Name:
Section:
Introductory Microbiology PreLab Assignment
Control of Microbial Growth: Alcohol Evaluation & Hand Scrubbing Effectiveness
1. (1pt.) Describe the contributions of Semmelweis and Lister to antiseptic procedures in
hospitals.
2. (0.5pt.) List two types of organisms alcohol is effective against.
3. (1pt.) When is alcohol not effective?
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4. (1pt.) Define puerperal fever.
5. (1pt.) What are the two most common alcohols used as antiseptics and in what
concentrations?
6. (0.5pt.) Define nosocomial infection.
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Lab Exercise
Control of Microbial Growth:
Alcohol Evaluation & Hand Scrubbing Effectiveness
Feb. 17, 2015
Between 1847 and 1850, the physician Ignaz Phillip Semmelweis, at the Lying-In
Hospital in Vienna, observed that medical students and physicians seemed to spread puerperal
fever (childbed fever: uterine streptococcal infection occurring after childbirth) at a much
higher rate than midwives. He noticed that it was routine for medical students and physicians,
directly after autopsies and with no attempt to cleanse their hands, to assist deliveries.
Midwives, on the other hand, did not attend autopsies. He deduced that contamination from
corpses led to the spread of the disease and ordered medical students to wash their hands before
delivering babies. Subsequently, the death rate due to puerperal fever fell in one year from 12%
to ~1%. Unfortunately, he died shortly after a nervous breakdown realizing that the majority of
the medical establishment did not accept the concept of obstetric antisepsis.
Soon after Semmelweis' death, Joseph Lister, an English surgeon much influenced by
Pasteur's research, published his work on antiseptic surgery techniques. He soaked dressings in
phenol (carbolic acid, C6H5OH) and sterilized instruments. His results were impressive, and
many surgeons began using his techniques to prevent infection during surgery.
Techniques such as hand washing and alcohol antisepsis are now commonplace in
medical settings. In this exercise, you will evaluate the effectiveness of alcohol swabs to kill
bacteria and hand washing to degerm the skin.
NO STUDENT SHOULD WASH HIS/HER HANDS
UNTIL INSTRUCTED TO DO SO!!!!!
HOWEVER….
READ!!!!!!! ONLY ONE STUDENT
in the class will actually be scrubbed during the
HAND WASHING experiment.
Other students will assist.
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Experiment 1: Alcohol Evaluation
Ethanol and isopropanol are alcohols whose mode of action is to denature proteins and
dissolve membrane lipids. They are widely used at a 70-80% concentration as bacteriocidal
and fungicidal agents. However, they are not effective against endospores and some viruses.
Day 1
Materials: group of 2 students
Alcohol swab
Blood agar plate
Procedure:
One student in each group will perform the experiment with the other student assisting. The
student who performs the experiment must not wash either hand preceding the exercise!
1. On the bottom of the plate (with the agar), mark the plate into 4 quadrants, and label the
quadrants "L1", "L2", "R1", and "R2".
2. The unwashed student will "roll" the pad of the left thumb against the agar surface in the
"L1" quadrant. Do this gently. Do not gouge the agar.
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3. The same student will then "roll" the left thumb on the "L2" quadrant. Do not touch
anything before you do this.
This will establish the amount of bacteria removed by simply touching the agar.
4. The same student will then "roll" the pad of the right thumb against the agar surface of the
"R1" quadrant. Do not touch anything after this step!
5. The student will then have the pad of the right thumb cleansed with the alcohol swab. The
assisting partner will do this. Be thorough.
6. The student will then "roll" the right thumb in the "R2" quadrant.
7. Incubate plates inverted at 37C for 48 hours.
Day 2
Colony counts in each of the quadrants will be taken. Record the number of colonies in
the lab report. Then calculate the percentages of reduction for both the left thumb and right
thumb using the formula below. A zero percent reduction is recorded if the number of colonies
increased in the second press.
Percent Reduction
=
(Colony Count 1st press) - (Colony Count 2nd press)
Colony Count 1st press
x 100
After all groups have tabulated their percent reductions, record the class results, and calculate
the average class percent reductions for the left and right thumbs.
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Experiment 2: Hand Scrubbing Effectiveness
Only one student in the class will actually be scrubbed. This student should not wash his/her
hands before the experiment or perform the alcohol evaluation. All other students will assist.
Hand scrubbing is routinely utilized in order to prevent nosocomial (hospital-acquired)
infections. Two types of microbes are removed during a surgical scrub, transient and resident.
Transient microbes (contaminating) are not deeply entrenched and are removed from the skin
fairly quickly during scrubbing. On the other hand, resident (constantly present) microbes,
consisting primarily of Staphylococcus, are deeply embedded and require a longer scrub time in
order to be removed.
Day 1
Materials:
Sterile surgical scrub brushes (6 total per class)
Basins containing 1000ml of sterile water (6 total per class)
Antibacterial soap
1.0ml pipet (1 per basin)
Pipet pump
TSA plates (18 total per class, 3/basin)
Bactispreaders
Bunsen burners
Striker
Safety Glasses
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Follow the procedures exactly as instructed.
