MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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Unit VIII: Natural Microbiota and Non-Specific Host Defense
Activities:
17.2 Effectiveness of Hand Scrubbing (Ex 23)
19.1 Lytic Effect of Tears and Saliva (Ex 24)
19.2 Antimicrobial Sensitivity Testing: Kirby Bauer Method (Ex 25)
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p5
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18.1 Effectiveness of Hand Scrubbing: (Ex 23)
The importance of hand disinfection in preventing the spread of disease is accredited to the
observations of Semmelweis at the Lying-In Hospital in Vienna in 1846 and 1847. He noted that the
number of cases of puerperal fever was closely related to the practice of sanitary methods. Until he
took over his assignment in this hospital, it was customary for medical students to go directly from the
autopsy room to a patient's bedside and assist in deliveries without scrubbing and disinfecting their
hands. When the medical students were on vacation, only the nurses, who were not permitted in the
autopsy room, attended the patients. Semmelweis noted that during this time, deaths due to puerperal
fever fell off markedly.
As a result of his observations, he established a policy that no medical students would be allowed
to examine obstetric patients or assist in deliveries until they had cleansed their hands with a solution
of chloride of lime. This ruling caused the death rate from puerperal infections to drop from 12% to
1.3% in one year.
Today it is routine practice to wash hands prior to the examination of any patient and to do a complete
surgical scrub prior to surgery. Scrubbing the hands involves the removal of transient (contaminant)
and resident microorganisms. Depending on the condition of the skin and the numbers of bacteria
present, it takes from seven to eight minutes of washing with soap and water to remove all transients;
and they can be killed with relative ease using suitable antiseptics. Residents, on the other hand, are
firmly entrenched and are removed slowly by washing. These organisms, which consist primarily of
staphylococci of low pathogenicity, are less susceptible than the transients to the action of antiseptics.
In this exercise, an attempt will be made to evaluate the effectiveness of the length of time in removal
of organisms from the hands using a surgical scrub technique. One member of the class will be
selected to perform the scrub. Another student will assist by supplying the soap, brushes, and basins, as
needed. During the scrub, at two-minute intervals, the hands will be scrubbed into a basin of sterile
water. Bacterial counts will be made of these basins to determine the effectiveness of the previous twominute scrub in reducing the bacterial flora of the hands. Members of the class not involved in the
scrub procedure will make the inoculations from the basins for the plate counts.
MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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Exercise 23:
Part A: Hand Scrubbing:
Materials:
5 sterile surgical scrub brushes, individually wrapped
5 sterile basins, covered to prevent contamination.
7 liters sterile water.
1000 mL sterile graduate cylinder, covered to prevent contamination.
dispenser of disinfecting soap
Procedure:
The two members of the class who are chosen to perform the surgical scrub will set up their materials
near a sink for convenience. As one student performs the scrub, the other will assist in reading the
instructions and providing materials as needed. Before beginning the scrub, both students should read
all the steps carefully.
Figure 1. Major steps in the hand scrubbing routine. Note steps 2 & 3 get repeated three more times
to generate basins C, D, and E.
1. To get some idea of the number of transient organisms on the hands, the scrubber will scrub all
surfaces of each hand with a sterile surgical scrub brush for 30 seconds into Basin A. No disinfecting
soap will be used for this step. The successful performance of this step will depend on spending the
same amount of time on each hand (30 seconds), maintaining the same amount of activity on each
hand, and.scrubbing under the fingernails, as well as, working over their surfaces. After completion of
this 60-second scrub, notify Group A that their basin is ready for use in inoculations of plates.
MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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2. Using the same brush as above, begin scrubbing with disinfecting soap for two minutes, using
cool tap water to moisten and rinse the hands. One minute is devoted to each hand. The assistant
will make one application of disinfecting soap to each hand as it is being scrubbed. Rinse both hands
for five seconds under tap water at the completion of the scrub. Discard the brush.
3. With a fresh sterile brush, scrub the hands into Basin B in a manner that is identical to #1, above.
Don't use soap. Notify Group B when this basin is ready.
