43038 CH10 0091

Part IVThe Spreadand Controlof Micro-organisms

utbreak! The word has become part of our modern lexicon offrightening words to describe a sudden and rapid spread of aninfectious disease like Ebola or hepatitis A in a limited area. An

outbreak represents a more limited form of an epidemic. Some out-breaks or epidemics (or even pandemics) may be the result of newlyemerging infectious diseases, such as severe acute respiratory syn-drome (SARS) or West Nile fever, the result of reemerging infectious dis-eases, such as malaria or tuberculosis, or from recurring infectiousdiseases like the flu.

Whatever the form, there are many ways a disease can be spread. It mightbe spread by aerosolized droplets resulting from a cough or sneeze. It may betransmitted via contaminated water or food. Many infectious diseases arecarried and spread by vectors, which include insects like mosquitoes and fleas.Epidemics also may be spread by pathogens on fomites; that is, inanimateobjects like doorknobs, eating utensils, and bed linens. Even currency likepaper money and coins can harbor and contribute to an epidemic. In fact, coins“contaminated” with yeast cells represent a good example to use in a simulatedepidemic to illustrate how disease might spread in a population. Often insuch epidemics, the nonliving source of the disease or the first individual(index patient) spreading the disease needs to be identified. All of these aspectscan be examined through a simulated epidemic (Exercise 10).

The ability of infectious diseases to spread in numerous ways througha population highlights the need to continually maintain high levels ofsanitation and strengthen public health measures to ensure potentiallyinfectious agents are controlled or destroyed.

Looking at bacterial diseases, physical agents are one way to keepinfectious agents under control. Physical agents include, among others,

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moist heat, ultraviolet radiation, and drying. Experiments can be designedto effectively show how such physical agents limit microbial growth and theopportunity to spread into a community or population (Exercise 11).

In many cases, the more common and familiar agents of control arechemical compounds. Supermarkets are full of products, many good andsome unnecessary, to keep microorganisms under control. Chemicalagents, such as antiseptics and disinfectants, are key to any publichealth initiative or good sanitation practice when it comes to a potential forspread of an infectious disease. These chemicals, as well as other com-mercial and biological substances can be examined for their antibacterialaction and as water disinfecting agents (Exercise 12).

The number of disinfectants available on the market is tremendous andcontinues to grow. How effective are these disinfectants? It depends onwhether they are designed for inanimate objects, as handwashing agents, oras mouthwashes (Exercise 13). The relative effectiveness of disinfectantscan be determined selecting one of several evaluation procedures. Perhapsthe most used procedure has been the phenol coefficient, where variousdisinfectants can be compared to a phenol standard (Exercise 13).

Historically, besides improving the standards of public health, themost influential event that has led to control or elimination of infectionsis the development of antibiotics. By definition, antibiotics are chemicalinhibitors produced by microorganisms. Many other therapeutic agentshave been developed and synthesized by chemists for use in the body andare referred to as chemotherapeutic agents. Here, we will lump themall under the umbrella of antibiotics. The effectiveness of such agents oninhibiting bacterial growth can be evaluated using the StandardizedDisk Susceptibility Test, often referred to as the Kirby-Bauer method(Exercise 14).

The effects of physical and chemical agents in controlling bacterialgrowth can be studied by carrying out a bacterial growth curve and deter-mining the generation time, which is the interval of time for the cellpopulation to double (Exercise 15).

earning Objectives

When you have completed the exercises in Part IV, you should be capable of:• Evaluating a simulated epidemic in terms of the nonliving source or the index

patient, and explaining the spread of the infectious agent.• Assessing the effects of heat, UV light, drying, and other physical agents on

bacteria.• Judging the susceptibility of bacteria to antiseptics and disinfectants, metals, and

other chemical substances, including lysozyme.• Evaluating the effectiveness of disinfectants on contaminated inanimate objects,

on the skin surface, and as mouthwashes. • Employing the phenol coefficient to compare the effectiveness of disinfectants. • Completing a Standardized Disk Susceptibility Test (Kirby-Bauer method) to

evaluate the sensitivity of bacteria to antibiotics.• Carrying out and analyzing a bacterial growth curve and estimating the genera-

tion time.

