Download Unit 12: Susceptibility of Bacteria to Antibiotics and Introduction to

Unit 12
Unit 12: Susceptibility of Bacteria to Antibiotics, Introduction
to Bacterial Identification and Streak Isolation
By Karen Bentz, Patricia G. Wilber and Heather Fitzgerald.
Copyright Central New Mexico Community College, 2015
Introduction
The term antibiotic generally refers to a chemical that inhibits the growth of microbes.
Medications that only inhibit the growth of one type of microbe are referred to as
antibacterials, antivirals, antifungals, or antiprotozoals, depending on which type of organism
they are effective against. The first widely used antibiotic, Penicillin, was isolated from a fungus
in the 1920s. During WWII (in the 1940s) it was manufactured en masse and used to treat
wound infections in injured soldiers. Since that time, a variety of antibiotics have been
developed to treat bacterial infections. Unfortunately for us, bacteria that are constantly
exposed to an antibiotic will develop resistance to that antibiotic, thus reducing the usefulness
of the antibiotic as a treatment.
The response of bacteria to various antibiotics can be judged as either susceptible/sensitive if
bacterial growth is inhibited by the antibiotic. If bacterial growth is unaffected by the presence
of the antibiotic, the bacteria is considered resistant. A common method to test the response
of bacteria to an antibiotic is to grow bacteria using a lawn pattern on a media plate, and then
to place a paper disc containing a specified amount of antibiotic on top of the bacteria. The
inoculated plate containing the disc is then incubated, and growth patterns of the bacteria is
observed. If the bacteria can grow up to or close to the disc, the bacteria is said to be resistant
to that particular antibiotic. If you were choosing an antibiotic to treat a patient with, an
antibiotic to which the bacteria is resistant would not be a good choice. If the bacteria is
sensitive to the antibiotic, it will not be able to grow close to the disc because the antibiotic has
killed the bacteria. This produces a clearing (a circular region without growth whose diameter
can be measured) around the disc, called the zone of inhibition. If the zone of inhibition is big
enough, the bacteria is considered to be sensitive to the antibiotic, and that antibiotic might be
an effective medication for your patient.
Factors affecting zone size include: susceptibility of the organism to the antibiotic, the
concentration of the bacteria used on the plate, the concentration of the antibiotic in the disc
and the temperature and length of incubation.
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Novobiocin (NB)
Novobiocin is an antibiotic produced by the soil bacterium Streptomyces niveus. Novobiocin is a
very effective inhibitor of the bacterial enzyme DNA gyrase, thus it inhibits DNA replication and
ATPase activity, resulting in death of susceptible bacteria.
Clinically, novobiocin is most frequently used to identify novobiocin-resistant Staphylococcus
saprophyticus, a common urinary tract pathogen in young, sexually active females. Other
common skin flora such as Staphylococcus aureus and Staphylococcus epidermidis are sensitive
to novobiocin. Novobiocin is also effective against MRSA (methicillin resistant Staph. aureus),
many Gram(-) species and some streptococcal species.
The zone of clearing around the Novobiocin disc must be more than 16 mm for the bacteria to
be considered sensitive to Novobiocin. A bacteria that produces a zone of 16 mm or less is
considered resistant to Novobiocin.
Bacitracin (A)
The antibiotic Bacitracin is produced by Bacillus subtilis and it disrupts peptidoglycan synthesis
in the cell walls of both Gram(+) and Gram(-) organisms. Bacitracin is used as a topical (skin)
preparation only, as it is highly toxic if taken internally. Its widespread use in triple antibiotic
ointments such as Neosporin has contributed to the emergence of antibiotic resistant bacteria
such as MRSA.
Clinically, bacitracin is used to distinguish between β-hemolytic streptococci (such as
Streptococcus pyogenes), which are sensitive to bacitracin, and various other Staphylococcal
and Streptococcal species which are resistant to bacitracin.
The zone of clearing around the Bacitracin must be greater than 10 mm in diameter to indicate
sensitivity. A bacteria that produces a zone of 10 mm or less is considered resistant to
Bacitracin.
