Principles of Microbiology and Molecular Biology PDF

Principals of microbiology and molecular biology (Different references) Collected and organized Ahmed Dewan PhD candidate in life sciences 2024  PArT 1 Basic Laboratory Techniques for Isolation, Cultivation, and Cultural Cha...

Principals of microbiology and molecular biology (Different references) Collected and organized Ahmed Dewan PhD candidate in life sciences 2024  PArT 1 Basic Laboratory Techniques for Isolation, Cultivation, and Cultural Characterization of Microorganisms LearnIng OBjeCTIves Once you have completed the experiments in this section, you should be familiar with 1. The types of laboratory equipment and culture media needed to develop and maintain pure cultures. 2. The types of microbial flora that live on the skin and the effect of hand washing on them. 3. The concept of aseptic technique and the procedures necessary for successful subculturing of microorganisms. 4. Streak-plate and spread-plate inoculation of microorganisms in a mixed microbial population for subsequent pure culture isolation. 5. Cultural and morphological characteristics of microorganisms grown in pure culture. Introduction medium. Basically, all culture media are liquid, semisolid, or solid. A liquid medium lacks a solidi- Microorganisms are ubiquitous. They are found fying agent and is called a broth medium. A broth in soil, air, water, food, sewage, and on body sur- medium is useful for the cultivation of high num- faces. In short, every area of our environment is bers of bacterial cells in a small volume of medium, replete with them. The microbiologist separates which is particularly helpful when an assay these mixed populations into individual species requires a high number of healthy bacterial cells. for study. A culture containing a single unadultera- A broth medium supplemented with a solidifying ted species of cells is called a pure culture. To agent called agar results in a solid or semisolid isolate and study microorganisms in pure culture, medium. Agar, an extract of seaweed, is a com- the microbiologist requires basic laboratory appa- plex carbohydrate composed mainly of galactose, ratus and the application of specific techniques, and is without nutritional value. Agar serves as as illustrated in Figure P1.1. an excellent solidifying agent because it liquefies at 100°C and solidifies at 40°C. Because of these Media properties, organisms, especially pathogens, can The survival and continued growth of microorgan- be cultivated at temperatures of 37.5°C or slightly isms depend on an adequate supply of nutrients higher without fear of the medium liquefying. A and a favorable growth environment. For survival, completely solid medium requires an agar concen- most microbes must use soluble low-molecular- tration of 1.5% to 1.8%. A concentration of less than weight substances that are frequently derived from 1% agar results in a semisolid medium. A semi- the enzymatic degradation of complex nutrients. solid medium is useful for testing a cell’s ability to A solution containing these nutrients is a culture grow within the agar at lower oxygen levels and 17  Broth Media Semisolid Agar slant Solid Agar deep Autoclave Agar plate Bunsen burner Microincinerator Culture tubes Petri dishes Equipment Wire loops and needles Transfer instruments Pipettes Waterbaths Cultivation chambers Incubators Refrigerators Streak plate Pure culture techniques Pour plate–loop dilution Isolation of pure cultures Spread plate Figure P1.1 Laboratory apparatus and culture techniques for testing the species’ motility. A solid medium area for microorganism growth while minimizing has the advantage that it presents a hardened the amount of medium required. Similar tubes surface on which microorganisms can be grown that, following preparation, are allowed to harden using specialized techniques for the isolation of in the upright position are designated as agar discrete colonies. Each colony is a cluster of cells deep tubes. Agar deep tubes are used primarily that originates from the multiplication of a single for the study of the gaseous requirements of cell and represents the growth of a single species microorganisms since gas exchange between of microorganism. Such a defined and well­isolated the agar at the butt of the test tube and the exter­ colony is a pure culture. Also, while in the lique­ nal environment is impeded by the height of the fied state, solid media can be placed in test tubes, agar. Liquid agar medium can also be poured into which are then allowed to cool and harden in a Petri dishes, producing agar plates, which provide slanted position, producing agar slants. These are large surface areas for the isolation and study of useful for maintaining pure cultures. The slanted microorganisms. The various forms of solid media surface of the agar maximizes the available surface are illustrated in Figure P1.2. Side view Front view (a) Agar slants (b) Agar deep tube (c) Agar plate Figure P1.2 Forms of solid (agar) media 18 Part 1  160° to 180°C for 11/2 to 3 hours; for Dry (hot air) empty glassware, glass pipettes, and glass syringes Free-flowing steam at 100°C (intermittent sterilization); for thermolabile solutions (e.g., sugars, milk) Heat Moist (wet heat) Autoclave, steam under pressure, temperatures above 100°C; for culture media, syringes, thermostable solutions, etc. Cellulose-acetate membrane filters Removal of organisms from thermolabile solutions Filtration with pore sizes in the range of 8.0 µm by passage through filters that retain bacteria; note, to less than 0.05 µm viruses are not removed by this procedure Ethylene oxide Plastic dishes and pipettes Chemicals Beta-propiolactone Living tissues Radiation Ionizing Plastic pipettes and Petri dishes Figure P1.3 Sterilization techniques In addition to nutritional needs, the environ­ sleeve­like caps (Morton closures) made of metal, mental factors must also be regulated, including such as stainless steel, or heat­resistant plastics. proper pH, temperature, gaseous requirements, and The advantage of these closures over the cotton osmotic pressure. A more detailed explanation is plug is that they are labor­saving and, most of all, presented in Part 4, which deals with cultivation of slip on and off the test tubes easily. microorganisms; for now, you should simply bear in Petri dishes provide a larger surface area for mind that numerous types of media are available. growth and cultivation. They consist of a bottom dish portion that contains the medium and a larger top portion that serves as a loose cover. Petri Aseptic Technique dishes are manufactured in various sizes to meet Sterility is the hallmark of successful work in the different experimental requirements. For routine microbiology laboratory, and sterilization is the purposes, dishes approximately 15 cm in diameter process of rendering a medium or material free are used. The sterile agar medium is dispensed to of all forms of life. To achieve sterility, it is manda­ previously sterilized dishes from molten agar deep tory that you use sterile equipment and employ tubes containing 15 ml to 20 ml of medium, or from aseptic techniques when handling bacterial a molten sterile medium prepared in bulk and con­ cultures. Using correct aseptic techniques mini­ tained in 250­, 500­, and 1000­ml flasks, depending mizes the likelihood that bacterial cultures will on the volume of medium required. When cooled be contaminated, and reduces the opportunity for to 40°C, the medium will solidify. Remember that students to be exposed to potential pathogens. after inoculation, Petri dishes are incubated in an Although a more detailed discussion is presented inverted position (top down) to prevent condensa­ in Part 9, which describes the control of microor­ tion formed on the cover during solidification from ganisms, Figure P1.3 is a brief outline of the routine dropping down onto the surface of the hardened techniques used in the microbiology laboratory. agar. For this reason, Petri dishes should be labeled