Physical Agents of Microbial Control

Preparation and Materials

per team (2 or 4 people)
    8 sterile tryptic soy broth tubes
    2 empty sterile petri dishes
    melted agar
    6 regular TSA plates
    5 TSA plates with varying salt concentrations
    5 sterile cotton swabs
    E. coli, B. subtilis cultures

Student instructions

A.  Temperature
    Label 8 of your tubes of TS broth with name, date, and code as follows
    (E= E. coli and B= B. subtilis):

        EC and BC to be inoculated and incubated for 48 hours, then observed as control tubes.

        ERT and BRT to be inoculated and kept at room temperature for 48 hours, then observed.

        E56 and B56 to be inoculated and set in a 56^o C water bath for 10 minutes, then incubated
        for 48 hours and observed.

        EB and BB to be inoculated and set in a boiling water bath for 10 minutes then incubated 48
        hours and observed.

    Move to the incubator the 4 tubes inoculated in the previous lab:

        EF and BF (frozen in previous lab) to be incubated for 48 hours and observed.

        ER and BR (refrigerated in previous lab) to be incubated for 48 hours and observed.

B.  Osmotic concentration

   Label the salt (NaCl) TSA plates with name, date, and meat (as assigned by the instructor).  Each group/pair should should have one plate each of the following concentrations: 0%, 5%, 10%, 15% and 20%.  Using a sterile cotton swab, swab the meat and then lawn the 0% TSA plate.  Repeat for the other four concentrations, using a new cotton swab for each plate.  Incubate for 48 hours. Observe growth and make comparisons.

C. Drying

Label 2 empty petri dishes EA and BA.  With a sterile cotton swab, smear the "E" plate with E. coli broth and the "B" plate with B. subtilis broth.  Retrieve the "D" plates inoculated in the previous lab from the drawer  Overlay the "A" and "D" plates with melted agar and incubate for 48 hours.

Next lab:  Observe and record results of the "A" and "D" plates. Did either organism survive drying period?

D. Effect of ultraviolet radiation

Two lab tables should label a set of agar plates each:

        Bacillus closed, UV   (10 min.)
        Bacillus opened, UV 
        Bacillus, cut-out, UV

        E. Coli, closed, UV
        E. coli, opened, UV
        E. coli, cut-out, UV

Two other lab tables should label a set of agar plates each:

        Bacillus closed, FL   (10 min.)
        Bacillus opened, FL 
        Bacillus, cut-out, FL

        E. coli, closed, FL
        E. coli, opened, FL
        E. coli, cut-out, FL

  Lawn the plates with either E. coli, or B. sub. as labeled.

The UV labeled plates will be placed under the sterile guard hood and exposed to the UV light for 10 minutes.  Those labeled "closed" will be exposed with the lids on, and those labeled "open" will be exposed with the lids off.  Those labeled "cut out" will be covered by a cardboard cut out in any shape design.  (these lids will also be removed at the start of exposure)

The FL labeled plates will be placed in the Sterile guard hood and exposed to the fluorescent work light for 10 minutes. 

All plates will be incubated for 48 hours, observed and then comparisons should be made.