4. Same as #2; scrub each hand for one minute using soap.
5. Same as #3; use a fresh brush and scrub each hand for 30 seconds in Basin C.
6. Same as #2; scrub each hand for one minute using soap.
7. Same as #3; use a fresh brush and scrub each hand for 30 seconds in Basin D.
8. Same as #2; scrub each hand for one minute using soap.
9. Same as #3; use a fresh brush and scrub each hand for 30 seconds in Basin E.
10. Dry hands and apply lotion if available.
Part B: Inoculating Pour Plates:
Materials:
30 veal infusion agar pours-6 per group (25 mL in each)
1 ml pipettes
30 sterile Petri plates-6 per group
70% alcohol
L-shaped glass stirring rod (optional)
Procedure:
While the scrub is being performed, the rest of the class will work collaboratively to determine make
five sets of pour plates for determining the bacterial count per milliliter in each scrub basin. In this way
we hope to determine, in a relative way, the effectiveness of scrubbing in bringing down the total
bacterial count of the skin. Perform the following for each of the five basins (A, B, C, D, and E)
MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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B
A
Figure 2. Mixing of basin sample (A) prior to use in inoculating pour plates (B).
1. Liquefy six pours of veal infusion agar and cool to 50° C. While the medium is being liquefied, label
two plates each: 0.1 ml, 0.2 ml, and 0.4 ml. Also, indicate the Basin designation on the plate.
2. As soon as the scrubber has prepared the basin, take it to your bench and make inoculations as
follows:
a. Stir the water in the basin with a pipette or an L-shaped stirring rod for 15 seconds.
For consistency of results, you should all use the same method of stirring.
b. Deliver the proper amounts of water from the basin to the six Petri plates. The
pipette used for stirring may be used for the deliveries.
c. Pour a tube of veal infusion agar, cooled to 50° C, into each plate, rotate to get good
distribution of organisms, and allow to cool.
d. Incubate the plates at 37° C for 24 hours, after the agar has solidified.
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3. After the plates have been incubated, select the pair that has the best colony distribution with no
fewer than 30 and no more than 300 colonies. Count the colonies on the two plates using the Quebec
Counter and record your counts on the chart on the chalkboard.
4. After all data is on the chalkboard, record the table into your notebook.
5. Make a “XY scatter” graph with symbols and lines for these results (CFU/mL to wash number) and
include a proper figure legend.
6. What was your expected result (“a steady decline in numbers with each successive wash”)? Offer
an explanation as to why you did or did not get your expected result.
7. After all that scrubbing, why should a surgeon still where sterile gloves for the patients sake?
MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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19.1 Lytic Effect of Tears and Saliva: (Ex 24)
Our defenses against microbial infection are collectively termed resistance. Resistance can either be
non-specific (act on all microbes) or specific (target a particular microbe).
Specific resistance, or immunity, involves the production by our immune system of specific proteins
called antibodies, which bind to distinct microorganisms. Binding of antibodies is a means of
hindering and identifying invading cells needing to be destroyed by other immune system functions. A
set of unique antibodies will be produced in response to every new strain of microorganism that has
breeched the non-specific defenses and causes an infection. For example, microbe A causes an
infection and the host responds by producing antibodies against microbe A; microbe B infects the body
and response is to produce antibodies against microbe B; antibodies against microbe A are ineffective
against microbe B, and visa versa. More specifically, antibodies target their binding to unique
biomolecules (proteins, polysaccharides, etc) often on the microbe cell surface, which are called
antigens. It is the presence of a new antigen in the body that induces antibody production against it,
and thereby against the microbe with that antigen.
Non-specific resistance includes defenses to protect against invasion by any microbe. These defenses
include physical barriers, such as epithelial (non-vascularized) tissues, i.e. skin and mucus membranes.
Inflammation and phagocytosis by white blood cells (WBC; leukocytes) also play a role in nonspecific resistance, as do certain groups of proteins called bactericidins. One such bactericidin is the
enzyme lysozyme, which catalyzes the breakdown of the peptidoglycan in the cell wall of grampositive and some gram-negative bacteria. Lysozyme is present in our saliva and tears, as well as in the
whites of bird eggs. In fact, lysozyme from eggs is commercially produced for its use in
biotechnology application requiring the destruction of bacterial cell walls (e.g., DNA extraction
protocols).
In this lab exercise, we will run some experiments to test for lysozyme presence in our tears and saliva.
