Hoeksema received his Ph. He held positions as a post-doctoral researcher at the University of California, Santa Cruz from , and as a postdoctoral fellow at the National Evolutionary Synthesis Center at Duke University from , before joining UM in Copp received his M.
Skip to main content. Toggle navigation. This study was carried out between July and August Overall, 20 volunteers were taken to a large, frequently visited British museum, or asked to travel on a bus or the underground.
They were asked to deliberately wipe their hands over hand contact surfaces such as handrails, door handles and seats with the aim of contaminating their hands with whatever bacteria were present. Using a pre-determined random sequence, not known to the participants during self-contamination, participants were then asked to wash their hands with soap, to use water only or not to wash at all. Each volunteer underwent this sequence 24 times, 8 times for each of the three hand hygiene approaches soap, water, no handwash.
Participants assigned to handwashing were asked to wash their hands as they would normally do, without instructions on length of time or thoroughness. The volunteers allocated to handwashing were then provided with a paper towel to dry their hands. A wet NaCl-soaked charcoal swab was then wiped across the fingers of the dominant hand of the participant. The swabs were returned to the laboratory within 5 hours of being taken. In total, samples were collected; after handwashing with plain soap, after handwashing with water alone and with no handwashing.
During the experimental phase we measured the amount of time taken to conduct handwashing with and without soap, once for each volunteer.
Upon arrival at the laboratory the swabs were immediately cut into a universal tube containing 10 mL of Purple MacConkey broth using aseptic techniques. All samples were then streaked onto the MacConkey agar No.
MacConkey agar No. For all other colonies produced on MacConkey agar No. Bile Aesculin agar is a differential medium for the isolation of Enterococcus spp. Enterococcus and Group D Streptococcus spp. Any white colonies on Bile Aesculin agar were presumed to be Staphylococcus spp. Agglutination indicated a positive result for Enterococcus spp. The prevalence of bacterial contamination in the three study arms soap, water, no handwash was compared using logistic regression.
Since the same volunteers repeatedly underwent testing, within-subject correlation was accounted for by the use of generalised estimating equations GEE with robust standard errors. If the cell numbers were too low for conducting regression analysis, Fishers exact test was used instead, ignoring clustering the design effect was found to be low, see results.
Table 1 shows the different organisms isolated in the three study arms. Enterococcus spp. Figure 1 shows the effect of handwashing with soap or water only on contamination, compared to no handwashing. Overall, handwashing with water alone reduced the prevalence of bacteria substantially.
Handwashing with soap was more effective in reducing the prevalence of contamination and specifically of Enterococcus spp. There was a trend that handwashing with soap was also more effective in reducing the prevalence of other species and of multiple isolates, but the statistical support was low Figure 1. Effect of handwashing with water alone or soap and water compared to no handwashing.
The design effect due to within-person clustering was low around 1. Note different y-axis scales in top vs. Organisms found after self-contamination of hands, and handwashing with either soap, water only, or no handwashing. The effect of repeated measurements in the same individual was low: the design effect the factor by which a sample size needs to be increased to achieve the same statistical power as an unclustered study ranged from 1.
Participants were asked to wash their hands as long and as thorough as they would normally do. The length of time required to carry out handwashing was measured once for each method in all volunteers. Participants took on average 12 seconds standard deviation 2. Thus, handwashing with soap took them only slightly longer than handwashing with water alone.
It seems unlikely that this small difference can explain the large difference in the removal of bacteria. Soap on its own appears to have an effect on the removal of bacteria of potential faecal origin, independent of the possibility that soap use may cause people to wash their hands longer.
Unlike the study by Hoque and colleagues our trial was conducted in an experimental albeit naturalistic setting, where volunteers deliberately contaminated their hands. It also improved control over the conduct of the experiment, but may affect generalisability, as the study primarily aimed at providing a proof of principle. However, we believe that the superior effect of soap on the removal of bacteria compared to water alone as the principal finding of our study is unlikely to depend on the setting.
Not all of the bacteria isolated in our study are known to cause disease in humans. Surprisingly, we found few E. Overall, the effect of soap appeared to be independent of the type of bacteria Figure 1 , a view which is supported by the study by Hoque and colleagues who found a similar effect of hand hygiene on unspecified faecal coliform bacteria [ 6 ].