Optochin (P)
Optochin is a chemical (ethylhydrocupreine hydrochloride) used to distinguish Streptococcus
pneumonia from other α-hemolytic streptococci. It entered medical use around 1914.
A zone of inhibition greater than 14 mm in diameter is interpreted as susceptibility or sensitivity
to Optochin. Less than 14 mm indicates resistance
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Identification of Unknown Bacteria
A patient who has a bacterial infection is often treated with antibiotics to eliminate the
presumptive pathogen. Even without actually identifying the pathogen, this often works
because the presentation of the symptoms suggests certain bacteria. But, antibiotics do not
work equally well against all bacteria, so if illness is very severe or if the first attempt at
treatment is ineffective, specific identification of the bacteria that is causing the disease may
occur so that a more effective antibiotic can be prescribed. Individual bacterial species are
characterized by unique physical and metabolic traits, and these traits can be used to identify
an unknown bacteria.
DAY 1: Inoculation
Video Links:
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Novobiocin https://www.youtube.com/watch?v=b3nd1RVLKzY
A / P Disk Video has this on a CNA blood plate but we will use the TSA with Blood.
https://www.youtube.com/watch?v=i01Awhrrp5g
Videos created by Corrie Andries.
Materials
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Five sterile cotton swabs
One tube of sterile water
Tweezers
Novobiocin (NB) discs
Bacitracin (A) discs
Optochin (P) discs

Media (per pair)
o Five blood agar plates
Bacteria Cultures
o Staphylococcus aureus (Sa) in broth
o Streptococcus pyogenes (Spy) in broth
o Streptococcus pneumoniae (Spn) in broth
o Proteus vulgaris (Pv) in broth
o Unknown Bacteria “A” on a petri dish
o Unknown Bacteria “B” on a petri dish

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Procedures
I.
Novobiocin, Optochin and Bacitracin Antibiotic Sensitivity Test
Figure 12-1: Use of the three antibiotic discs, Novobiocin (NB), Optochin (P) and Bacitracin (A) on
blood plates.
Antibiotic discs containing either: Novobiocin (NB), Optochin (P), or Bacitracin (A)
Bacteria: Sa lawn
Spy lawn
Spn lawn
Pv lawn
1. Label the bottom of four of your blood agar plates with your name, date, and the name of
the bacteria that you’ll be testing for antibiotic sensitivity. Save one plate for part II, unknown
identification.
2. Create a lawn of bacteria on each blood plate. See Unit 2 for a review of the technique.
Dispose of your swab in the biohazard bucket on the table top.
3. Sterilize the end of a pair of tweezers by holding it in the micro incinerator for a few seconds.
4. Let tweezers cool before using.
5. Use the sterile tweezers to carefully grab one of the novobiocin (NB) discs from the
container.
5. Carefully place the NB disc on your bacterial lawn as shown in Fig. 21.1, on top of the
bacteria you have just spread.
6. Press the disc gently, but firmly onto the plate. Do not break the agar below it. You want the
disk to stick when you invert the plate, but not be embedded in the agar.
7. Re-sterilize and cool the tweezers.
8. Grab an Optochin (P) disc and place it on another part of the plate. Tap the disc to make sure
it is stuck on the plate.
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9. Re-sterilize and cool the tweezers.
10 Grab a Bacitracin (A) disc and place it on an open area of the plate. Tap gently to make sure
it sticks.
11. Re-sterilize and cool the tweezers.
12. Repeat steps one through eleven for your other three plates, using a different type of
bacteria for each plate.
13. Place your four inoculated plates upside down in a candle jar for incubation.
14. Return the tweezers to the canister.
Note: The antibiotic discs can also be tested on bacterial lawns made on T-soy agar. Using blood
agar will show you bacterial hemolysis as well as antibiotic sensitivity.
Precautions
o Be sure to sterilize tweezers before and after each use to prevent cross contamination.
o Make sure that your antibiotic discs are spaced far apart on the blood plate so that any
resulting zones of inhibition do not overlap.
o Tap the disc gently with the tweezers to make sure the disc sticks to the agar, but do not
push the disc into the agar or results will be inconclusive.
