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The Effectof ChemicalAgents onBacteria

any chemical agents exert an inhibitory effect on bacteria.Among the best known are the antiseptics and disinfectantsused in medicine, industry, and the home. Metals may also be

employed as disinfecting agents because they combine with microbial pro-teins. Mercury chloride, silver nitrate, and copper sulfate are among theuseful compounds of heavy metals. In addition to these, several antimicrobialsubstances of a chemical nature exist in foods and in the body.

The paper disk method will be employed to test the effect of chemicalagents on bacteria. In this procedure, chemical substances are adsorbed ontodisks of special paper, which are then applied to an inoculated growthmedium. Although convenient for demonstrating antibacterial activity, themethod does not take into account such things as the diffusion rate of thechemical or the effect of the growth medium. Also, the bacterial concen-tration and chemical concentration are not standardized. The results musttherefore be interpreted with these factors in mind.

Antiseptics and Disinfectants

A broad assortment of antiseptics and disinfectants is available for the con-trol and destruction of bacteria. Antiseptics are commonly used on the skinsurface, while disinfectants are applied to lifeless objects such as tabletops,equipment, and instruments.

In this section, saturated paper disks will be used to demonstrate theeffect of chemical agents on two species of bacteria. The bacteria will beplaced onto nutrient agar medium, and various antiseptics and disinfectantswill be applied to the surface of the medium. The destruction of bacteria willbe noted by the appearance of a clear zone of inhibition where the chemi-cal agent has been applied.

A.

T H E E F F E C T O F C H E M I C A L A G E N T S O N B A C T E R I A 12 111

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PURPOSE: to determine thesusceptibility of bacteria toantiseptics and disinfectants.

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pecial Materials

• Two selected bacterial species• Plates of nutrient agar or materials• Beakers of various disinfectants and antiseptics, such as iodine, Merthiolate,

formaldehyde, Lysol, 70% ethyl alcohol, phenol, and crystal violet• Sterile paper disks• Forceps in beakers of ethyl alcohol• Millimeter rulers

rocedure

1. Obtain or prepare two plates of nutrient agar, label the bottom sides withyour name and the date, and designate one plate for each of two bacteria tobe tested. The depth of the agar should be about 4 millimeters. Obtainbroth cultures of two species of bacteria and two sterile swabs.

2. Aseptically, dip a swab into the broth culture, press it to the inside of the tubeto express the excess fluid, and inoculate one of the nutrient plates bymaking a lawn of bacteria. The swabbing should be performed at threedifferent angles, and a final sweep should be made around the rim of theplate as shown in Figure 8.2 in Exercise 8. The instructor may demon-strate this “3 + 1” inoculation method. Discard the swab in the beaker of dis-infectant after use. Inoculate the second plate with the second organism inthe same manner.

3. Allow the plates to dry at room temperature for several minutes so that theexcess moisture is absorbed. Then, use a wax marking pencil to mark thebottom side and divide each plate into four sectors to test four chemicalagents. Label the bottom side of each sector with the name of the agent tested.

4. Lightly flame the forceps and obtain a sterile disk. Impregnate the disk witha chemical agent by touching it gently to the surface of the chemical, asshown in Figure 12.1. Dip half the disk into the solution, and allow the chem-ical to adsorb onto the disk. This will avoid excessive amounts of chemicalagent on the disk. Place the disk on the agar of the appropriate sector, and presslightly to achieve firm contact with the agar surface. Saturate three otherdisks with three other chemicals for the other three sectors. An alternative

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112 12 T H E E F F E C T O F C H E M I C A L A G E N T S O N B A C T E R I A

!Chemical disinfectants could be caustic to the skin.Be sure to handle them with care.

F I G U R E 1 2 . 1Method for impregnating the paper disk with the chemicalagent.

Quick ProcedureEffect ofChemical Agentson Bacteria

1. Inoculate a nutrient agarplate with bacteria.

2. Dip sterile disks into va-rious chemical agents.

3. Place disks on the agarsurface.

4. Incubate.

5. Observe and measurezones of inhibition.

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method of saturating disks is to place them on a sheet of paper toweling andadd drops of the chemical agent onto the disks. The paper toweling willabsorb the excess chemical so it is not carried over to the surface of the plate.