Because the smears are not heat fixed and the stain used does not penetrate into the cells, the organisms remain viable. As bacterial smear preparations for negative staining differ to some extent from conventional staining procedures, students should be reminded not to heat fix the smear.
Also it may be advisable to demonstrate the technique for spreading the smear with the aid of a second glass slide. As the bacteria are not killed during the negative-staining procedure, students should be instructed in the importance of discarding the slides into a beaker containing disinfectant following their microscopic examination.
The experimental procedure may be modified to include the staining of an organism by both simple and negative staining to allow students to compare the observed results. The instructor should emphasize that these organisms are not heat fixed and thus are viable. Students should be given the option to use disposable gloves. Some labs reuse slides for negative staining. Only new and clean slides should be used in this experiment. Baradkar, V.
Prevalence and clinical presentation of Cryptococcal meningitis. Methylene blue as a basic, cationic dye cannot be used in negative staining. An acidic stain, such as nigrosin, is required so that it does not bind to the negatively charged cell surface. Negative staining allows the visualization of living microbial cells that have not undergone distortion by heat fixation.
The nigrosin is an anionic acidic stain and does not have an affinity for the negatively charged cell surfaces. As such, the dye colors the background, and the cells remain unstained. The Gram stain is one of the first procedures to be performed for the identification of microorganisms. In the classroom setting, it serves as the prototype for a variety of other differential staining procedures. As the Gram stain is the most frequently performed differential staining technique, the instructor should explain the functions of the chemicals used in differential staining as well as the chemical basis of this procedure.
The most critical step in all differential staining procedures, including the Gram stain, is the decolorization process. Students should be cautioned that the density of the smear will be a major factor in determining the amount of decolorizing agent necessary for proper decolorization of the smear. Thus, the method used by the instructor should be explained and demonstrated. When the water bubble clinging to the edge of the slide is almost clear, decolorization is complete.
Simple staining uses a single dye and stains all cells and their cytological components the same color. Thus, these procedures can be used only to determine cell morphology. Differential staining utilizes two stains of contrasting colors that allow for the separation of bacteria into groups, e. The mordant is a chemical that acts as an intensifier in the Gram staining procedure. It forms a complex with the crystal violet, which cannot be easily removed from gram-positive cells with the decolorizing agent.
The decolorizing reagent functions to remove the primary stain only from some cell types or cell structures, thus allowing for their differentiation, on the basis of color, following the application of the counterstain. It should again be stressed that thorough washing of the slides under running water between the applications of all staining reagents is essential to remove excess chemicals.
The counterstain is the second, contrasting-color stain that is applied. This stain will be absorbed only by decolorized cells. Considering bacteria cannot be separated on the basis of differences in cell morphology, differential staining, using dyes of contrasting colors, allows for the microscopic separation of organisms into groups based on a difference in color.
Decolorization is the most crucial step. The basis of the Gram stain is the ease with which the primary stain can be removed by the decolorizing agent. Therefore, over-decolorization will remove the primary stain from gram-positive organisms, causing many cells to appear to be gram negative. Insufficient decolorization fails to remove the primary stain from organisms that are gram negative, thereby resulting in a gram-positive reaction.
With increasing age of a culture, the ability of organisms to absorb the stain becomes variable because of changes in cell wall structure. Thus, a uniformly colored preparation is not possible and results in a gram-variable reaction with the B. This phenomenon of gram variability is noted more frequently with grampositive organisms.
Included among these are members of the genus Bacillus. Fresh cultures, 18—24 hours old, are necessary for optimum Gram staining reactions. Older cultures tend to produce gram-variable results. Washing of stained smears should be done carefully. Overwashing should be avoided so as not to overdecolorize the preparation. Considering this is the first time students are performing a differential stain, the instructor may wish to demonstrate the method for the class. Uehara, Y. Impact of reporting gram stain results from blood culture bottles on the selection of antimicrobial agents.