We will also compare the effectiveness of lysozyme on three different bacteria. We will test grampositive (Micrococcus luteus) and gram-negative bacteria (Escherichia coli). In both experiments we
will use a commercial stock of egg lysozyme as a positive control and sterile water as a negative
control.
Exercise 24:
Lysozyme Activity Assay:
Materials:
2x Nutrient agar plates
Culture of either: M. luteus and E. coli
2x Sterile cotton swab
Sterile blotting paper pieces
Wax pencil
Bunsen burner
Forceps
Sterile water
Egg Lysozyme
MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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Onion
Procedure:
1. Divide both your plates into quadrates using a wax pencil, and label as illustrated below:
+ = positive control
-
(egg lysozyme)
T
- = negative control
(water)
+
S
T = tears
S = saliva
2. Each plate will be inoculated with one of the two bacteria, so label plates accordingly.
3. Swab-inoculate each plate with culture of the appropriate bacterium for lawn growth.
a) Dip a sterile swab into the culture, and then gently squeeze excess culture drops from the
swab by rolling the swab on the inside wall of the culture container.
b) Use the swab to inoculate the entire surface area of the plate. Work the swab back and
forth once and then again at 90 degree from the first swabbing.
4. Expectorate saliva (i.e. spit) into the bottom half of a sterile Petri plate.
5. Generate tears, via the sliced onion technique, and collect in the lid of a sterile Petri plate.
6. Wick the test solution and controls into respective pieces of sterile paper, and place in the center of
the appropriately labeled quadrate of the inoculated plates. In doing this step, it is important not to
overload the paper piece – listen to your instructor’s suggestions in this regard.
7. Incubate at 37 ºC for 24 h to 48 hours.
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8. Measure the distance for any zone of growth inhibition (like in the disinfectant lab), and record
results on the chalk board for class average calculations. Record the table in your notebook.
9. Calculate class averages for each bacterium and treatment.
MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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10. Did you get the results you expected for negative and positive controls?
11. Was there lysozyme in your tears and saliva? Which had a greater effect, tears or saliva?
Assuming you loaded equal volumes of body fluid to the paper wicks, why might there be a difference
in the level of activity between tears and saliva?
12. What are at other factors that contribute to the non-specific resistance of your eyes? Of your
mouth?
13. Look up another group of proteins involved in our non-specific resistance to microbes
(textbook/literature search assignment). Describe its function and where it is located in the body.
19.2 Antimicrobial Sensitivity Testing: Kirby Bauer Method: (Ex 25)
The principal drugs used in the treatment of infectious disease fall into three categories: antibiotics,
sulfonamides, and chemotherapeutics. Collectively, they may be referred to as antimicrobics. Once
the causative organism of a specific disease has been isolated, the physician needs to know, as soon as
possible, which antimicrobic will be most effective. The use of sensitivity disks can readily provide
this information.
Antimicrobic impregnated disks were first introduced in the late 1940s as penicillin came into
widespread use. As the decades rolled by and a multitude of new drugs were discovered, a great deal
of experimentation took place with the hope of developing a test method that would accommodate the
large variety of antimicrobics with a high degree of reliability. Many problems were encountered.
The effectiveness of an antimicrobic in sensitivity testing is based on the size of the zone of inhibition.
The zone of inhibition, however, varies with the diffusibility of the agent, the size of the inoculum, the
type of medium, and many other factors. Only by taking all these variables into consideration could a
reliable method be worked out. The Kirby-Bauer method, which is described here, is such a method
and is the accepted procedure in use today. It is sanctioned by the U.S. FDA and the Subcommittee on
Antimicrobial Susceptibility Testing of the National Committee for Clinical Laboratory Standards.
Although time is insufficient here to consider all facets of the test, the basic procedure will be
followed.
The recommended medium in this test is Mueller-Hinton agar. Its pH should be between 7.2 and 7.4,
and it should be poured to a uniform thickness of 4 mm in the Petri plate. This requires 60 ml in a 150
mm plate and 25 ml in a 100 mm plate. For certain fastidious microorganisms, 5% defibrinated animal
blood (sheep, horse, or other) is added to the medium.
Inoculation of the surface of the medium is made with a cotton swab from a broth culture. In clinical
applications, the broth turbidity has to match a defined standard. Care must also be taken to express
the excess broth from the swab prior to inoculation.
MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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High potency disks are used in this test. The disks may be placed on the agar with a mechanical
dispenser or sterile forceps. Regardless of how they are placed, it is desirable to press down on each
disk to ensure close contact of the disk to the medium.
After 16 to 18 hours incubation, the plates are examined and the diameters of the zones of growth
inhibition are measured to the nearest millimeter. Resistance or sensitivity of the microbe to the
antimicrobic is based on this inhibition zone diameter (Table 1).
In this exercise we will work with four microorganisms: Staphylococcus epidermidis, Proteus vulgaris
and two unknowns you isolated from last week. Each student will inoculate four plates with the four
organisms and then place the disks on the medium using the dispenser.
Exercise 25:
Kirby Bauer Method:
Materials:
4 Petri plate of Mueller-Hinton agar
nutrient broth cultures (with swabs) of S. epidermidis, P. vulgaris, and two unknowns
disk dispenser (BBL or Difco)
cartridges of high-potency antimicrobic disks (BBL or Difco)
forceps and Bunsen burner
Step 1
Step 2
Step 3
Figure 3. The three major steps in performing the Kirby Bauer Methof for assessing antimicrobial
sensitivity: step 1, inoculation of a lawn of bacterial growth; step 2, dispensing antimicrobial disks;
and step 3, measuring zones of no growth.
Procedure:
1. Label your plates with the name of the organism or isolate.
2. Inoculate the surface of the medium with the swab after expressing excess fluid from the swab by
pressing and rotating the swab against the inside walls of the tube above the fluid level. Cover the
MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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surface of the agar evenly by swabbing in three directions. A final sweep should be made of the agar
rim with the swab. Perform this on one plate for each bacterium.
3. Allow 3 to 5 minutes for the agar surface to dry before applying disks.
4. Dispense disks from the automatic dispenser by removing the lid, placing the dispenser over the
plate, and pushing down firmly on the plunger. Note which antimicrobials you used and their code.
5. With the sterile tip of forceps tap each disk lightly to secure it to medium.
6. Invert and incubate the plate for 16 to 18 hours at 37° C. Recall, longer incubations at this
temperature may denature antimicrobics, and cause growth of surviving cells.
7. You will need to construct a table for results in your notebook for each bacteria and its reaction for
each antimicrobial.
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8. After incubation, measure the zone diameters with a metric ruler to the nearest whole millimeter.
The zone of complete inhibition is determined without magnification. Ignore faint growth or tiny
colonies that can be detected by very close scrutiny. Large colonies growing within the clear zone
might represent resistant variants or a mixed inoculum, and may require reidentification and retesting
in clinical situations. Ignore the "swarming" characteristic of Proteus, measuring only to the margin of
heavy growth. Record the zone measurements on the table you constructed in your notebook.
9. Use Table 1 to determine the degree of resistance (R, I, or S) and note in your notebook and in the
class table constructed on the chalk board.
10. What were the class results for S. epidermidis and P. vulgaris? Should there be variation in the
results for either bacterium? What may explain any variation in either set of results?
11. Regarding results for putative enteric bacterial isolates (ENDO agar “coliform” colonies), was
there any consistent antimicrobial resistance profile? To which antimicrobial were these isolates most
resistant? To which antimicrobial(s) was there variability in results among isolates? Can you
speculate, or formulate a hypothesis to test, to explain any variation based on the source of inoculum?
12. Plot, as a stacked bar graph, the frequency of resistance (Y-axis) in enteric isolates and those from
other sources for all antimicrobials tested (X-axis). Include a proper figure legend. Was there greater
variation in resistance profiles among enteric isolates or among all other isolates of varied sources?
13. To which antibiotic were most isolates (enteric and others together) resistant? What is the action
of this antimicrobial agent (textbook/literature search assignment)?
MICRB 202: Introductory Microbiology Lab
Unit VIII: Natural Microbiota and Non-Specific Host Defense
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Table 1. The degree of resistance (R, I, or S) to an antimicrobial agent is interpreted from the diameter
of the zone of growth inhibition from Kirby Bauer method results.
NOTE: All three labs were taken from Benson, H. J. 2002. Microbiological Applications: Laboratory
Manual in General Microbiology, Short V ersion, 8th ed. McGraw Hill, New York. p 381.