on the bottom of the dish. This makes it easier to Culture Tubes and Petri Dishes read the label and minimizes confusion if two Petri dish covers are interchanged. Figure P1.4 illustrates Glass test tubes and glass or plastic Petri dishes some of the culture vessels used in the laboratory. are used to cultivate microorganisms. A suitable Built­in ridges on tube closures and Petri dishes pro­ nutrient medium in the form of broth or agar may vide small gaps necessary for the exchange of air. be added to the tubes, while only a solid medium is used in Petri dishes. A sterile environment is maintained in culture tubes by various types of Transfer Instruments closures. Historically, the first type, a cotton plug, Microorganisms must be transferred from one was developed by Schröeder and von Dusch in the vessel to another or from stock cultures to various nineteenth century. Today most laboratories use media for maintenance and study. This transfer Part 1 19  A B C D E (b) Petri dish A. Bacteriological tube D. Metal closure B. Screw cap E. Nonabsorbent cotton C. Plastic closure (a) Test tube rack with tubes showing various closures (c) DeLong shaker flask with closure Figure P1.4 Culture vessels is called subculturing and must be carried out (commonly called a “pipetter”) with a disposable, under aseptic conditions to prevent possible single­use plastic tip is useful for transferring small contamination. volumes of liquid (less than …1 ml). Wire loops and needles are made from inert Figure P1.5 illustrates these transfer instru­ metals such as Nichrome or platinum and are ments. Your instructor will demonstrate the proper inserted into metal shafts that serve as handles. procedure for using pipettes. They are extremely durable instruments and are easily sterilized by incineration in the blue (hottest) portion of the Bunsen burner flame. A wire loop is Pipetting by mouth is not permissible! useful for transferring a small volume of bacteria Pipetting must be performed with mechanical onto the surface of an agar plate or slant. A needle pipette aspirators. is used primarily to inoculate a culture into a broth medium or into an agar deep tube. A pipette is another instrument used for aseptic transfers. Pipettes are similar in function to straws; that is, they draw up liquids. They are Cultivation Chambers glass or plastic and drawn out to a tip at one end, The specific temperature requirements for growth with a mouthpiece forming the other end. They are discussed in detail in Part 4. However, a prime are calibrated to deliver different volumes depend­ requirement for the cultivation of microorganisms ing on requirements. Pipettes may be sterilized is that they be grown at their optimum tempera­ in bulk inside canisters, or they may be wrapped ture. An incubator is used to maintain optimum individually in brown paper and sterilized in temperature during the necessary growth period. an autoclave or dry­heat oven. A micropipette It resembles an oven and is thermostatically 20 Part 1  Loop No etched ring on mouthpiece Needle Etched ring (to deliver) on mouthpiece (blow out) TD 1 IN 1/100 ml Identification and graduations 0.1 ml: major division 0.01 ml each: Shaft minor divisions Handle Final few drops must be blown out to deliver indicated volume (a) Transfer (b) Transfer (c) Blow-out (d) To-deliver needle loop pipette pipette Mechanical Pipette Aspirators (f) Plastic (g) Rubber (e) Micropipette pump bulb Figure P1.5 Transfer instruments Part 1 21 controlled so that temperature can be varied Many laboratories also use shaking incuba­ depending on the requirements of specific micro­ tors that utilize dry air incubation to promote organisms. Most incubators use dry heat. Moisture aeration of the broth medium. This method has is supplied by placing a beaker of water in the a distinct advantage over a shaking waterbath incubator during the growth period. A moist envi­ since there is no chance of cross contamination ronment retards dehydration of the medium and from microorganisms that might grow in the thereby avoids misleading experimental results. waterbath. A thermostatically controlled shaking waterbath is another piece of apparatus used to cultivate microorganisms. Its advantage is that Refrigerator it provides a rapid and uniform transfer of heat A refrigerator is used for a wide variety of purposes to the culture vessel, and its agitation provides such as maintenance and storage of stock cultures increased aeration, resulting in acceleration of between subculturing periods, and storage of ster­ growth. The primary disadvantage of this instru­ ile media to prevent dehydration. It is also used as ment is that it can be used only for cultivation a repository for thermolabile solutions, antibiotics, of organisms in a broth medium. serums, and biochemical reagents. 22 Part 1  E xP E R IMEnT Techniques for Isolation of Pure Cultures 2 In nature, microbial populations do not segregate themselves by species, but exist with a mixture Principle of many other cell types. In the laboratory, The techniques commonly used for isolation of these populations can be separated into pure discrete colonies initially require that the number cultures. These cultures contain only one type of organisms in the inoculum be reduced. The of organism and are suitable for the study of resulting diminution of the population size ensures their cultural, morphological, and biochemical that, following inoculation, individual cells will properties. be sufficiently far apart on the surface of the In this experiment, you will first use one of the agar medium to separate the different species. techniques designed to produce discrete colonies. The following are techniques that can be used Colonies are individual, macroscopically visible to accomplish this necessary dilution: masses of microbial growth on a solid medium 1. The streak-plate method is a rapid qualitative surface, each representing the multiplication isolation method. It is essentially a dilution of a single organism. Once you have obtained technique that involves spreading a loopful these discrete colonies, you will make an aseptic of culture over the surface of an agar plate. transfer onto nutrient agar slants for the isolation Although many types of procedures are per­ of pure cultures. formed, the four­way, or quadrant, streak is described. Refer to Figure 2.1, which schematically illustrates this technique. PA RT A Isolation of Discrete a. Place a loopful of culture on the agar Colonies from a Mixed Culture surface in Area 1. Flame the loop, cool it by touching an unused part of the agar surface close to the periphery of the plate, LearnIng OBjeCTIve and then drag it rapidly several times across the surface of Area 1. Once you have completed this experiment, you should be able to b. Reflame and cool the loop, and turn the Petri dish 90°. Then touch the loop to a 1. Perform the streak-plate and/or the corner of the culture in Area 1 and drag spread-plate inoculation procedure it several times across the agar in Area 2. to separate the cells of a mixed culture The loop should never enter Area 1 again. so that discrete colonies can be c. Reflame and cool the loop and again; turn isolated. the dish 90°. Streak Area 3 in the same manner as Area 2. Turn Turn Turn plate 90. plate 90. plate 90. 