American Journal of Clinical Pathology, 1 — The acid-fast stain is a highly specialized diagnostic staining procedure that is used to identify members of the genus Mycobacterium. Its application in the clinical setting is for the diagnosis of tuberculosis and leprosy. Considering that mycobacteria have a tendency to clump, students should be instructed to vigorously spread the inoculum on the slide to separate the organisms.
When preparing the mixed-culture smear, students should be cautioned to use a more concentrated sample of M. In order to obtain a satisfactory acid-fast reaction using the heat method, the following points should be stressed: a. The carbol fuchsin—covered smear must be heated for the required period of time.
The carbol fuchsin must be maintained at a steaming rather than a boiling temperature to prevent rapid evaporation of the stain. Additional applications of carbol fuchsin will be required during the heating process even though the slide is maintained at a steaming temperature. Following the application of heat, the slide preparations must be allowed to cool prior to their vigorous washing with water to prevent breakage of the slides. Students should be reminded to blot the stained smear with bibulous paper but not to rub the bibulous paper over the wet slide.
Three- to 4-day cultures of M. Specialized media, such as the LowensteinJensen medium, may be used to culture Mycobacterium sp. If a broth medium is used, the addition of 0.
Wilmer, A. The role of the third acid-fast bacillus smear in tuberculosis screening for infection control purposes: A controversial topic revisited.
The application of heat or a surface-active agent is essential to soften the waxy cell wall components to facilitate the penetration of the primary stain into the cells.
The acid-fast staining procedure is used for the diagnosis of leprosy and tuberculosis, both of which are caused by members of the genus Mycobacterium. Application of heat or a surface-active agent is not required during the application of the counterstain. The acid-fast organisms, because of the waxy nature of their cell walls, are not decolorized, and the red stain remains trapped inside the cells. The non—acid-fast organisms lack the lipoidal cell wall components.
Therefore, the primary stain is easily removed during decolorization, and the colorless cells are readily stained by the counterstain. The presence of acid-fast bacilli in the gastric washing suggests that the tubercle bacilli, released from the lungs, were swallowed by the child rather than eliminated by coughing.
This evidence is suggestive of a tuberculosis infection. If the heatless modification of the ZiehlNeelsen method is used, add 2 drops of Triton X per ml of carbol fuchsin. These differential staining procedures are used to demonstrate anatomical structures that may be present in bacteria, namely the endospore and the capsule.
The procedures, although of academic interest, are not frequently performed. Because of the impervious nature of the protein spore coats, the stain-covered smear is heated to ensure penetration of the stain into the spore. The function of water is to remove excess primary stain from the spore. The vegetative cells lack an affinity for this stain; thus it is removed by water, rendering the vegetative cells colorless.
Acid-alcohol would not decolorize the stained spore, and the final observations would be the same as with the use of water. Reemphasize the precautions outlined in Experiment 8 for the application of dyes with heat.
As in the acid-fast staining procedure, the absorption of the primary stain requires the application of sufficient heat. Be sure to tell students not to allow malachite green to evaporate from the smear during heating. Be careful not to wash more than 35—45 seconds with tap water or the malachite green stain will overdecolorize. Overdecolorization is a common mistake made by students. If safranin is applied with heat, both the endospore and the vegetative cell will accept the stain and appear red in color.
Tap water will not remove the stain, and therefore, malachite green would not be accepted. Both the endospore and the vegetative cell will be red. Caution students to avoid vigorous spreading with the loop or needle during smear preparation because of the fragile nature of the capsular material. Also, remind students that water is not used in this procedure for washing.
Remind the students not to heat fix the capsule smears. Failure to apply heat with the primary stain will not allow the stain to penetrate into the endospore. The vegetative cell will be red, and the endospore will be colorless and refractile. It is of medical significance as its presence renders the cell resistant to the phagocytic activities of WBCs, thereby increasing the virulence of the organism.
The capsule is nonionic and as such will not bind with the cationic primary stain, crystal violet. In this method, copper sulfate is used rather than water to wash out excess stain from the cell. During this process, the copper sulfate is absorbed into the capsule, giving it a light blue color in contrast to the deep purple color of the cell.
Optional Procedural Additions or Modifications Projected slides, commercially prepared slides, or colored transparencies can be used to acquaint students with these cytological structures.
