1. Exp 1: Culture Transfer Techniques , with organisms
MICROBIOLOGY LAB, 156
Day 3
Microorganism: a living organism too small to be seen with the naked eye, include bacteria, fungi,protozoa, microscopic algae, and viruses
Exp 1: Culture Transfer Techniques , with organisms
Bacteria: simple, single celled organisms, prokaryotes ( no nuclear membrane) measured in um (10-6m), 1-10 um
Exp 2A, Isolation of Pure Cultures
Streak Plate Dilution Technique , SPS
Spread Plate Technique
5/9/2005

2. MICROBIOLOGY LAB, 156
Day 3
Exp 2A, Isolation of Pure Cultures
Streak Plate Dilution Technique , SPS
Spread Plate Technique
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.
Terms: pure culture, sterilization, sub culturing, aseptic technique, media, streak plate dilution technique (and theory), spread plate technique
5/9/05

3. Flaming- prevents contamination of culture
Hold Inoculating loop
Insert in flame until loop glows red
Allow to cool

4. 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
   

5. 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

6. 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.

7. Broth to streak plate Procedure for Streaking a Plate for Isolation:
  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.    

8. Streak plate

9. Growth of organism over plate

11. 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

12. 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

13. Organisms SM, Serratia marcescens ML, Micrococcus luteus
BS, Bacillus subtilis

14. Exp 1: Culture Transfer Techniques , w/ organisms Materials: BC
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 SM
RF,SM, 1/27

15. Streak Plate Dilution Technique , SPD Spread Plate Technique
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
Spread Plate Technique
SM, Serratia marcescens
ML, Micrococcus luteus
BS, Bacillus subtlus
SM/ ML
BS/SM
cultures SP
SM/ ML
BS/SM
cultures
SPD
SPD,SM/ML RF
SPD,bs/sm RF
SM/ML RF
Bs/sm RF
Incubation, 25C, 24 hrs

16. Exp 2A, Isolation of Pure Cultures
Streak Plate Dilution Technique , SPD
Spread Plate Technique
SM/ ML
cultures SP
SM/ ML
BS/SM
cultures
SPD
BS/SM
22C/24hr
SPD,SM/ML RF
SPD,BS/SM RF
SM/ML RF
BS/SM RF
Materials: mixed cultures: SM/ML & BS/SM , one per table, 4 TSA Plates per person

17. 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

18. 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)

19. Comparison of E. coli and Micrococcus luteus

20. Colony morphology

21. Colony morphology

22. Margin of the colonies

23. Elevation

24. Broth culture( refer to supplement)
Cloudy
Turbid( Flocculent)
Sediment formation
Pellicle formation

25. 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

26. 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

27. New work( supply table)
Eight Organisms for Study/Table
8 Plates
8 Deeps( if available)
8 Slants
8 Broths

28. 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

29. Organisms for study 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