2 3 4 Flame 1 Flame Flame loop. loop. loop. 2 1 3 1 1 2 Figure 2.1 Four-way streak-plate technique 31  yet to master the necessary lab skills that would allow them to use the rapid method Heavy confluent listed above. This alternative method involves growth spreading a loopful of culture over the surface of an agar plate that has the quadrants laid out Heavy growth visibly for quick reference. Refer to Figure 2.3, which illustrates this technique. Discrete colonies a. Using a marker, draw two bisecting lines on the bottom of the Petri dish to divide the plate into 4 equal parts. Label each Light growth quadrant 1 through 4, starting with the top right quadrant and labeling counter­ clockwise. Sterilizing the loop at the points Figure 2.2 Four-way streak-plate inoculation indicated is to dilute the culture due to with Serratia marcescens fewer organisms available to be streaked into each area, resulting in the final desired separation. d. Without reflaming the loop, again turn the dish 90° and then drag the culture b. Turn the Petri dish over and place a loopful from a corner of Area 3 across Area 4, of culture on the agar surface in quadrant 1. using a wider streak. Don’t let the loop Using the edge of the loop and holding touch any of the previously streaked the loop at a shallow angle so as not to areas. The flaming of the loop at the points gouge the agar, quickly spread the bacteria indicated is to dilute the culture so that throughout the quadrant. fewer organisms are streaked in each area, c. Reflame and cool the loop, and turn resulting in the final desired separation. the Petri dish 90°. Then touch the loop A photograph of a streak­plate inoculation into an area that has been streaked in is shown in Figure 2.2. quadrant 1 and drag it across the agar 2. An alternative streak­plate method is for into quadrant 2, repeat this twice without students new to the laboratory who have flaming the loop. (a) Label bottom of dish View through agar 2 1 4 3 2 1 4 3 (b) 3 4 1 2 1 2 2 3 2 1 4 3 4 3 1 4 Figure 2.3 Alternate streak-plate method 32 Experiment 2  d. Reflame and cool the loop and again turn the dish 90°. Streak the bacteria into quadrant 3 C L I n I C A L A P P L I C AT I o n in the same manner used for quadrant 2. Isolation of Cultures as a Diagnostic e. Reflame and cool the loop and again turn the Technique dish 90°. Streak the bacteria into quadrant 4 The isolation of pure cultures is the most important in the same manner used for quadrant 3. diagnostic tool used in a clinical or research 3. The spread-plate technique requires that laboratory to uncover the cause of an infection a previously diluted mixture of microorgan­ or disease. Before any biochemical or molecular isms be used. During inoculation, the cells techniques may be used to identify or characterize are spread over the surface of a solid agar the causative organism, an individual bacterial medium with a sterile, L­shaped bent glass rod colony must be isolated for testing. The isolation while the Petri dish is spun on a “lazy Susan” of Staphylococcus aureus from cultures taken from turntable. The step­by­step procedure for this abscesses or Streptococcus pyogenes from a throat technique is as follows: culture are two examples of clinical applications a. Place the bent glass rod into a beaker and of this technique. add a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion. b. Place an appropriately labeled nutrient agar plate on the turntable. With a sterile pipette, place one drop of sterile water on the center of the plate, followed by a sterile loopful of Micrococcus luteus. Mix gently AT T hE BE nCh with the loop and replace the cover. c. Remove the glass rod from the beaker, and pass it through the Bunsen burner flame Materials with the bent portion of the rod pointing downward to prevent the burning alcohol Cultures from running down your arm. Allow the 24­ to 48­hour nutrient broth cultures of a alcohol to burn off the rod completely. mixture of one part Serratia marcescens and Cool the rod for 10 to 15 seconds. three parts Micrococcus luteus and a mixture d. Remove the Petri dish cover and spin the of one part Escherichia coli and ten parts turntable. Micrococcus luteus. e. While the turntable is spinning, lightly touch Sources of mixed cultures from the environ­ the sterile bent rod to the surface of the ment could include cultures from a tabletop, agar and move it back and forth. This will bathroom sink, water fountain, or inside of an spread the culture over the agar surface. incubator. Each student should obtain a mixed culture from one of the environmental sources f. When the turntable comes to a stop, replace listed above. the cover. Immerse the rod in alcohol and reflame. g. In the absence of a turntable, turn the Media Petri dish manually and spread the culture Three Trypticase™ soy agar plates per designated with the sterile bent glass rod. student group for each inoculation technique to be performed. 4. The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette. The diluted inoculum is Equipment then added to a molten agar medium in a Microincinerator or Bunsen burner, inoculating Petri dish, mixed, and allowed to solidify. loop, turntable, glassware marking pencil, culture The serial dilution and pour­plate procedures tubes containing 1 ml of sterile water, test tube are outlined in Experiment 18. rack, and sterile cotton swabs. Experiment 2 33 Procedure Lab One Tips for SucceSS 1. Following the procedures previously described, 1. an isolation plate has isolated distinct, prepare a spread­plate and/or streak­plate individual colonies. If your technique results inoculation of each test culture on an in isolated colonies in a quadrant that was not appropriately labeled plate. the last one to be streaked, that is okay. The 2. Prepare an environmental mixed culture. point of using this method is to get those indi- vidual colonies somewhere on the plate. a. Dampen a sterile cotton swab with sterile 2. Pay attention to how well you sterilize your water. Wring out the excess water by loop and maintain your aseptic technique. pressing the wet swab against the walls If the loop is not properly sterilized between of the tube. streaks, or your aseptic technique is not b. With the moistened cotton swab, obtain maintained, the resulting plate will not exhibit your mixed­culture specimen from one of a decrease in bacteria leading to individual the selected environmental sources listed colonies. With that in mind, if a plate you have in the section on cultures. streaked or poured does not exhibit a decrease c. Place the contaminated swab back into in bacterial colonies area-to-area, you may the tube of sterile water. Mix gently and let want to re-examine your technique for main- stand for 5 minutes. taining sterilization. d. Perform spread­plate and/or streak­plate inoculation on an appropriately labeled plate. 3. Incubate all plates in an inverted position PART B Isolation of Pure for 48 to 72 hours at 25°C. Cultures from a Spread-Plate or Streak-Plate Preparation Procedure Lab Two 1. Examine all agar plate cultures to identify the LearnIng OBjeCTIve distribution of colonies. In the charts provided Once you have completed this experiment, in Part A of the Lab Report, complete the you should be able to following: 1. Prepare a stock culture of an organism a. Draw the distribution of colonies appearing on each of the agar plate cultures. using isolates from mixed cultures prepared on an agar streak plate and/or b. On each of the agar plate cultures, spread plate. select two discrete colonies that differ in appearance. Using Figure 3.1 on page 42 as a reference, describe each colony as to its Form: Circular, irregular, or spreading. Principle Elevation: Flat, slightly raised, or markedly Once discrete, well­separated colonies develop raised. on the surface of a nutrient agar plate culture, each may be picked up with a sterile needle and Pigmentation. transferred to separate nutrient agar slants. Each Size: Pinpoint, small, medium, or large. of these new slant cultures represents the growth 2. Retain the mixed­culture plates to perform of a single bacterial species and is designated as Part B of this experiment. a pure or stock culture. 34 Experiment 2  C L I n I C A L A P P L I C AT I o n Media Four Trypticase™ soy agar slants per designated Transferring a Colony of Bacteria student group. Daughter Cells For identification of a bacterial pathogen, a discrete Equipment bacterial colony must be transferred from a streak Microincinerator or Bunsen burner, inoculating or spread plate to the new testing media. This needle, and glassware marking pencil. new culture will consist of daughter cells that are genetic and metabolic clones of the original bacte- rial cells that were transferred to the plate. This Procedure Lab One will allow for identification of the unknown bacte- 1. Aseptically transfer, from visibly discrete rial species through its biochemical and molecular colonies, the yellow M. luteus, the white characteristics. E. coli, the red S. marcescens, and a discrete colony from the environmental agar plate specimen to the appropriately labeled agar slants as shown in Figure 2.4. 