II. Characterizing and Identifying Unknown Bacteria
A. Novobiocin, Optochin and Bacitracin Antibiotic Sensitivity Test
1. Choose either Unknown Bacteria A or Unknown Bacteria B to identify.
2. Label the bottom of a blood plate with your name, date, and the Unknown letter.
3. Use a moistened swab to make a lawn of your unknown bacteria on a blood plate. Leave a bit
of an edge of the plate unswabbed so you can see the original plate color after incubation.
4. Using sterile, cooled tweezers, carefully place a novobiocin, optochin, and bacitracin disc on
the plate, keeping the discs far apart on the plate.
5. Sterilize the tweezers between discs and when finished.
6. Place your inoculated plate upside down in the rack for incubation.
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B. Gram Stain of Unknown Bacteria
Perform a Gram stain on the Unknown Bacteria you have chosen to identify. Refer to Unit 5 for
the technique if need be. Use your assigned microscope to view your cells at a total
magnification of 1000X. Be sure to use oil.
What color are your cells? _____________ What shape are your cells? ________________
Are your cells arranged in a particular pattern? ______ If so what is the arrangement?
In addition, calculate cell size (as a review for the midterm). See Unit 2.
C. Catalase Test of Unknown Bacteria
Perform a catalase test on your Unknown bacteria.
Record your observations: ______________________________________________
III. Streak Isolation from a broth
Materials
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1 T-soy plate per student.
Bacterial Culture in a broth: Choose from Sa, Spy, or Pv. Do not use Spn. It does not
always grow well enough.
Procedure
1. Refer to Unit 3 to review the streak isolation procedure from a broth. A broth tends
to be easier to streak from than a plate.
2. Perform your streak isolation on your T-soy plate.
3. Be sure to label your plate correctly.
General procedure for the Streak Isolation Technique:
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B, 10 streaks through A
A, 1 cm
smear
C
E
D
Figure by Patricia G. Wilber
DAY 2: Results and Interpretation
I. Antibiotic Disc Results and Interpretation
Pick up your blood plates and a metric ruler. You will use the ruler to measure the diameter (in
mm) of the zone of inhibition around each antibiotic disc.
Carefully observe the growth of the bacteria around each antibiotic disc. Does the bacteria
grow right up to the disc? If so, the bacteria is resistant to that antibiotic, and that antibiotic
would not be a good choice to treat an infection caused by that particular bacteria.
Figure 12-2: Antibiotic sensitivity test for bacteria grown on blood agar.
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Unit 12
Accessed 8/9/2015 from
https://commons.wikimedia.org/wiki/File:M._cat_BSAC.JPG#filelinks GNU
Free Documentation License
If
there is a clear space on top of the media all around the antibiotic disc, this indicates that the
bacteria is sensitive to that antibiotic, and was not able to grow in the area where it came in
contact with the antibiotic. If a bacteria is sensitive to an antibiotic, that antibiotic might be a
good choice to treat an infection caused by that particular bacteria.
There are specific parameters for judging a bacteria sensitive or resistant to and antibiotic. One
of the parameters is a certain size of the “zone of inhibition” or diameter of inhibition around
the antibiotic disc. This is specific for each type of antibiotic. (See table below)
To measure a zone of inhibition, use a metric ruler and measure the diameter of the circle of
inhibition in mm. Compare your measurement with the metric measurements in the table to
determine if your bacteria is sensitive or resistant to the antibiotic.
Figure 12-3: How to measure the zone of inhibition using a metric ruler
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Blood Plate
Antibiotic disc
Lawn of Bacterial Growth
Measure the diameter of the zone of inhibition in mm. This zone measures 43 mm and
the bacteria would be sensitive to the antibiotic in the disc.
Figure by Karen Bentz and Patty Wilber
Table 12-1: Required Zones of Inhibition for Specific Antibiotics
Antibiotic
Required Diameter in mm
for Bacterial Sensitivity
Novobiocin
Greater than 16 mm
Optochin
Greater than 14 mm
Bacitracin
Greater than 10 mm
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1. Use the metric ruler to measure the zone of inhibition for all three of the discs on each of
your five plates. If the bacteria have grown right up to the disc, record the bacteria as resistant
to the antibiotic. Record your results in the table below.