Procedure: The Scrub
1. The student scrubbing his/her hands will begin at Basin 1. Students at the table will assist by
opening the brush package and timing the scrub. Do not use soap at this time.
The student, him/herself, will scrub each hand with the scrub brush for 30 seconds into Basin
1 (60 seconds total). Scrub all surfaces of the hands including under the fingernails.
2. The student will move to the sink and utilize the same brush as was used in Basin 1.
Assistants will apply a drop of soap to each hand and turn on the water. The student will scrub
each hand under running tap water for 1 minute (total 2 min.). Assistants will time the scrub.
After the 2 minute scrub, rinse each hand for 5 seconds under the tap water.
Assistants will discard the brush and turn off the water.
3. The student will then move to Basin 2. With a new, sterile brush (supplied by the assistants
at Basin 2), scrub the hands into Basin 2 in a manner identical to Basin 1 (30 seconds each
hand). Do not use soap!
Assistants should time the scrub.
4. Repeat the tap water scrub (one minute each hand) with assistants turning water on and off,
supplying soap to each hand at the beginning, and timing the scrub. Each hand then is rinsed
for 5 seconds.
5. Repeat this procedure at Basins 3, 4, 5, and 6: Basin scrub with no soap for 60 total seconds.
Follow with a tap water scrub with soap for 2 minutes total and a 10 second rinse.
Assistants should be ready to supply soap and fresh brushes,
turn the water on and off, and time the scrubs!
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Procedure: The Inoculations
Each group corresponding to each basin (1, 2, 3, 4, 5, & 6) will inoculate 3 Trypticase Soy
Agar (TSA) plates.
Therefore, there will be a total of 18 agar plates for the entire class.
1. Label the three agar plates 0.1ml, 0.2ml, and 0.4ml, respectively. Also, indicate the basin
number on each plate.
2. Stir the water thoroughly with a pipet for 60 seconds.
3. Pipette the appropriate amount of water from the basin to the center of each of the 3 plates.
4. Before spreading each plate, briefly flame the bactispreader, and cool. Spread the water
evenly over the agar.
5. After a few minutes, invert the plates, and incubate at 37C for 48 hours.
Day 2
1.Count the colonies on all three of the plates.
2. Calculate the number of organisms per milliliter using the dilution factors of the plate from
which the colonies were counted.
3. Record the number of organisms per ml in the appropriate column (either 0.1ml, 0.2ml, or
0.4ml) in the lab report. Also record the class results in the lab report.
4. Average the number of organisms per milliliter for each basin and record the average in the
lab report.
AMOUNT OF WATER ON PLATE
DILUTION FACTOR
0.1ml (1/10)
10
0.2ml (2/10)
5
0.4ml (4/10)
2.5
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Name:
Section:
Introductory Microbiology Lab Report
Control of Microbial Growth:
Alcohol Evaluation & Hand Scrubbing Effectiveness
1. (O.5pt) Results: Alcohol Evaluation-Your Results
Left Thumb: no alcohol
Right Thumb: alcohol treated
Colony Count Colony Count Percent
(L1 quadrant) (L2 quadrant) Reduction
Colony Count Colony Count Percent
(R1 quadrant) (R2 quadrant) Reduction
2. (0.5pt) Results: Alcohol Evaluation-Class Results
Left Thumb Percent Reductions: no alcohol
Right Thumb Percent Reductions: alcohol
treated
Class average, left thumb:
Class average, right thumb:
3. (0.5pt.) What is the mode of action of alcohol?
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4. (0.5pt.) Contrast transient and resident microbes.
5. (0.5pt) Results: Effectiveness of Hand Scrubbing - Class Results
To calculate organisms per ml, multiply the colony count by the dilution factor.
Dilution factors:
0.1 ml = 10
0.2 ml = 5
0.2 = 2/10 (must determine lowest possible denominator
= 1/5 ; Therefore, dilution factor is 5)
0.4 ml = 2.5
0.4 = 4/10 = 2/5.
Now some simple algebra. 2/5 =1/x (Need to find how
much 1 unit of the substancehas been diluted; The
denominator x is the dilution factor).
2x/5 = 1.
2x = 5.
x = 5/2 = 2.5 (dilution factor)
Place each group's results ONLY in the appropriate column and row.
Group (Basin)
# organisms per
ml on 0.1 ml
plate
# organisms per
ml on 0.2 ml
plate
1
2
3
4
5
6
10
# organisms per
ml on 0.4 ml
plate
Average #
organisms per ml
6. (1pt.) Hand scrubbing experiment: Not including contamination, why might there be more
organisms in later basins compared to earlier basins?
7. (0.5pt.) Give an example of a resident skin microbe.
8. (1pt.) Lister used phenol to soak sutures and during surgical procedure. Although phenol
derivatives (phenolics) are commonly used today, pure phenol is not. Using your textbook or
other sources, give an explanation of why phenol is rarely used today as an antiseptic.
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