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.    

Streak plate

Growth of organism over plate

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

Organisms

SM, Serratia marcescens ML, Micrococcus luteus BS, Bacillus subtilis

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)

Comparison of E. coli and Micrococcus luteus

Colony morphology

Colony morphology

Margin of the colonies

Elevation

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

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