2. Incubate all slants at 37°C for 18 to 24 hours. AT T h E B E n C h Procedure Lab Two 1. In the chart provided in Part B of the Lab Materials Report, complete the following: a. Draw and indicate the type of growth of Cultures each pure­culture isolate, using Figure 3.1 on page 42 as a reference. Mixed­culture, nutrient agar streak­plate and/or spread­plate preparations of S. marcescens and b. Observe the color of the growth and record M. luteus, M. luteus and E. coli, and the environ­ its pigmentation. mental specimen plate from Part A. c. Indicate the name of the isolated organisms. Experiment 2 35  PROCEDURE 1 Flame the straight needle until the entire 2 After isolating a discrete colony on the agar streak wire is red. plate, touch the straight needle to the surface of the selected colony. 3 Uncap the agar slant and pass the neck of the 4 Inoculate the slant by drawing the needle upward tube rapidly over the Bunsen burner flame. in a zigzag motion along the surface of the agar. Do not dig into the agar. 5 Flame the neck of the tube and recap. 6 Flame the inoculating needle. Figure 2.4 Procedure for the preparation of a pure culture 36 Experiment 2  E xP E R IMEnT Cultural Characteristics of Microorganisms 3 3. Optical characteristics: Optical charac­ LearnIng OBjeCTIve teristics may be evaluated on the basis of Once you have completed this experiment, the amount of light transmitted through the you should be able to growth. These characteristics are described as opaque (no light transmission), translucent 1. Determine the cultural characteristics of (partial transmission), or transparent (full microorganisms as an aid in identifying transmission). and classifying organisms into taxonomic 4. Form: The appearance of the single­line groups. streak of growth on the agar surface is designated as: a. Filiform: Continuous, threadlike growth with smooth edges. b. Echinulate: Continuous, threadlike Principle growth with irregular edges. When grown on a variety of media, microorgan­ c. Beaded: Nonconfluent to semiconfluent isms will exhibit differences in the macroscopic colonies. appearance of their growth. These differences, d. Effuse: Thin, spreading growth. called cultural characteristics, are used as a e. Arborescent: Treelike growth. basis for separating microorganisms into taxo­ nomic groups. The cultural characteristics for all f. Rhizoid: Rootlike growth. known microorganisms are contained in Bergey’s 5. Consistency: Manual of Systematic Bacteriology. They are a. Dry: Free from moisture. determined by culturing the organisms on nutri­ ent agar slants and plates, in nutrient broth, and b. Buttery: Moist and shiny. in nutrient gelatin. The patterns of growth to be c. Mucoid: Slimy and glistening. considered in each of these media are described below, and some are illustrated in Figure 3.1. nutrient Agar Plates These demonstrate well­isolated colonies and are nutrient Agar Slants evaluated in the following manner: These have a single straight line of inoculation 1. Size: Pinpoint, small, moderate, or large. on the surface and are evaluated in the following manner: 2. Pigmentation: Color of colony. 3. Form: The shape of the colony is described 1. Abundance of growth: The amount of as follows: growth is designated as none, slight, moderate, or large. a. Circular: Unbroken, peripheral edge. 2. Pigmentation: Chromogenic microorganisms b. Irregular: Indented, peripheral edge. may produce intracellular pigments that are c. Rhizoid: Rootlike, spreading growth. responsible for the coloration of the organisms 4. Margin: The appearance of the outer edge as seen in surface colonies. Other organisms of the colony is described as follows: produce extracellular soluble pigments a. Entire: Sharply defined, even. that are excreted into the medium and also produce a color. Most organisms, however, are b. Lobate: Marked indentations. nonchromogenic and will appear white to gray. c. Undulate: Wavy indentations. 41  Crateriform Napiform Infundibuliform Saccate Stratiform (a) Gelatin liquefaction Uniform fine turbidity Flocculent growth Forms Circular Rhizoid Irregular Flat Entire Raised Lobate Undulate Convex Pellicle Sediment Serrate Filamentous Umbonate Margins Elevation (b) Colonies on agar plates (c) Growth in broth media Filiform Echinulate Beaded Effuse Arborescent Rhizoid (d) Growth on agar slants Figure 3.1 Cultural characteristics of bacteria 42 Experiment 3  d. Serrate: Toothlike appearance. e. Filamentous: Threadlike, spreading AT T hE BE nCh edge. 5. Elevation: The degree to which colony growth is raised on the agar surface is described as follows: Materials a. Flat: Elevation not discernible. Cultures b. Raised: Slightly elevated. Twenty­four–hour nutrient broth cultures of c. Convex: Dome­shaped elevation. Pseudomonas aeruginosa BsL -2 , Bacillus d. Umbonate: Raised, with elevated convex cereus, Micrococcus luteus, and Escherichia coli. central region. Seventy­two– to 96­hour Trypticase™ soy broth culture of Mycobacterium smegmatis. nutrient Broth Cultures These are evaluated as to the distribution and Media appearance of the growth as follows: Per designated student group: five each of nutri­ ent agar slants, nutrient agar plates, nutrient broth 1. Uniform fine turbidity: Finely dispersed tubes, and nutrient gelatin tubes. growth throughout. 2. Flocculent: Flaky aggregates dispersed Equipment throughout. Microincinerator or Bunsen burner, inoculating 3. Pellicle: Thick, padlike growth on surface. loop and needle, and glassware marking pencil. 4. Sediment: Concentration of growth at the bottom of broth culture may be granular, flaky, or flocculent. Procedure Lab One 1. Using aseptic technique, inoculate each of the nutrient Gelatin appropriately labeled media listed below in This solid medium may be liquefied by the the following manner: enzymatic action of gelatinase. Liquefaction a. Nutrient agar slants: With a sterile needle, occurs in a variety of patterns: make a single­line streak of each of the 1. Crateriform: Liquefied surface area is cultures provided, starting at the butt and saucer­shaped. drawing the needle up the center of the slanted agar surface. 2. Napiform: Bulbous­shaped liquefaction at surface. b. Nutrient agar plates: With a sterile loop, pre­ pare a streak­plate inoculation of each of the 3. Infundibuliform: Funnel­shaped. cultures for the isolation of discrete colonies. 4. Saccate: Elongated, tubular. c. Nutrient broth cultures: Using a sterile 5. Stratiform: Complete liquefaction of the loop, inoculate each organism into a tube upper half of the medium. of nutrient broth. Shake the loop a few times to dislodge the inoculum. d. Nutrient gelatin: Using a sterile needle, prepare a stab inoculation of each of the C L I n I C A L A P P L I C AT I o n cultures provided. Examining Colony Growth Characteristics 2. Incubate all cultures at 37°C for 24 to 48 hours. to Aid Identification Bacterial species each have a characteristic pattern of colony growth in a liquid culture or on Procedure Lab Two a solid medium. While not truly a diagnostic tool, 1. Before beginning observation of all the cul­ recognition of these patterns of characteristics will tures, place the gelatin cultures in a refrigera­ aid in a clinical lab setting by helping to minimize tor for 30 minutes or in a beaker of crushed the list of potential bacterial species to test for. ice for a few minutes. The gelatin culture will be the last to be observed. Experiment 3 43 2. Refer to Figure 3.1 on page 42 and the descrip­ Record your observations in the chart pro­ tions presented in the introductory section vided in the Lab Report. of Experiment 3 while making the following c. Nutrient broth cultures: Observe each of the observations: nutrient broth cultures for the appearance a. Nutrient agar slants: Observe each of of growth (flocculation, turbidity, sediment, the nutrient agar slant cultures for the or pellicle). Record your observations in the amount, pigmentation, form, and consis­ chart provided in the Lab Report. tency of the growth. Record your obser­ d. Nutrient gelatin: Remove gelatin cultures vations in the chart provided in the Lab from the refrigerator or beaker of crushed Report. ice, and observe whether liquefaction of b. Nutrient agar plates: Observe a single, the medium has developed and whether the well­isolated colony on each of the nutri­ organism has produced gelatinase. Record ent agar plate cultures and identify its size, your observations in the chart provided in elevation, margin, form, and pigmentation. the Lab Report. 