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A SimulatedEpidemic

pidemic diseases (or epidemics) break out suddenly within apopulation. Often such epidemics are caused by microorgan-isms transmitted by lifeless objects known as fomites. It often

happens that several people handling the same fomite come in contact withthe same organism, or one person transmits a disease organism to several oth-ers via a fomite. For example, if a dollar bill were contaminated with staphy-lococci (such as from an abscess), the staphylococci could be transmitted toother people handling that dollar bill. An open wound such as an abrasion orpimple could then provide the staphylococci with an entryway to deepertissues.

To locate the responsible fomite, public health officials commonly takeswab samples from various objects and environmental surfaces and culti-vate any microorganisms collected on the swabs. Usually they wait for themicroorganisms to grow and form colonies (and hope they have used thecorrect growth media). In some cases, however, a marker enzyme is avail-able. In this exercise, the enzyme catalase is used as a marker. Catalasebreaks down hydrogen peroxide (H2O2) into water and oxygen, and causesthe solution to bubble as the oxygen gas is released. Catalase is produced bya wide variety of microorganisms, including the harmless yeast cells usedin brewing and baking (Exercise 9). Thus, the enzyme can be used to trackyeast cells and demonstrate how a true pathogen could move through a setof individuals.

Microbial Transmission via Fomites

Fomites are a widely known mechanism for transmitting microorgan-isms during epidemics. Such things as bed linens, for example, can trans-mit intestinal microorganisms such as salmonellae and amoebic cysts,and contaminated syringes may be the source of bloodborne viruses. In thissection, we shall demonstrate how yeast cells are transmitted among var-ious individuals by contaminated coins, and we shall use catalase as theexperimental marker for the yeast cells’ presence.

A.

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10

PURPOSE: to simulate thespread of a disease-causingorganism by fomites and toidentify the index patient.

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pecial Materials

• Several washed coins• Suspension of yeast cells (commercial yeast such as “active dry” yeast or yeast

cake, or from a broth culture of Saccharomyces cerevisiae)• Sterile swabs and forceps• Sterile saline for moistening swabs• Tubes of hydrogen peroxide (H2O2)

rocedure

I. Simulation of Disease Spread1. The fomites used in this exercise will be a set of coins. To prepare for the

exercise, wash several coins with soap and water until they are clean; drythe coins with paper toweling before using them.

2. To demonstrate that a contaminated fomite can transmit microorganismsto a series of individuals, proceed as follows: Using a forceps, dip a coin intoa heavy suspension of yeast cells (Saccharomyces cerevisiae). Place the coinon a clean paper towel to dry. (Drying may be hastened by placing thepaper toweling on a warming tray.) Now, one student should take the con-taminated coin and rub it between the thumb and forefinger to simulatehandling the coin. The coin should then be passed to a series of fellowstudents, who will also handle the coin before passing it on. After the coinhas been passed to a designated number of individuals, it should bereturned to the instructor.

3. Having simulated an epidemic, the object will be to verify which individ-uals have been contaminated. To do this, obtain a sterile swab and tubes ofhydrogen peroxide and sterile saline. Moisten the swab with sterilesaline and swab the fingertips of a person who handled the coin. Thendrop the swab into a tube of hydrogen peroxide.

4. Observe the hydrogen peroxide for signs of oxygen release, signified by bubbling. If yeast cells were present on the subject’s fingertips, theywill release catalase, which breaks down H2O2 to water and oxygen gas(Figure 10.1). The bubbling will be vigorous if many yeast cells are recov-ered and mild if there are few yeast cells. Thus, you may estimate the

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94 10 A S I M U L A T E D E P I D E M I C

Yeastcells

Cotton swab

Catalase

O2

H2O2

Hydrogenperoxide(H2O2)

F I G U R E 1 0 . 1Yeast cells produce catalase, which reactswith hydrogen peroxide (H2O2) to produce oxygen (O2) and water (H2O).

!The yeast culture used inthis exercise should befreshly made. If it has remained for too manydays, it may have becomecontaminated with bacteriaand be potentially dangerous.

Quick ProcedureFomite Transmission

1. Contaminate a coinwith harmless yeast cells.

2. Pass the coin amongseveral individuals.

3. Swab the fingertips andplace the swabs in hydrogen peroxide.

4. Observe the tube forbubbling.

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relative concentration of microorganisms “infecting” the subject. A “neg-ative control” tube should be prepared using a swab sample taken froma known uncontaminated person. A “positive control” tube should con-tain a swab that has been dipped into the yeast suspension. Note yourobservations in the Results section, and draw your conclusions on theimportance of the fomite in microbial transmission.