Name of Bacteria Tested
Unknown Bacteria ____
Antibiotic
Tested
Measurement
Is the bacteria
of the diameter
sensitive to or
of inhibition
resistant to the
(mm)
antibiotic tested?
Novobiocin (NB)
Optochin (P)
Bacitracin (A)
Novobiocin (NB)
Optochin (P)
Bacitracin (A)
Novobiocin (NB)
Optochin (P)
Bacitracin (A)
Novobiocin (NB)
Optochin (P)
Bacitracin (A)
Novobiocin (NB)
Optochin (P)
Bacitracin (A)
2. Now look at the media under your bacterial lawns and record your bacteria’s ability to
hemolyze blood.
Bacteria
Blood Hemolysis:
no hemolysis = gamma,
partial hemolysis = alpha
complete hemolysis = beta
Unknown Bacteria ____
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3. Which antibiotic was most effective at inhibiting the growth of:
Staphylococcus aureus
Streptococcus pyogenes
Streptococcus pneumoniae
Proteus vulgaris
Unknown Bacteria ____
Insert Photos of Results Here:
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II. Unknown Bacterial Identification
Record all of the results you have for your unknown bacteria here:
Gram stain:
Catalase result:
Hemolysis
Type:
Zone of
Inhibition
Sensitive? Or
Resistant?
Novobiocin:
Bacitracin:
Optochin:
Insert Photos of Results Here:
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Unit 12
Your Unknown bacteria is one of the four bacteria used in this lab:
Staphylococcus aureus
Streptococcus pyogenes
Streptococcus pneumoniae
Proteus vulgaris
Using the four types of information you have obtained (cell wall type, presence of catalase,
ability to hemolyze blood, and optochin, bacitracin and novobiocin sensitivity/resistance), make
a dichotomous key that keys out and identifies each of the four bacteria listed above.
Dichotomous Key:
Use the dichotomous key you created above and the data you have collected to identify your
Unknown bacteria. Write the name here: ____________________________
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1. Sometimes some evidence suggests pathogenicity and other evidence suggests a nonpathogen, but if there are any test results that indicate pathogenicity, you should
consider the organism a possible pathogen.
a. Based on your results, do you think your Unknown bacterium is a pathogen? (Write
“yes” or “no”)
b. List all of the evidence that suggests your Unknown bacteria is a pathogen.
Post-Activity Questions
1. Given the picture shown here, how would you
decide which of these antibiotics would be best
for use on a patient?
2. Was the purpose of the catalase test for the unknown identification you performed To
split G(+) from G(-)?
3. When might an antibiotic sensitivity test be ordered by a medical professional?
4. List the indicators of pathogenicity we examined in this lab.
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III. Streak Isolation
This was your last streak isolation prior to the midterm and the final project. If you have not
mastered the technique, please consider how to remedy this with some tips below!!
Insert a photo of your streak isolation here.
Post-Activity Questions
1. Do you have 8-10 isolated colonies?
2. If not, why not? Circle all that apply.
a. Bad pattern?
b. Too much bacteria?
c. No sterilizing between streaks?
d. Too much overlap?
Four ideas to help fix your pattern if it did not work:
1. Have you mastered the basic pattern? YES or NO.
a. If no, look at the pattern in U3, look up streak isolations online and practice the
pattern on a piece of paper to develop muscle memory.
2. Are you using too much bacteria? YES or NO.
a. If yes, USE LESS! Touch your loop to a solid colony if using a plate; do not scoop
it. Tap the excess liquid off your loop if using a broth.
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3. Did you sterilize your loop between sections? YES or NO.
a. If no, just remember to use that incinerator/ Bunsen burner!
4. Are you overlapping too much? YES or NO.
a. If yes practice the pattern with (see 1) less overlap between sections. You must
overlap some, though. No overlap = no bacteria to spread to the next section.
The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms,
our associate dean Linda Martin for many kinds of aid, Mike Jillson and Alex Silage for IT support, and our dean John Cornish .
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