Clostridium difficile in raw products of animal origin. International Journal of Food Microbiology, 1—2 —5. Martin, M. An outbreak of conjunctivitis due to atypical Streptococcus pneumoniae.
New England Journal of Medicine, 12 — The purpose of this experiment is twofold. First, it will evaluate synthetic chemically defined media, complex chemically undefined media, and enriched media for their ability to support microbial growth. Second, students will ascertain the degree of fastidiousness of selected microorganisms.
Absorbance is directly proportional to the amount of microbial growth, whereas percent T is inversely proportional to the number of cells present. Uninoculated media tubes, representative of the media in which the cultures have been grown, are used as blanks. Artificial media are used for the routine cultivation of microorganisms as the peptones and beef extract are sufficient to provide the nutritional growth requirements for most microorganisms.
Thus, knowledge of the specific nutritional needs of the organism is not needed. Heterotrophic organisms require the use of organic carbon sources and, in some cases, organic nitrogen sources and vitamin supplements.
These organisms would not grow in an inorganic medium. If the organism showed minimal growth in a basic artificial medium, yeast extract could be added as a supplement, as it contains all the B vitamins. As this is the first time students will be using a spectrophotometer, they should be given a complete explanation and a demonstration of its use. Ancillary information should include the following reminders: a.
The organisms in each culture must be resuspended. However, the cultures must be allowed to stabilize until the bubbling subsides prior to the determination of the A readings. Otherwise, erroneous readings will be obtained. The outside of all culture tubes must be wiped with lens paper to remove finger marks before their insertion into the test tube well. All culture tubes must be inserted into the test tube well in the same position. The etched marking on the test tube may be used as a guide.
The test tube well cover must be closed prior to obtaining A readings. Lindqvist, R. Estimation of Staphylococcus aureus growth parameters from turbidity data: characterization of strain variation and comparison of methods.
To determine the specific vitamin needs of the organism, a vitamin assay is required. In performing the assay, the control would contain all the vitamins. Each of the remaining assay culture media would contain all the vitamins present in the control culture with the deletion of one different vitamin from each test tube.
Culture tubes lacking growth in the absence of a particular vitamin would indicate that this vitamin is an essential growth factor. The purpose of this experiment is to demonstrate the functions of special-purpose media used for the isolation and the identification of specific groups of microorganisms. In the clinical laboratory, these media are frequently used to facilitate the rapid detection and isolation of possible pathogens from mixed microbial populations in biological specimens.
High salt concentration in the mannitol salt agar medium is used to inhibit the growth of organisms other than halophiles. Students should be reminded of the necessary precautions to prevent exogenous contamination when performing multiple inoculations on a single plate. Lactose is a major microbial carbon source. In MacConkey agar medium, it serves to differentiate between lactose fermenters and nonfermenters on the basis of their ability to produce acid.
Students should be cautioned to confine the line of inoculation of each organism well within its designated section of the plate. Phenylethyl alcohol in the phenylethyl alcohol agar medium partially inhibits growth of gram-negative organisms; thus, the number and size of gram-negative colonies is markedly reduced.
Although blood agar is not truly classified as a differential or selective medium, it can be used as such in the separation and classification of the streptococci on the basis of their hemolytic patterns alpha, beta, and gamma on blood agar. It is a good opportunity for students to become familiar with this, considering they will see it again in Experiment Gram-positive organisms are very sensitive to the basic dye crystal violet.
The exact mechanism of action by which crystal violet acts is still unclear. When incorporated into a medium, 7. This medium is excellent for the selection and differentiation of different species of staphylococci, which are halophilic organisms. A boil is usually the result of a staphylococcal or a streptococcal infection.
The exudate should first be cultured in a broth medium, followed by streak-plate inoculations on blood and mannitol salt agar plates for the isolation of discrete colonies. If the etiological agent of the boil is Staphylococcus aureus, a yellow halo will be present surrounding some of the colonies on the mannitol salt agar plate, and beta-hemolysis will be evident on the blood agar plates.
