory), spread plate technique
Flaming- prevents contamination of culture
Hold Inoculating loop Insert in flame until
loop glows red Allow to cool
Broth to slant
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 6. Pick up the slant in your non dominant hand
Transfer of broth to broth
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
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 Procedure for Streaking a Plate for Isolation:
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.
Journal Entry
Exp 2. Exp 1 Culture Transfer Tech. date
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:
Pg #
signed
Journal Entry
Exp 3. Exp 2A. Isolation of Pure Cultures date
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:
Pg #
signed
Exp 1: Culture Transfer Techniques , w/ organisms
Materials:
•per table: cultures: one BS broth, SM broth, ML broth and slants
•per person : media: 3 broth, 3slant,3 deeps, 3 plate ( min)
Procedure:
•properly label each medium
• aseptically transfer, inoculate each organism to the three different media.
• Prep for incubation at 25C, /24hrs
RF,BC, 1/27
BC
SM
RF,SM, 1/27
Exp 2A, Isolation of Pure Cultures
•Streak Plate Dilution Technique , SPD
•Spread Plate Technique
SM/ ML BS/SM
cultures
SPD
SPD,SM/ML RF SPD,bs/sm RF SM/ML RF Bs/sm RF
SM, Serratia marcescens
ML, Micrococcus luteus
BS, Bacillus subtlus
Incubation, 25C, 24 hrs
Materials: mixed cultures: SM/ML & BS/SM , one per table, 4 TSA Plates per person
SM/ ML BS/SM
cultures
SP
Exp 2A, Isolation of Pure Cultures
•Streak Plate Dilution Technique , SPD
•Spread Plate Technique
SM/ ML BS/SM
cultures
SPD
SM/ ML
cultures
SP
SPD,SM/ML RF SPD,BS/SM RF SM/ML RF BS/SM RF
BS/SM
Materials: mixed cultures: SM/ML & BS/SM , one per table, 4 TSA Plates per person
22C/24hr
Culture characteristics
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
Color production( chromogenesis). An example of this is the pink color of Serratia
Growth pattern and characteristics Amount of growth( scant or heavy)
Broth culture( refer to supplement)
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 wordsAerobicAnaerobicFacultative
Isolation of Pure culture
Observe your dilution streak of your mixed culture
On the bottom of your Petri dish circle colonies of two organisms
ExampleML/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
New work( supply table)
Eight Organisms for Study/Table 8 Plates 8 Deeps( if available) 8 Slants 8 Broths
Preparation
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