II. Identification of the Index Patient1. It often happens that one individual is the index patient in an epidemic. The

index patient is the first person to transmit microorganisms to other indi-viduals. To investigate this principle, proceed as follows: One person shoulddip the thumb and forefinger into the yeast suspension, then pick up a washed,uncontaminated coin. After rubbing the coin, the person should pass the cointo a series of individuals who will become “infected” by handling the coin. Thecoin should be returned after the transfers have been completed. Now the cata-lase tests can be performed (section I, steps 3 and 4). Determine which indi-viduals acquired the yeast cells from the fomite and were thus “infected” bythe index patient. Record your conclusions in the Results section.

III. Other Variations1. Many other variations to the above procedures may be employed to study

the principle of fomite transmission. One variation is to use several differentcoins (e.g., a penny, a nickel, and a dime) and contaminate only certainones. An epidemiological study may then be instituted to determine whichpersons became infected by handling which coin.

2. Another variation of the procedure is to test the effectiveness of hand-washing to interrupt the epidemic. To perform this exercise, several of theinfected individuals should wash their hands after handling the “infected”coins. Swab samples are then taken, and a study is conducted to determinewhether washing reduced or eliminated the microbial population.

A Microbial Hunt

During the course of an epidemiological investigation, public health officialsoften are called on to sample various surfaces or objects to determinewhether they were the source of a disease outbreak. For example, recov-ering microorganisms from eating utensils may indicate unsanitary con-ditions exist and persons eating with that batch of utensils are at risk forcontracting intestinal disease.

One method for testing objects and surfaces is to use moistened sterileswabs to recover microorganisms from a surface, then cultivate the micro-organisms under laboratory conditions. Another method is to use a dis-tinctive marker enzyme to identify the microorganism. In this section, testswill be conducted to locate the source of microorganisms responsible for the“epidemic” that is currently taking place. As in Part A, the microorganisminvolved will be yeast cells, and the marker enzyme will be catalase.

B.


PURPOSE: to identify thesource of a disease outbreakor epidemic.

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pecial Materials

• Suspension of yeast cells (commercial yeast such as “active dry” yeast or yeastcake, or from a broth culture of Saccharomyces cerevisiae)

• Sterile swabs and forceps• Sterile saline for moistening swabs• Tubes of hydrogen peroxide (H2O2)

rocedure

1. Prior to the laboratory session, the instructor has painted several surfacesand objects in the lab with a suspension of yeast cells, the test organismused in the study. The surfaces painted could include a door handle, apiece of equipment, a telephone, a table top, or any other object. Theinstructor may wish to set the stage for your investigation by pointing outcertain known facts about the epidemic. Alternatively, you may wish tomake inquiries or ask certain salient questions.

2. Once you have decided on the surface or object to be sampled, obtain a ster-ile swab, a tube of sterile saline, and a tube of hydrogen peroxide. Take theswab and moisten it with sterile saline. Then use the swab to rub the sur-face vigorously. Now drop the swab into a tube of hydrogen peroxide(H2O2).

3. Observe the tubes for signs of oxygen gas release, which will be mani-fested by bubbling as Figure 10.2 shows. The bubbling results from catalasereleased by yeast cells on the swab. (Nothing will happen in the tube if yeastcells were not picked up on the swab.) The bubbling may be vigorous if alarge number of yeast cells are recovered, or it may be mild if there arefewer cells. Therefore, you may ascertain whether the surface was heav-

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(a) (b)When the swab contains no yeast, no changes occur in the hydrogen peroxide.

When the swab contains yeast cells, the latter produce catalase that breaks down the hydrogen peroxide andreleases oxygen bubbles.

Oxygen bubbles

Swab without yeast cells

Hydrogenperoxide(H2O2)

Swab withyeast cells

F I G U R E 1 0 . 2The catalase test for detecting yeast cells.

