· Web viewPlace your inoculated plate upside down in the rack for incubation. B. Gram Stain of Unknown Bacteria Perform a Gram stain on the Unknown Bacteria you have chosen to identify.

Unit 12

Unit 12: Susceptibility of Bacteria to Antibiotics, Introduction to Bacterial Identification and Streak IsolationBy 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

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=i01Awhrrp5gVideos created by Corrie Andries.

Materials

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 brotho Streptococcus pyogenes (Spy) in brotho Streptococcus pneumoniae (Spn) in brotho Proteus vulgaris (Pv) in brotho Unknown Bacteria “A” on a petri disho Unknown Bacteria “B” on a petri dish

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Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links:

Bacteria: Sa lawn Spy lawn Spn lawn Pv lawn    L

Antibiotic discs containing either: Novobiocin (NB), Optochin (P), or Bacitracin (A)

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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.

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

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

D

E

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Figure by Patricia G. Wilber

DAY 2: Results and Interpretation

I. Antibiotic Disc Results and InterpretationPick 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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Accessed 8/9/2015 from https://commons.wikimedia.org/wiki/File:M._cat_BSAC.JPG#filelinks GNU Free Documentation License

Unit 12

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

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.

Antibiotic disc

Figure by Karen Bentz and Patty Wilber

Unit 12

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 Antibiotic Tested

Measurement of the diameter

of inhibition (mm)

Is the bacteria sensitive to or

resistant to the 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)

Unknown Bacteria ____ 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.

BacteriaBlood 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 IdentificationRecord 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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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 non-pathogen, 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 Questions1. 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 IsolationThis 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 Questions1. 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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