5. Repeat the above procedure for the second plate.

6. Incubate the plates in the inverted position at 37° C for 24 to 48 hours, andthen refrigerate until observed.

7. Examine the nutrient agar plates for evidence of clear halos or zones ofinhibition surrounding the disks. A zone indicates that the organismsare susceptible to the chemical agent. The word “susceptible” should benoted in Table 12.1 in the Results section. The degree of inhibition may bedetermined by measuring the diameter of the zone in millimeters, in-cluding the disk. Use the underside of the plate for this procedure, andrecord your results in the table, together with the names of organisms andchemical agents used. If growth appears up to the edge of the disk, the or-ganism is resistant to the chemical agent, and the word “resistant” maybe entered in the chart. Compare the activity of the various chemicals on theorganisms tested, and note the differences in your observations. Labeled rep-resentations of the plates may be included with the results.

Metals

Some metals exert an oligodynamic activity on bacteria. This means thata small amount of the metal has a significant effect on the growth of theorganism. This effect is generally due to interference with enzyme activity inthe metabolizing cell. The following procedure demonstrates the phenome-non of oligodynamic action. It is performed in basically the same way asPart A.

pecial Materials

• Plates of nutrient agar or materials

• Selected bacterial species

• Forceps in beakers of ethyl alcohol

• Samples (disks) of various metals, such as copper, zinc, aluminum, lead, and silver

rocedure

1. Prepare two nutrient agar plates and label one for each of the organismstested. Carefully inoculate each plate by the “3 � 1” method explained inPart A.

2. Clean the metals as follows: First wash the metal disks in hot soap and water.Rinse and, using sterile forceps, dip the metal in alcohol. Allow the alcohol toevaporate.

3. Using sterile forceps, place samples of different metals in different sectorsof the agar, pressing gently to ensure firm contact. Do not turn the plates

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B.

PURPOSE: to examine theeffects of metals on bacterialgrowth.

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over. Treat both plates similarly. Incubate the plates in the upright positionfor 24 to 48 hours at 37° C and then refrigerate until observed.

4. Examine the plates for zones of inhibition as explained in Part A. Measurethe diameters of the zones, and enter your results in Table 12.2. Note whichmetals were inhibitory to which organisms in your observations. Diagramsof the plates may be included.

Other Chemical Substances

In addition to antiseptics, disinfectants, and metals, many other chemicalagents may inhibit the growth of bacteria. Certain of these are pharma-ceutical substances; others occur in foods; still others are produced by thebody. The inhibitory effects of these miscellaneous agents may be testedby using the basic procedures outlined in Parts A and B. It is importantthat observations be made after a brief incubation period of 24 hours or lessbecause the inhibition often is overcome by rapidly multiplying bacteria.

pecial Materials

• Plates of nutrient agar or materials• Selected bacterial species• Forceps in beakers of ethyl alcohol• Samples of miscellaneous products, such as mouthwashes, hand sanitizers, tooth-

paste, various foods, body substances, and other substances as noted in theprocedure

rocedure

1. Prepare inoculated plates of bacteria in nutrient agar using the “3 + 1”method outlined in Part A. The materials to be examined for antimicrobialactivity will be noted by the instructor, as explained below.

2. Mouthwashes and other liquid pharmaceutical products may be tested bydipping sterile paper disks into the product and placing the disks on the sur-face of the agar. Semisolid products such as toothpaste or hand sanitiz-ers may be tested by placing a small sample of the product onto the surfaceof the agar as described in Part A.

3. The chemicals in newsprint generally keep the pages of a newspaper freeof bacteria. This concept may be tested by placing a small square of fresh,untouched newspaper onto the agar surface. Plants are also a source of anti-microbial chemicals. To demonstrate the effect of plant antimicrobials, cuta square from a plant leaf, and place it onto the surface of the inoculated agar.

4. Tears contain the inhibitory enzyme lysozyme, which kills gram-positivebacteria. To obtain tears, rub a piece of fresh onion under the eye or squirtlemon in the eye. Now collect the tear drops on a sterile disk and place it onan inoculated agar plate for testing.

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C.

PURPOSE: to test the effec-tiveness of other chemicalagents to inhibit bacterialgrowth.

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5. Foods such as garlic, horseradish, mustard, onion, and whole clovesmay be tested for antimicrobial substances by placing a small piece oninoculated agar plates.