If the causative agent is a pathogenic streptococcus, evidence of betahemolysis will be present on the blood agar plate; however, none of the colonial growth on the mannitol salt agar plate will exhibit a yellow halo. Craven, R. Evaluation of a chromogenic agar for detection of group B streptococcus in pregnant women.
Journal of Clinical Microbiology, 48 9 —1. Crystal violet in MacConkey agar medium is an inhibitor to suppress the growth of grampositive organisms. Blood serves to enrich an agar medium to support the growth of fastidious organisms and to differentiate microorganisms, particularly streptococcal species, on the basis of their hemolytic activities.
The eosin and methylene blue in the EMB agar medium is used to identify E. The large amount of acid produced by these organisms causes the dyes to precipitate out onto the surface of the colonies, thereby producing a green coloration to the growth. Methylene blue also partially inhibits growth of grampositive organisms.
ExpErimEnts 16, 17, and 18 Physical Factors: Temperature Physical Factors: pH of the Extracellular Environment Physical Factors: Atmospheric Oxygen Requirements These experiments are designed to demonstrate the microbial diversity as it relates to the specific environmental requirements essential to support microbial growth.
This diversity is dependent upon the enzymatic capabilities of specific microorganisms. Students should be made aware of the presence of a Durham tube in each of the carbohydrate broth medium tubes and cautioned not to accidentally introduce air into this gas collection vial.
Rapidly rotate the test tubes between the palms of the hands for the even distribution of the organisms throughout the medium. Atmospheric Oxygen Stab culture preparations in nutrient agar deep tubes may be substituted for the molten agar shake-tube procedure. Konishi, T. Influence of temperature on growth of Legionella pneumophila biofilm determined by precise temperature gradient incubator. Journal of Bioscience and Bioengineering, 6 — Stingl, K.
Energetics of Helicobacter pylori and its implications for the mechanism of urease-dependent acid tolerance at pH 1. Journal of Bacteriology, 11 — Das, D. Staphylococcal catalase protects intracellularly survived bacteria by destroying H2O2 produced by the murine peritoneal macrophages.
Microbial Pathogenesis, 47 2 — Enzymes will be denatured above the maximum growth temperature. Below the minimum growth temperature, enzymes are inactivated but not destroyed. It is not possible for obligate thermophiles to induce infections in warm-blooded animals because they require temperatures that exceed body temperature for growth.
Microbial metabolic activities will generate shifts in pH within the culture. For example, if carbohydrates are primarily utilized, the accumulation of acidic end products will lower the pH of the medium. If protein compounds are metabolized, alkaline end products are released into the environment, thereby raising the pH.
Facultative anaerobes possess the most extensive bioenergetic enzyme systems because of their dual capability to respire aerobically and anaerobically. Aerobes cannot grow in the absence of atmospheric oxygen, as these organisms can utilize this molecule only as the final electron acceptor in aerobic respiration.
Microaerophiles have specific atmospheric oxygen needs, requiring a reduced oxygen tension that exists in a narrow zone beneath the surface of the agar preparation. Facultative anaerobes, which grow throughout the entire test tube culture, are capable of anaerobic and aerobic respiration.
However, they preferentially use the aerobic pathway, which is more efficient, and thus exhibit more growth toward the surface of the culture. Clostridium sporogenes is a strict anaerobe and therefore grows in the depth of the agar culture. This organism generates copious amounts of gas that fractures or elevates the medium from the bottom of the test tube.
Streptococci are aerotolerant organisms facultative anaerobes that have the enzymatic capacity to survive in the presence or absence of oxygen.
These organisms do not use oxygen in their metabolism, and use other molecules as a final electron acceptor. They are homofermentative and are able to obtain their energy from fermenting sugars glucose to lactic acid via the hexose monophosphate pathway. Differences in pH requirements among microorganisms are dependent upon the susceptibility of their individual enzyme systems to denaturing at various pH levels.
Not all organisms grow optimally at a neutral pH, as their enzyme systems have already adapted for existence in their natural environment. For example, many fungi grow best in an acidic environment, while many species of soil bacteria prefer an alkaline pH. In the chemically defined E.
