Microbiology Lab Notes 9/8/09

- heat lid of bottle of sterile broth and put 1mL into sterile test tube after heating the opening of the test tube as well
- suspend 1 colony from handprint plate in broth byt inflaming inoculating loop, letting it cool, and getting an isolated colony from the streak plate
- swirl inoculating loop with bacteria on it in the sterile test tube full of sterile broth

Kirby-Bauer Technique
- Mueller-Hinton Agar: used to test for antibiotic (abx) susceptibility
- make "lawn" of bacteria using swab & broth w/ bacteria in it by the following
1. get swab and 2 Mueller-Hinton Agar plates and open tube with bacteria in it
2. heat the test tube opening
3. put swab into broth and swirl
4. smear 1/2 way across Mueller-Hinton Agar overlapping smears
5. turn 90 degrees and repeat
6. turn another 90 degrees and repeat again
Repeat 1-6 on second plate as well
7. make sure whole plate is covered; cannot overdo this
- Place antibiotic discs on agar using flame/alcohol-sterilization tweezers
1. take alcohol jar and put tweezers in it
2. put tweezers into the fire and allow the alcohol to burn off
3. repeat this several times to ensure sterilization
4. use tweezers to gently grab antibiotic disc from tube
5. put on agar and tap it into it gently
6. label antibiotic on back of plate immediately so you know which is which antibiotic
7. use 5 antibiotics and 1 negative control (3 on one plate 3 on the other)

- Streak test tube sample from test tube containing bacteria onto EMB (eosin-methylene blue) agar and onto MSA (manitol-salt agar)

Serial Dilution Technique
- tells how many bacteria in a sample
- enables us to count live bacteria in a sample
- start w/ test tube w/ bacteria suspended w/in
- put .9 mL of sterile broth into 6 sterile test tube using best aspetic technique and label them 1-6
- take .1 mL out of original sample of broth with bacteria suspended in it
- flick tube to mix
- take .1mL out of tube labeled #1 that you just put the .1 mL in from the original sample
- put .1mL from test tube #1 and put into #2
- flick and take .1 mL from test tube #2 and put it into #3
-flick again to mix
- continue until get to test tube #6
-this is the process of dilution

Spread Plate Technique
- lapel 7 plates by dilution for example
- 1:1 = originial sample 1:10 1:100 and so on until 1:1,000,000 dilution
- start with the plate labeled 1,000,000 and go backwards starting with test tube #6
- place .1 mL drop from #6 on center of plate labeled 1:1,000,000
- get glass hockey stick dip in alcohol and heat so alcohol burns off
- gently run glass hockey stick back and forth over the plate while simultaneously turning the plate as well
- contine these steps until you get done with the 1:1 dilution plate

Put all plates done in this lab into the incubator and interpret results in lab on Thursday, September 10.

Amy Blass

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