44 Experiment 3  PArT 3 Bacterial staining LearnIng OBjeCTIves Once you have completed the experiments in this section, you should be familiar with 1. The chemical and theoretical basis of biological staining. 2. Manipulative techniques of smear preparation. 3. Procedures for simple staining and negative staining. 4. The method for performing differential staining procedures, such as the Gram, acid-fast, capsule, and spore stains. Introduction The ability of a stain to bind to macromo- lecular cellular components such as proteins or Visualization of microorganisms in the living nucleic acids depends on the electrical charge state is quite difficult, not only because they are found on the chromogen portion, as well as on the minute, but also because they are transparent cellular component to be stained. and practically colorless when suspended in an Acidic stains are anionic, which means that, aqueous medium. To study their properties and to on ionization of the stain, the chromogen portion divide microorganisms into specific groups exhibits a negative charge and therefore has a for diagnostic purposes, biological stains strong affinity for the positive constituents of the and staining procedures in conjunction with cell. Proteins, positively charged cellular compo- light microscopy have become major tools in nents, will readily bind to and accept the color of microbiology. the negatively charged, anionic chromogen of an Chemically, a stain (dye) may be defined as acidic stain. Structurally, picric acid is an example an organic compound containing a benzene ring of an acidic stain that produces an anionic chro- plus a chromophore and an auxochrome group mogen, as illustrated in Figure P3.3. (Figure P3.1). Basic stains are cationic, because on ioniza- The stain picric acid may be used to illustrate tion the chromogen portion exhibits a positive this definition (Figure P3.2). Benzene: Organic colorless solvent + Chromogen: Chromophore: Chemical group that imparts Colored compound, + color to benzene not a stain Stain Auxochrome: Chemical group that conveys the property of ionization to the chromogen, enabling it to form salts and bind to fibers or tissues Figure P3.1 Chemical composition of a stain 63  H H OH H H O2N NO2 O2 N NO2 + 3NO2– + OH– H H H H H H H NO2 NO2 Benzene Nitro groups Trinitrobenzene Auxochrome Trinitrohydroxybenzene colorless chromophore chromogen, yellow (picric acid) yellow stain in color due to the presence of chromophores Figure P3.2 Chemical formation of picric acid – OH O O2N NO2 O2N NO2 + Ionization + H H H H H NO2 NO2 Picric acid Anionic chromogen Figure P3.3 Picric acid: an acidic stain + N N – Ionization + Cl (CH3 )2 N S N(CH3 )2 Cl (CH3 )2 N N(CH3 )2 S Methylene blue Cationic chromogen Figure P3.4 Methylene blue: a basic stain charge and therefore has a strong affinity for the Basic stains are more commonly used for bac- negative constituents of the cell. Nucleic acids, terial staining. The presence of a negative charge on negatively charged cellular components, will read- the bacterial surface acts to repel most acidic stains ily bind to and accept the color of the positively and thus prevent their penetration into the cell. charged, cationic chromogen of a basic stain. Numerous staining techniques are available Structurally, methylene blue is a basic stain that for visualization, differentiation, and separation of produces a cationic chromogen, as illustrated in bacteria in terms of morphological characteristics Figure P3.4. and cellular structures. A summary of commonly Figure P3.5 is a summary of acidic and basic used procedures and their purposes is outlined in stains. Figure P3.6. 64 Part 3  Sodium, potassium, calcium, or ammonium salts of colored acids Acidic ionize to give a negatively charged chromogen Stains The chloride or sulfate salts of colored bases ionize to give a Basic positively charged chromogen Figure P3.5 Acidic and basic stains Simple staining: For visualization of morphological shape (cocci, Use of single stain bacilli, and spirilli) and arrangement (chains, clusters, pairs, and tetrads) Types of staining techniques Gram stain Separation into groups Acid-fast stain Differential staining: Use of two contrasting Flagella stain stains Visualization of Capsule stain structures Spore stain Nuclear stain Figure P3.6 Staining techniques Part 3 65  E xP E r iMEnt Preparation of Bacterial Smears 6 Note: Smears require only a small amount LearnIng OBjectIve of the bacterial culture. A good smear is Once you have completed this experiment, one that, when dried, appears as a thin whit- you should be able to ish layer or film. The print of your textbook should be legible through the smear. Different 1. Prepare bacterial smears for the techniques are used depending on whether the microscopic visualization of bacteria. smear is made from a broth- or solid-medium culture. a. Broth cultures: Resuspend the culture by tapping the tube with your finger. Depending on the size of the loop, one or Principle two loopfuls should be applied to the center Bacterial smears must be prepared prior to the of the slide with a sterile inoculating loop execution of any of the staining techniques listed and spread evenly over an area about the in Figure P3.6 on page 65. Although not dif- size of a dime. Set the smears on the labora- ficult, the preparation requires adequate care. tory table and allow to air-dry. Meticulously follow the rules listed below. b. Cultures from solid medium: Organisms 1. Preparation of the glass microscope cultured in a solid medium produce thick, slide: Clean slides are essential for the dense surface growth and are not ame- preparation of microbial smears. Grease or oil nable to direct transfer to the glass slide. from the fingers on slides must be removed These cultures must be diluted by placing by washing the slides with soap and water or one or two loopfuls of water on the cen- scouring powders such as Bon Ami®, followed ter of the slide in which the cells will be by a water rinse and a rinse of 95% alcohol. emulsified. Transfer of the cells requires After cleaning, dry the slides and place them the use of a sterile inoculating loop or a on laboratory towels until ready for use. Note: needle, if preferred. Only the tip of the Remember to hold the clean slides by their loop or needle should touch the culture edges. to prevent the transfer of too many cells. 