6. The acidity in orange juice, lemon juice, sauerkraut juice, and cran-berry juice contributes to the growth inhibition of many microorgan-isms. This phenomenon may be tested by soaking paper disks in the juiceand applying them to the inoculated agar surface.

7. At various times, newspaper and journal reports relate the effectiveness ofalcoholic beverages as antimicrobial agents. The paper disk methodmay be used to validate these reports. Samples of different beers and winesare tested by soaking paper disks in the sample and placing the disks on theinoculated agar surface. It might be valuable to test white and red wines,light and regular beers, and various types of spirits.

8. All plates should be incubated at 37° C and should be observed for zones ofinhibition in 24 hours or less. Refrigeration may be used to preserve theplates until they are to be observed. Results may be entered in Table 12.3. Adiagram of a plate may be included.

Effect of Lysozyme on Bacteria

Lysozyme is an antibacterial substance found in human tears and saliva.It breaks cell wall linkages of gram-positive bacteria, thereby killing them.Lysozyme is also found in the white of an egg, where it provides natural pro-tection against staphylococci, micrococci, and other gram-positive bacteria.In this exercise, various dilutions of egg white lysozyme are tested for theirantibacterial qualities.

pecial Materials

• Nutrient agar plates or materials for their preparation• Cultures of Micrococcus luteus and/or other bacteria• Straws and applicator sticks• Sterile swabs and beakers of disinfectant• Fresh egg• Pipettes and mechanical pipetters• 99-ml water blank

rocedure

1. Prepare or pour a plate of nutrient agar, filling the Petri dish about two-thirds full. Permit the agar to harden thoroughly and label the plate onthe bottom side with your name, the date, and the name of the test organ-ism used. Micrococcus luteus is recommended for this exercise because ofits high susceptibility to lysozyme. For comparison, other plates may beused with gram-positive and gram-negative bacteria.

2. Dip a sterile swab into a culture of M. luteus and swab the plate in the “3 � 1” pattern described in Exercise 8 (Figure 8.2), being sure to cover

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D.

PURPOSE: to examine theeffects of lysozyme onbacterial growth.

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the entire agar surface. If other organisms and plates are to be used, theymay be swabbed in this manner.

3. Using a straw, carefully remove four plugs of the inoculated agar fromeach of the four sectors as shown in Figure 12.1. Use the applicator stick topush the plugs out of the straw into the beaker of disinfectant. Deposit thestraw and the stick into the disinfectant when you have finished withthem. Removing the plugs forms a number of wells in the agar.

4. Over a clean beaker, carefully crack open a fresh egg and separate the yolkfrom the white. Do this by moving the yolk back and forth between the eggshells while allowing the white to fall into the beaker.

5. Using a mechanical pipetter, pipette 1 ml of the egg white into 99 ml ofsterile water to make a 1% concentration of egg white. Additional concen-trations of 0.5% and 0.1% should also be prepared. To prepare a 0.5% con-centration, mix 5 ml of the 1% concentration with 5 ml of sterile water. Fora 0.1% concentration, mix 1 ml of the 1% concentration with 9 ml of sterilewater. Other concentrations may be prepared as directed by the instructor.Label the plate bottom with the concentration(s) used.

6. Using fresh pipettes, fill various wells in the inoculated agar with samplesof the undiluted and diluted egg white prepared above. Do not overfill thewells, and be sure to label the plate with the concentrations used. Incubatethe plate at room temperature in the upright position for approximately 48 hours or longer.

7. Observe the plate for the presence of clear rings of inhibition surroundingthe wells. The rings indicate that lysozyme in the egg white has killed thetest bacterium. Record your observations in Table 12.4 of the Results sectionand draw an illustration of the plate indicating the areas of inhibition. If var-ious bacteria have been used, draw conclusions on the effect of lysozymeon different organisms. Note any variations in the size of the ring owing tothe concentration of lysozyme used, including whether the higher or lowerconcentration caused more inhibition. Suggest ways to modify this exerciseto learn more properties of lysozyme.