Quick ProcedureHunting ForMicroorganisms

1. Contaminate a surfacewith harmless yeast cells.

2. Swab the surface.

3. Place the swab inhydrogen peroxide.

4. Observe the tube forbubbling.

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ily or mildly contaminated. This may help you decide which surface wasa primary source of microorganisms and which ones were secondary. A“negative control” should be prepared with moistened swabs and a“positive control” with swabs dipped into the yeast suspension. Noteyour observations in the Results section, and draw your conclusions.

4. Your findings of contaminated surfaces may be confirmed in several ways.For example, it may be possible to cultivate the yeast cells on Sabouraud dex-trose agar or potato dextrose agar. To do this, swab the surfaces with moist-ened swabs and rub the swabs on plates on these media. Cultivation atroom temperature for several days should reveal yeast colonies. Micro-scopic observations can be performed to confirm the presence of yeasts. Itmay also be possible to take scrapings from the suspected surface and pre-pare slides for microscopic observation even before yeasts are cultivated.These options should be discussed with the instructor before you proceed.

Transmission of Microorganisms through Toilet Paper

Infectious diseases such as hepatitis A, viral gastroenteritis, typhoid fever,shigellosis, and numerous others can be transmitted by infected handswhen a person fails to wash thoroughly after using the toilet. One of thedrawbacks of toilet paper is its porosity, a factor that permits microbes toreach the finger surface even though the skin surface is not touched. In thisexercise, yeast cells will be used as a test organism and the presence of cata-lase will be used as a marker to detect them. A simulated “wiping” will beperformed, followed by a test for “infectious organisms.”

pecial Materials

• Roll of toilet paper• Yeast suspension• Plate of any type of agar• Sterile swabs and saline• Tubes of hydrogen peroxide

rocedure

1. Obtain a plate of agar of any type and pour onto its surface about 10 ml ofthe yeast suspension. The plate should be tilted to cover the surface with theyeast. If there is excess yeast fluid, pour it off into a beaker of disinfectant.The “infected” plate will represent the anal area after using the toilet.

2. Wash your hands thoroughly and completely to remove all traces of yeastcells contacted during preparation of the plate. To ensure that your handsare clean perform the following procedure. Obtain a sterile swab, a tube ofsterile water, and a tube of hydrogen peroxide. Moisten the swab with thesterile water, and vigorously rub your fingers where they were in contactwith the yeast suspension or agar plate. Drop the swab into a tube ofhydrogen peroxide and watch for the presence or absence of bubbling.

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C.


PURPOSE: to simulate thespread of disease-causingorganisms through handcontact.

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No bubbling should occur, an indication that there are no yeast cells on thefinger surface.

3. Now obtain several sheets of toilet paper and hold them as you might doafter using the toilet. Wipe the surface of the agar (try not to gouge theagar) to simulate wiping the anal area. Discard the toilet paper in the dis-infectant designated by the instructor.

4. A fellow student may assist you in the following. Obtain another sterileswab, tube of sterile water, and tube of hydrogen peroxide. Moisten theswab with the sterile water, then vigorously rub the fingers where they heldthe toilet paper. Drop the swab into a tube of hydrogen peroxide and watchfor the presence or absence of bubbling.

5. If the tube begins to bubble, then there is evidence for the presence ofyeasts because these organisms produce the catalase responsible for thebubbles. Assuming there were no yeasts present before you began the“wiping,” the data points to the transmission of yeasts through the toiletpaper. On the other hand if there is no bubbling, then you may determinethat yeasts were not transmitted through the toilet paper. Write your con-clusions in the Results section. How can one increase or decrease the abil-ity of toilet paper to transmit organisms?

uestions

1. What characteristics of yeasts make this organism suitable for use in anexercise such as this?

2. Why is it unnecessary to use sterile hydrogen peroxide in the proceduresof this exercise?

3. Name several microbial diseases whose agents are transmitted byfomites, and specify which fomites might be involved.

4. Hypothesize why yeast cells naturally produce catalase.

5. What steps might be taken to interrupt the spread of infectious micro-organisms in an epidemic even before the microorganisms have beenidentified?

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Name

Date Section

Exercise Results

A Simulated Epidemic

A. Microbial Transmission by FomitesI. Simulation of Disease Spread

10

II. Identification of Index Patient

Observations and Conclusions:


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B. A Microbial Hunt


C. Transmission of Microorganisms through Toilet Paper

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43038 CH10 0091