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

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

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

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

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

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

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 words Aerobic Anaerobic Facultative

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

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