In the nutrient broth medium, which contains natural buffers, there was no pH shift, and growth was sustained in the presence of adequate nutrients. The inoculated molten agar cultures require rapid solidification to prevent the diffusion of atmospheric oxygen into the depth of the cultures and to maintain the even distribution of the organisms throughout the medium. The purpose of this experiment is to introduce students to the use of specialized techniques for the cultivation of anaerobic microorganisms.
In clinical medicine, the use of anaerobic culture procedures has gained significance over the years. Currently, pseudomembranous enterocolitis may be caused by the anaerobe Clostridium difficile following certain types of antibiotic therapy. Students should be provided with a thorough demonstration in the use of the GasPak system. Furthermore, they should be cautioned to strictly adhere to the steps as outlined under the procedure section of this exercise to ensure the development of anaerobiosis.
Handle the test tubes of thioglycollate carefully, with minimal agitation, to prevent the introduction of oxygen into the medium. Introduce the inoculum into the bottom of the test tube. Slightly loosen screw-cap tubes incubated in the GasPak about a half of a turn to neutralize the oxygen inside the tubes by the generated hydrogen gas.
These media contain meat products that reduce the redox potential of the medium, thus establishing conditions that favor anaerobiosis. In the GasPak system, the gas generator component serves to replace the oxygen in the vessel with hydrogen gas and carbon dioxide.
The indicator strip is used to indicate the development of anaerobic conditions in the system. Heroin is usually diluted with a redox reducing agent such as quinine. Upon injection, if a spore-laden needle misses the vein, leakage of the heroin into the tissues establishes a focal point for an infection such as tetanus by the spore-forming Clostridium. The infections of concern are gas gangrene and tetanus, whose etiological agents are spore forms that are present in the upper layers of the soil.
The deep puncture wound caused by the tine would result in conditions favorable for spore germination. The absolute anaerobic condition is generated by the elimination of oxygen from the deep wound by aerobic and facultatively anaerobic contaminants.
In addition, the breakdown of dead tissues provides the essential nutritional source for rapid germination. A drain is inserted into a deep puncture wound to ensure that healing occurs from the depth of the wound outwards. Also, the drain provides an outlet for the passage of dead tissue debris, a contributory factor in spore germination, and it serves as an inlet for oxygenation of the infected area. Optional Procedural Additions or Modifications To reduce laboratory expenses, have students perform the GasPak procedure in groups of four students.
Two groups may share a single GasPak. The GasPak jar not only provides excellent anaerobic conditions, but it also provides increased concentrations of CO2 that can be used for enrichment alone. If time and equipment are available, some other methods Figure Fluid thioglycollate, dispensed in screw-cap culture tubes, must be freshly prepared to show a pink coloration in the upper third of the medium. Donelli, G. Biofilm-growing intestinal anaerobic bacteria.
The purpose of this experiment is to allow students to count the number of viable cells in a culture. As part of this procedure, students will be introduced to two new methodologies, namely the pour-plate preparation and pipetting techniques that are essential for the serial dilution of cultures.
As numerous dilution tubes and Petri dishes are used in this exercise, students should be cautioned to carefully label and organize all the glassware before proceeding with the experimental procedure. Students should be reminded to deposit all contaminated pipettes into a disinfectantcontaining receptacle.
These are never placed on the bench top. The pour-plate method should be demonstrated with emphasis on the gentle swirling of the molten agar so as to prevent the agar from sloshing onto the cover of the plate. Dilution refers to varying the concentration of a substance. The dilution factor is expressed mathematically as the reciprocal of the dilution.
The advantage of the serial dilution—agar plating procedure is that the cell count represents only viable cells. The disadvantage of this method is that it requires an incubation period that precludes the ability to obtain immediate results. A variety of parameters can be used to measure cell growth chemically: for example, by an increase in protein and DNA concentrations or by determination of dry cell mass.
Metabolic parameters can also be used: for example, the measurement of oxygen uptake by aerobic cells or the amount of carbon dioxide used by anaerobic organisms. Dilution plates showing spreading colonial growth should be eliminated from the experimental data because they may be obscuring the presence of small colonial forms, and they represent the growth of more than a single colony.
The spread-plate technique Experiment 3 may be substituted for the pour-plate preparation.
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