2. Labeling of slides: Proper labelling of the Suspension is accomplished by spreading slide is essential. The initials of the organism the cells in a circular motion in the drop of can be written on either end of the slide with water with the loop or needle. This helps a glassware marking pencil on the surface on to avoid cell clumping. The finished smear which the smear is to be made. Ensure that the should occupy an area about the size of a label does not come into contact with staining nickel and should appear as a translucent, reagents. or semitransparent, confluent whitish film Figure 6.1. At this point the smear should 3. Preparation of smear: It is crucial to avoid be allowed to dry completely. Note: Do not thick, dense smears. A thick or dense smear blow on slide or wave it in the air. occurs when too much of the culture is used in its preparation, which concentrates a large 4. Heat fixation: Unless fixed on the glass number of cells on the slide. This type of prep- slide, the bacterial smear will wash away dur- aration diminishes the amount of light that can ing the staining procedure. This is avoided pass through and makes it difficult to visualize by heat fixation, during which the bacte- the morphology of single cells. rial proteins are coagulated and fixed to the 67  At t hE BE nCh Materials Cultures Twenty-four–hour nutrient agar slant culture of Bacillus cereus and a 24-hour nutrient broth cul- Figure 6.1 A bacterial smear following fixation ture of Staphylococcus aureus BSL -2. Equipment Glass microscope slides, microincinerator or glass surface. Heat fixation is performed by Bunsen burner, inoculating loop and needle, and the rapid passage of the air-dried smear two glassware marking pencil. or three times over the flame of the Bunsen burner or in front of a microincinerator. While many texts will discuss the use of a Bunsen burner for sterilization and heat fixation, gov- Procedure erning bodies such as the American Society for Microbiology (ASM) have changed the pro- Smears from a Broth Medium scribed methods for heat fixation and bench Label three clean slides with the initials of the top sterilization to utilize a microincinerator organism, and number them 1, 2, and 3. Resuspend instead of a Bunsen burner to reduce the pos- the sedimented cells in the broth culture by tap- sibility of aerosolization of bacteria on the ping the culture tube with your finger. The next slide or loop. four steps of this procedure are illustrated in The preparation of a bacterial smear is illus- Figure 6.2a and c: trated in Figure 6.2. 1. With a sterile loop, place one loopful of culture on Slide 1, two loopfuls on Slide 2, and three loopfuls on Slide 3, respectively. 2. With a circular movement of the loop, spread C l i n i C A l A P P l i C At i o n the cell suspension into an area approximately the size of a dime. Proper Slide Preparation 3. Allow the slide to air-dry completely. This may Before any staining or visualization of a bacterial be done by placing the slide on a drying tray sample can take place, a proper smear must be attached to a microincinerator or by placing prepared. A smear that is too thick may give a false the slide on the bench. result due to retention of dye that should have been 4. Heat fix the preparation. Note: Pass the air- rinsed away or because the thickness may prevent dried slide in front of the entrance to the dye penetration. A smear that is too thin may have microincinerator or pass the slide through too few cells, increasing the time and energy to the outer portion of the Bunsen flame to pre- find the bacteria under magnification. Inconclusive vent overheating, which can distort the mor- results due to improperly prepared slides may have phology through plasmolysis of the cell wall. an impact on patient treatment and outcomes. Good smears are those that allow newsprint to be read Examine each slide for the confluent, whitish through the smear. film or haze and record your results in the Lab Report. 68 Experiment 6  PROCEDURE (a) From broth medium (b) From solid medium 1 Place one to two loopfuls of the cell suspension 1 Place one to two loopfuls of water on on the clean slide. the center of the slide. 2 With a circular movement of the loop, spread the 2 Transfer a small amount of the bacterial inoculum suspension into a thin area approximately the size from the slant culture into the drop of water. Spread of a dime. both into a thin area approximately the size of a nickel. (c) Fixation for solid and broth media 3 Allow the smear to air-dry. 4 While holding the slide at one end, quickly pass the smear over the flame of the Bunsen burner two to three times. Figure 6.2 Bacterial smear preparation Experiment 6 69 Smears from a Solid Medium Tips for SucceSS Label four clean slides with the initials of the 1. the bacterial smear should be heavy enough to organism. Label Slides 1 and 2 with an L for loop, leave a slight film but not so heavy that you can and Slides 3 and 4 with an N for needle. The next plainly see the bacteria without a microscope. four steps of this procedure are illustrated in Students sometimes err on the side of adding Figure 6.2b and c: too much bacteria to a slide to make sure there 1. Using a loop, place one to two loops of water will be “enough” bacteria there for later visual- on each slide. ization. This has the potential to interfere with 2. With a sterile loop, touch the entire loop to the later staining procedures and produce false culture and emulsify the cells in water on Slide results. 1. Then, with a sterile loop, just touch the tip 2. Heat fixing should warm the slide until it is of the loop to the culture and emulsify it in the hot to the touch but not to the point of burning. water on Slide 2. Repeat Steps 1 and 2 using a Overheating the slide during this step increases sterile inoculating needle on Slides 3 and 4. the potential for damaging the cells. Damaged cells do not retain stains and produce inconclu- 3. Allow all slides to air-dry completely. This may sive staining results. Underheating of the slide be done by placing the slide on a drying tray does not allow the cells to affix to the glass. attached to a microincinerator or by placing Resulting washes or stains will rinse the bac- the slide on the bench. teria off the glass, leaving few if any bacteria 4. Heat fix the preparation. Note: Pass the air- present for later viewing. dried slide in front of the entrance to the microincinerator or pass the slide through the outer portion of the Bunsen flame to prevent overheating, which can distort the morphology through plasmolysis of the cell wall. Examine each slide for the confluent, whitish film or haze and record your results in the Lab Report. 70 Experiment 6  E xP E r iMEnt Simple Staining 7 LearnIng OBjectIveS At t hE BE nCh Once you have completed this experiment, you should be able to 1. Perform a simple staining procedure. Materials 2. Compare the morphological shapes and arrangements of bacterial cells. Cultures Twenty-four–hour nutrient agar slant cultures of Escherichia coli and Bacillus cereus and a 24-hour nutrient broth culture of Staphylococcus aureus BSL -2. Alternatively, use the smears pre- pared in Experiment 6. Principle In simple staining, the bacterial smear is stained with a single reagent, which produces a distinc- Cocci are spherical in shape. tive contrast between the organism and its back- (a) Diplococcus Diplo = pair ground. Basic stains with a positively charged chromogen are preferred because bacterial (b) Streptococcus Strepto = chain nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the cationic chromogen. The purpose of (c) Staphylococcus Staphlyo = cluster simple staining is to elucidate the morphology and arrangement of bacterial cells (Figure 7.1). The most commonly used basic stains are methylene blue, crystal violet, and carbol fuchsin. (d) Tetrad Tetrad = packet of 4 (e) Sarcina Sarcina = packet of 8 C l i n i C A l A P P l i C At i o n Bacilli are rod-shaped. Quick and Simple Stain (a) Diplobacillus Diplo = pair Simple stains are relatively quick and useful meth- ods of testing for the presence of, determining the (b) Streptobacillus Strepto = chain shape of, or determining the numbers of bacteria present in a sample. Generally involving a single Spiral bacteria are rigid or flexible. staining step, simple staining methods are not (a) Vibrios are curved rods. considered differential or diagnostic and will have (b) Spirilla are helical and rigid. limited uses. However, this is a quick procedure for determining whether a clinical sample has the pres- (c) Spirochetes are helical and flexible. ence of a foreign bacterial pathogen. Figure 7.1 Bacterial shapes and arrangements 73 reagents PROCEDURE Methylene blue, crystal violet, and carbol fuchsin. Equipment Microincinerator or Bunsen burner, inoculating loop, staining tray, microscope, lens paper, bibu- lous (highly absorbent) paper, and glass slides. Procedure 1. Prepare separate bacterial smears of the organisms following the procedure described in Experiment 8. Note: All smears must be heat fixed prior to staining. 