Microbiology Microbiology TechniquesTechniques

20082008

Media TypesMedia Types

How to hold an Inoculating How to hold an Inoculating LoopLoop

Flaming the LoopFlaming the Loop

Streak PlateStreak Platehttp://www.sumanasinc.com/webcontent/anisamples/microbiology/strhttp://www.sumanasinc.com/webcontent/anisamples/microbiology/streakplate.htmleakplate.html

Triple streakTriple streak

Streaking and flamingStreaking and flaming Flame the loop to sterilize it and let cool. Flame the loop to sterilize it and let cool. Position the plate so that the spot of inoculum is nearest the hand not Position the plate so that the spot of inoculum is nearest the hand not

holding the loop (the opposite hand). holding the loop (the opposite hand). Lift the plate lid with the opposite hand; just enough to get the loop inside Lift the plate lid with the opposite hand; just enough to get the loop inside

and touch the loop to the inoculum spot. It is often helpful to treat the and touch the loop to the inoculum spot. It is often helpful to treat the inoculating loop as if it were a pencil - steadying the loop by resting the inoculating loop as if it were a pencil - steadying the loop by resting the heel of the hand against the lab bench. heel of the hand against the lab bench.

Move the loop back and forth across the spot and then gradually continue Move the loop back and forth across the spot and then gradually continue toward the center of the plate as you sweep back and forth. Use a very toward the center of the plate as you sweep back and forth. Use a very gentle and even pressure. gentle and even pressure.

When creating each phase, do not worry about keeping each pass across When creating each phase, do not worry about keeping each pass across the plate separate from previous ones. the plate separate from previous ones.

When about 30% of the plate has been covered by the first streaking When about 30% of the plate has been covered by the first streaking phase, remove the loop and flame sterilize it. phase, remove the loop and flame sterilize it.

Repeat the above procedure for the second phase, but this time pick up Repeat the above procedure for the second phase, but this time pick up some inoculum by crossing into the first phase 2-3 times and then not some inoculum by crossing into the first phase 2-3 times and then not passing into it again (Figure 1-5). passing into it again (Figure 1-5).

Repeat as necessary for the third and fourth phases. After streaking the Repeat as necessary for the third and fourth phases. After streaking the plate, flame sterilize the loop before setting it down.plate, flame sterilize the loop before setting it down.

Transfer to tubesTransfer to tubes

Flaming tubesFlaming tubes

Streaking a slantStreaking a slant

Transfer 2Transfer 2 1. Wrap fingers of non dominant hand around the culture tube 1. Wrap fingers of non dominant hand around the culture tube

containing broth for transfer containing broth for transfer         2. Using the pinkie finger of your dominant hand twist the red 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 cap from the tube.  Hold in your pinkie and do not place it on the counter counter

3.     Pass the mouth of the culture tube across the flame 3.     Pass the mouth of the culture tube across the flame 4.     Direct the inoculating needle into the broth. 4.     Direct the inoculating needle into the broth. 5.     Flame the mouth of your broth culture tube and replace the 5.     Flame the mouth of your broth culture tube and replace the

cap.  Place it in your rack cap.  Place it in your rack 6.     Pick up the slant in your non dominant hand 6.     Pick up the slant in your non dominant hand 7.     Twist off the red cap 7.     Twist off the red cap 8.     Flame the mouth of the slant tube 8.     Flame the mouth of the slant tube 9.     Direct the inoculating needle into the tube and “ stab” the 9.     Direct the inoculating needle into the tube and “ stab” the

agar in the base( butt) agar in the base( butt) 10. Withdraw on the entry line and when you reach the surface 10. Withdraw on the entry line and when you reach the surface

make a simple streak along the face. make a simple streak along the face. 11.  Flame the mouth of the tube and replace the cap. 11.  Flame the mouth of the tube and replace the cap. 12. Flame your inoculating needle and replace in your rack. 12. Flame your inoculating needle and replace in your rack.

Transfer 3Transfer 3 Steps for Transfer of Broth to BrothSteps 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

Colony MorphologyColony Morphology

Colony MorphologyColony Morphology

Colony morphologyColony morphology

ColorColor ShapeShape MarginMargin ElevationElevation

The SmearThe Smear Using aseptic technique remove a colony from a Using aseptic technique remove a colony from a

plate or cells from your slant.  Be carefully to gently plate or cells from your slant.  Be carefully to gently touch the surface of your culture with the touch the surface of your culture with the inoculating loop. inoculating loop.

Make a circular motion in the middle of the circle to Make a circular motion in the middle of the circle to spread the cells equally in this region of the slide spread the cells equally in this region of the slide

Add a drop of water in the middle Add a drop of water in the middle Mix again Mix again Let Air dry Let Air dry Run the slide through the flame until the slide is Run the slide through the flame until the slide is

warm ( The frosted side should be down)  This fixes warm ( The frosted side should be down)  This fixes the bacteria to the slide the bacteria to the slide

Let the slide cool Let the slide cool Place in the metal tray or in the rack Place in the metal tray or in the rack

Gram StainGram Stain All staining work is to be done at the sink All staining work is to be done at the sink Care should be taken to work directly over the sink Care should be taken to work directly over the sink Place 1 drop of crystal violet stain on the smear ( 1 Place 1 drop of crystal violet stain on the smear ( 1

minute) minute) Rock or roll the slide to cover the area Rock or roll the slide to cover the area Use the water bottle to drip water down the slide Use the water bottle to drip water down the slide Place 1 drop of iodine on the slide ( 1 minute) Place 1 drop of iodine on the slide ( 1 minute) Place 1 drop of alcohol on the slide 10 seconds ( KEY – do Place 1 drop of alcohol on the slide 10 seconds ( KEY – do

not leave on longer than 10 seconds or it will decolorize) not leave on longer than 10 seconds or it will decolorize) Place 1 drop of saffranin on the slide for 1 minute Place 1 drop of saffranin on the slide for 1 minute Rinse with water from the bottle Rinse with water from the bottle Let the slide air dry Let the slide air dry   

Review of Bacterial Cell Review of Bacterial Cell MorphologyMorphology

http://http://science.nhmccd.edscience.nhmccd.edu/biol/wellmeyer/bu/biol/wellmeyer/bacteria/bacmorph.hacteria/bacmorph.htmtm

StreptococcusStreptococcus

Staphylococcus aureusStaphylococcus aureus

Gram negative bacilliGram negative bacilli