Disinfection of Drinking Water

Clear water suspected of containing bacteria can be disinfected for drinkingpurposes by using a commercially available tincture of iodine. The Centersfor Disease Control and Prevention (CDC) recommends 5 drops of tinctureof iodine per quart of water, with 30 minutes contact time before consumption.In this section, the efficiency of water disinfection with iodine will be tested.

pecial Materials

• 1 quart of clear water• Fresh soil sample• Commercial tincture of iodine solution• 1-ml pipettes and mechanical pipetters• Sterile Petri dishes and melted nutrient agar

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Quick ProcedureEffect ofLysozymes onBacteria

1. Inoculate plate of nutrient agar with bacteria.

2. Remove plugs of agar toform wells.

3. Add various concentra-tions of egg white to thewells.

4. Incubate.

5. Observe for inhibition of agar.

PURPOSE: to test the effec-tiveness of a tincture ofiodine to disinfect drinkingwater.

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rocedure

1. Obtain three sterile Petri dishes, and label each on the bottom side with yourname and the date. Designate one Petri dish “pretreatment,” another “post-treatment,” and the third “control.”

2. In a clean vessel, obtain 1 quart of clear water such as might be used fordrinking. Using a sterile pipette and a mechanical pipetter, transfer 1 ml ofthe water to the Petri dish marked “control.” Add sufficient melted nutrientagar to cover the bottom of the plate. Mix the water and agar by rotating theplate in a wide arc, and allow the agar to harden. Discard the pipette in theavailable disinfectant.

3. Contaminate the water with a small pinch of fresh soil. Mix thoroughly bystirring and shaking so that the water remains relatively clear and thecontamination is not evident to the casual observer.

4. Using another sterile pipette and mechanical pipetter, transfer 1 ml of thewater to the Petri dish designated “pretreatment.” Again, add sufficientmelted nutrient agar to cover the bottom of the plate. Mix the water and agarby rotating the plate in a wide arc, and allow the agar to harden. Discard thepipette in the available disinfectant.

5. Add 5 drops of commercial tincture of iodine to the remaining contaminatedwater, and allow the water to stand at room temperature for 30 minutes.

6. Using a fresh sterile pipette, transfer 1 ml of the water to the Petri dish des-ignated “post-treatment.” Add sufficient agar to cover the bottom of the plate,and mix it with the water. Allow the agar to harden.

7. Incubate the three plates inverted at 37° C for 24 to 48 hours, then refrigerateuntil observed.

8. Examine the plates for bacterial colonies, and prepare representations of theplates in the appropriate spaces of the Results section. Observe the numberof colonies in each plate, and determine whether the iodine reduced the bac-terial content of the water by expressing the number of bacterial coloniesper milliliter of water. Express your opinion on the value of iodine as a dis-infectant in water.

uestions

1. List several organic materials other than blood that may affect the dis-infecting ability of a chemical agent under practical conditions.

2. How do metals interfere with the growth of bacterial organisms?

3. What might be the result of dipping paper disks into the chemical testedrather than lightly touching the disks to the surface?

4. Explain several variables that influence the outcome of the experiments per-formed in this exercise.

5. List a number of miscellaneous products, other than those mentioned in thisexercise, that may exert an inhibitory effect on bacterial growth.

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Table 12.1. Susceptibility or Resistance of Bacteria on Nutrient Agar

Organisms Tested

1. 2.

Zone Diameter Sus. or Resist. Zone Diameter Sus. or Resist.


Name

Date Section

Exercise Results

The Effect of Chemical Agents on Bacteria

A. Antiseptics and Disinfectants

12

Antiseptic or

Disinfectant

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Nutrient Agar Plate # 1 Nutrient Agar Plate # 2

Organism:

Observations and Conclusions:

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Table 12.2. Susceptibility or Resistance of Bacteria

Organisms Tested

1. 2.



B. Metals

Nutrient Agar Plate


Metal

Nutrient Agar Plate

Organism:

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Organisms Tested

1. 2.



C. Other Chemical Substances

Nutrient Agar Plate

Organism:


ChemicalSubstance

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Organisms Tested

1. 2.



D. Effect of Lysozyme on Bacteria


Plate # 1 Plate # 2

Organism:

Concentrationof

Lysozyme

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E. Disinfection of Drinking Water

Nutrient Agar PlatePretreatment

Nutrient Agar PlatePosttreatment

Colonies/ml:


Notes:

Nutrient Agar PlateControl

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