1 Place slide on the staining tray and flood the smear Simple Staining with methylene blue. Allow 1 to 2 minutes of exposure to the stain. The following steps are illustrated in Figure 7.2. 1. Place a slide on the staining tray and flood the smear with one of the indicated stains, using the appropriate exposure time for each: car- bol fuchsin, 15 to 30 seconds; crystal violet, 20 to 60 seconds; methylene blue (shown in Figure 7.2), 1 to 2 minutes. 2. Gently wash the smear with tap water to remove excess stain. During this step, hold the slide parallel to the stream of water; in this way you can reduce the loss of organisms from the preparation. 3. Using bibulous paper, blot dry, but do not wipe the slide. 2 Gently wash the smear with tap water. 4. Repeat this procedure with the remaining two organisms, using a different stain for each. 5. Examine all stained slides under oil immersion. 6. In the chart provided in the Lab Report, com- plete the following: a. Draw a representative field for each organ- ism. Refer to page 16 for proper drawing procedure. b. Describe the morphology of the organ- isms with reference to their shapes (bacilli, 3 Blot the slide dry with bibulous paper. cocci, spirilla) and arrangements (chains, clusters, pairs). Refer to the photographs in Figure 7.3. Figure 7.2 Simple staining procedure 74 Experiment 7  Diplobacilli (a) Bacilli and diplobacilli (rod-shaped) bacteria (b) Spirilla (spiral-shaped) bacteria (c) Cocci (spherical-shaped) bacteria: Staphylococcus Figure 7.3 Micrographs showing bacteria morphology Experiment 7 75  E xP E r iMEnt negative Staining 8 LearnIng OBjectIveS C l i n i C A l A P P l i C At i o n Once you have completed this experiment, Detecting Encapsulated invaders you should be able to The principle application of negative staining is to 1. Perform a negative staining procedure. determine if an organism possesses a capsule (a 2. Understand the benefit obtained from gelatinous outer layer that makes the microorgan- ism more virulent), although it can also be used visualizing unstained microorganisms. to demonstrate spore formation. The technique is frequently used in the identification of fungi such as Cryptococcus neoformans, an important infectious Principle agent found in bird dropping that is linked to menin- geal and lung infections in humans. Negative staining requires the use of an acidic stain such as India ink or nigrosin. The acidic stain, with its negatively charged chromogen, will not pen- etrate the cells because of the negative charge on the surface of bacteria. Therefore, the unstained cells are At t hE BE nCh easily discernible against the colored background. The practical application of negative staining is twofold. First, since heat fixation is not required and the cells are not subjected to the distorting Materials effects of chemicals and heat, their natural size and shape can be seen. Second, it is possible to observe Cultures bacteria that are difficult to stain, such as some spi- Twenty-four–hour agar slant cultures of rilla. Because heat fixation is not done during the Micrococcus luteus, Bacillus cereus, and other staining process, keep in mind that the organisms alternate bacterial cultures. are not killed and slides should be handled with care. Figure 8.1 shows a negative stain of bacilli. reagent Nigrosin. Equipment Microincinerator or Bunsen burner, inoculating loop, staining tray, glass slides, lens paper, and microscope. Procedure Steps 1–4 are illustrated in Figure 8.2. 1. Place a small drop of nigrosin close to one end of a clean slide. 2. Using aseptic technique, place a loopful of inoculum from the M. luteus culture in the Figure 8.1 negative staining: Bacilli (1000×) drop of nigrosin and mix. 79 3. Place a slide against the drop of suspended 5. Repeat Steps 1–4 for slide preparations of organisms at a 45° angle and allow the drop to the remaining cultures. spread along the edge of the applied slide. 6. Examine the slides under oil immersion, 4. Push the slide away from the drop of and record your observations in the Lab suspended organisms to form a thin smear. Report. Air-dry. Note: Do not heat fix the slide. PROCEDURE 1 Place a drop of nigrosin at one end of the slide. 2 Place a loopful of the inoculum into the drop of stain and mix with the loop. 45° 3 Place a slide against the drop of suspended organisms 4 Push the slide away from the previously spread drop at a 45° angle and allow the drop to spread along the of suspended organisms, forming a thin smear. Air-dry edge of the applied slide. the slide. Figure 8.2 negative staining procedure 80 Experiment 8  E xP E r iMEnt gram Stain 9 based on the difference in the chemical com- LearnIng OBjectIveS position of bacterial cell walls. Gram-positive Once you have completed this experiment, cells have a thick peptidoglycan layer, whereas you should understand the peptidoglycan layer in gram-negative cells is much thinner and surrounded by outer lipid- 1. The chemical and theoretical basis for containing layers. Peptidoglycan is mainly a differential staining procedures. polysaccharide composed of two chemical 2. The chemical basis for the Gram stain. subunits found only in the bacterial cell wall. 3. The procedure for differentiating between These subunits are N-acetylglucosamine and N-acetylmuramic acid. With some organisms, as two principal groups of bacteria: gram the adjacent layers of peptidoglycan are formed, positive and gram negative. they are cross-linked by short chains of peptides by means of a transpeptidase enzyme, resulting in the shape and rigidity of the cell wall. In the case Principle Differential staining requires the use of at least four chemical reagents that are applied sequen- tially to a heat-fixed smear. The first reagent is called the primary stain. Its function is to impart its color to all cells. The second stain is a mor- dant used to intensify the color of the primary stain. In order to establish a color contrast, the third reagent used is the decolorizing agent. Based on the chemical composition of cellular compo- nents, the decolorizing agent may or may not remove the primary stain from the entire cell or only from certain cell structures. The final reagent, the counterstain, has a contrasting color to that of the primary stain. Following decolorization, (a) Gram-positive stain of streptococci if the primary stain is not washed out, the coun- terstain cannot be absorbed and the cell or its components will retain the color of the primary stain. If the primary stain is removed, the decolor- ized cellular components will accept and assume the contrasting color of the counterstain. In this way, cell types or their structures can be distin- guished from each other on the basis of the stain that is retained. The most important differential stain used in bacteriology is the Gram stain, named after Dr. Hans Christian Gram. It divides bacterial cells into two major groups, gram positive and gram negative, which makes it an essential tool for classification and differentiation of microorgan- (b) Gram-negative stain of E. coli isms. Figure 9.1 shows gram-positive and gram-negative cells. The Gram stain reaction is Figure 9.1 Gram-stained cells 83 of gram-negative bacteria and several Decolorizing Agent of the gram-positive such as the Bacillus, the Ethyl Alcohol, 95% This reagent serves a dual cross-linking of the peptidoglycan layer is direct function as a protein-dehydrating agent and as a because the bacteria do not have short peptide lipid solvent. Its action is determined by two fac- tails. Early experiments have shown that a tors, the concentration of lipids and the thickness gram-positive cell denuded of its cell wall by of the peptidoglycan layer in bacterial cell walls. the action of lysozyme or penicillin will stain In gram-negative cells, the alcohol increases the gram-negative. porosity of the cell wall by dissolving the lipids in The Gram stain uses four different reagents. the outer layers. Thus, the CV-I complex can be Descriptions of these reagents and their mecha- more easily removed from the thinner and less nisms of action follow. Figure 9.2 shows the micro- highly cross-linked peptidoglycan layer. Therefore, scopic appearance of cells at each step of the the washing-out effect of the alcohol facilitates the Gram staining procedure. release of the unbound CV-I complex, leaving the cells colorless or unstained. The much thicker Primary Stain peptidoglycan layer in gram-positive cells is Crystal Violet (hucker’s) This violet stain is used responsible for the more stringent retention of the first and stains all cells purple. CV-I complex, as the pores are made smaller due to the dehydrating effect of the alcohol. Thus, the Mordant tightly bound primary stain complex is difficult to remove, and the cells remain purple. Note: Be care- Gram’s iodine This reagent serves not only as a ful not to over-decolorize the smear with alcohol. killing agent but also as a mordant, a substance that increases the cells’ affinity for a stain. The reagent does this by binding to the primary stain, Counterstain thus forming an insoluble complex. The resultant Safranin This is the final reagent, used to stain crystal-violet–iodine (CV-I) complex serves to pink those cells that have been previously decolor- intensify the color of the stain. At this point, all ized. Since only gram-negative cells undergo decol- cells will appear purple-black. orization, they may now absorb the counterstain. Primary stain Mordant Decolorizing agent Counterstain (a) Application (b) Application of (c) 95% alcohol wash: (d) Application of of crystal violet: Gram’s iodine: Gram-positive cells are safranin: All cells are purple. All cells are purple-black. purple; gram-negative Gram-positive cells are cells are colorless. purple; gram-negative cells are pink. Figure 9.2 Microscopic observation of cells following steps in the Gram staining procedure 84 Experiment 9 Gram-positive cells retain the purple color of the primary stain. The preparation of adequately stained smears At t hE BE nCh requires the following precautions: 1. The most critical phase of the procedure is the decolorization step, which is based on the ease with which the CV-I complex is released from Materials the cell. Remember that over-decolorization will result in loss of the primary stain, causing Cultures gram-positive organisms to appear gram nega- Twenty-four–hour nutrient agar slant cultures of tive. Under-decolorization, however, will not Escherichia coli, Staphylococcus aureus BSL -2 , completely remove the CV-I complex, caus- and Bacillus cereus. ing gram-negative organisms to appear gram positive. Strict adherence to all instructions will help remedy part of the difficulty, but indi- reagents vidual experience and practice are the keys to Crystal violet, Gram’s iodine, 95% ethyl alcohol, correct decolorization. and safranin. 2. It is imperative that, between applications of the reagents, slides need to be thoroughly washed under running water or water applied Equipment with an eyedropper. This removes excess Microincinerator or Bunsen burner, inoculating reagent and prepares the slide for application loop or needle, staining tray, glass slides, bibulous of the subsequent reagent. paper, lens paper, and microscope. 3. The best Gram-stained preparations are made with fresh cultures (i.e., not older than 24 hours). As cultures age, especially in the case of gram- positive cells, the organisms tend to lose their Procedure ability to retain the primary stain and may appear to be gram-variable; that is, some cells Smear Preparation will appear purple, while others will appear pink. 1. Obtain four clean glass slides. 2. Using aseptic technique, prepare a smear of C l i n i C A l A P P l i C At i o n each of the three organisms and on the remain- ing slide prepare a smear consisting of a mix- ture of S. aureus BSL -2 ,and E. coli. Do this Gram Staining: the First Diagnostic test by placing a drop of water on the slide, and The Gram stain is a diagnostic staining procedure then transferring each organism separately to that can be done on body fluids, tissue biopsies, the drop of water with a sterile, cooled loop. throat cultures, samples from abscesses when Mix and spread both organisms by means infection is suspected, and more. Clinically impor- of a circular motion of the inoculating loop. tant results are obtained much more rapidly from (Note: If bacteria are taken from a broth cul- staining than from culturing the specimen. The ture, the drop of water is not required. Place results of the Gram stain will aid a clinical lab in a loop of bacterial suspension directly on the determining which additional tests may be required glass slide.) for identification of the bacterial strain in question. 3. Allow smears to air-dry and then heat fix in the Once the bacterial gram type, shape, and orienta- usual manner. tion are determined, it expedites the appropriate choice of antibiotic needed to treat the patient. Experiment 9 85  PROCEDURE 1 Gently stain with crystal violet for 2 Gently wash off the stain with 3 Gently apply Gram's iodine for 1 minute. tap water. 1 minute. 4 Gently wash off the Gram's iodine 5 Add 95% alcohol drop by drop 6 Gently wash off the 95% alcohol with tap water. until the alcohol runs almost clear. with tap water. 7 Counterstain with safranin for 8 Gently wash off the safranin with 9 Blot dry with bibulous paper. 45 seconds. tap water. Figure 9.3 Gram staining procedure 86 Experiment 9 Gram Staining Tips for SucceSS The following steps are shown in Figure 9.3. 1. Proper slide preparation is key to successful 1. Gently flood smears with crystal violet and let staining. Incorrect heat fixation will affect the stand for 1 minute. number of bacteria that will be present during 2. Gently wash with tap water. staining. Fixation that was not hot enough or was too short will not allow the cells to adhere 3. Gently flood smears with the Gram’s iodine to the glass slide properly, and the cells will be mordant and let stand for 1 minute. rinsed away during the multiple stain and rinse 4. Gently wash with tap water. steps. Conversely, overheating will result in the 5. Decolorize with 95% ethyl alcohol. Note: Do destruction of the cells and cell debris adhered not over-decolorize. Add reagent drop by drop to the cell. Few, if any, cells will remain intact until the alcohol runs almost clear, showing for the staining process. only a blue tinge. 2. Timing of the decolorizing step may be the most 6. Gently wash with tap water. important step of the procedure. Over decolor- izing with an incorrect alcohol solution or from 7. Counterstain with safranin for 45 seconds. allowing the slide to decolorize too long, will 8. Gently wash with tap water. remove the CV-I complex by causing extensive 9. Blot dry with bibulous paper and examine damage to the cell membrane and cell wall, under oil immersion. even on a gram-positive cell. Alternatively, 10. As you observe each slide under oil immer- decolorizing for too short a time period will not sion, complete the chart provided in the Lab remove enough CV-I complexes. The safranin- Report. stained cells will appear to be darker in color, and could be mistaken for a light purple, gram- a. Draw a representative microscopic field. positive stained cell. b. Describe the cells according to their mor- 3. the age of the culture or colony being stained phology and arrangement. may impact the Gram stain results. The best c. Describe the color of the stained cells. Gram-stained preparations are made with fresh d. Classify the organism as to the Gram reac- cultures that are not older than 24 hours. As tion: gram positive or gram negative. cultures age, especially in the case of gram- positiv