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Stippling may be used to depict different aspects of cell structure e. The types of laboratory equipment and culture media needed to develop and maintain pure cultures. The types of microbial flora that live on the skin and the effect of hand washing on them.
The concept of aseptic technique and the procedures necessary for successful subculturing of microorganisms. Streak-plate and spread-plate inoculation of microorganisms in a mixed microbial population for subsequent pure culture isolation. Cultural and morphological characteristics of microorganisms grown in pure culture.
Introduction Microorganisms are ubiquitous. They are found in soil, air, water, food, sewage, and on body surfaces. In short, every area of our environment is replete with them.
The microbiologist separates these mixed populations into individual species for study. A culture containing a single unadulterated species of cells is called a pure culture. To isolate and study microorganisms in pure culture, the microbiologist requires basic laboratory apparatus and the application of specific techniques, as illustrated in Figure P1. Media The survival and continued growth of microorganisms depend on an adequate supply of nutrients and a favorable growth environment.
For survival, most microbes must use soluble low-molecular-weight substances that are frequently derived from the enzymatic degradation of complex nutrients. A solution containing these nutrients is a culture medium. Basically, all culture media are liquid, semisolid, or solid. A liquid medium lacks a solidifying agent and is called a broth medium.
A broth medium supplemented with a solidifying agent called agar results in a solid or semisolid medium. Agar, an extract of seaweed, is a complex carbohydrate composed mainly of galactose, and is without nutritional value.
Because of these properties, organisms, especially pathogens, can be cultivated at temperatures of A completely solid medium requires an agar concentration of about 1. A solid medium has the advantage that it presents a hardened surface on which microorganisms can be grown using specialized techniques for the isolation of discrete colonies.
Such a defined and well-isolated colony is a pure culture. Also, while in the liquefied state, solid media can be placed in test tubes, which are then allowed to cool and harden in a slanted position, producing agar slants. These are useful for maintaining pure cultures. Similar tubes that, following preparation, are allowed to harden in the upright position are designated as agar deep tubes. Agar deep tubes are used primarily for the study of the gaseous requirements of microorganisms.
The various forms of solid media are illustrated in Figure P1. In addition to nutritional needs, the environmental factors must also be regulated, including proper pH, temperature, gaseous requirements, and osmotic pressure. A more detailed explanation is presented in Part 4, which deals with cultivation of microorganisms; for now, you should simply bear in mind that numerous types of media are available.
Filtration Cellulose-acetate membrane filters with pore sizes in the range of 8. To achieve sterility, it is mandatory that you use sterile equipment and employ aseptic techniques when handling bacterial cultures.
Although a more detailed discussion is presented in Part 9, which describes the control of microorganisms, Figure P1. Culture Tubes and Petri Dishes Glass test tubes and glass or plastic Petri dishes are used to cultivate microorganisms.
A suitable nutrient medium in the form of broth or agar may 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 closures. Today most laboratories use sleevelike caps Morton closures made of metal, such as stainless steel, or heat-resistant plastics. The advantage of these closures over the cotton plug is that they are labor-saving and, most of all, slip on and off the test tubes easily. Petri dishes provide a larger surface area for 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 dishes are manufactured in various sizes to meet different experimental requirements.
For routine purposes, dishes approximately 15 cm in diameter are used. The sterile agar medium is dispensed to previously sterilized dishes from molten agar deep tubes containing 15 to 20 ml of medium, or from a molten sterile medium prepared in bulk and contained in , , and ml flasks, depending on the volume of medium required. Remember that after inoculation, Petri dishes are incubated in an inverted position top down to prevent condensation formed on the cover during solidification from dropping down onto the surface of the hardened agar.
Figure P1. Built-in ridges on tube closures and Petri dishes provide small gaps necessary for the exchange of air. Transfer Instruments Microorganisms must be transferred from one vessel to another or from stock cultures to various media for maintenance and study.
Such a transfer is called subculturing and must be carried out under aseptic conditions to prevent possible contamination. Wire loops and needles are made from inert metals such as Nichrome or platinum and are inserted into metal shafts that serve as handles.
They are extremely durable instruments and are easily sterilized by incineration in the blue hottest portion of the Bunsen burner flame. Bacteriological tube B. Screw cap C. Plastic closure D. Metal closure E. Nonabsorbent cotton a Test tube rack with tubes showing various closures c DeLong shaker flask with closure Figure P1. Pipettes are similar in function to straws; that is, they draw up liquids.
They are made of glass or plastic drawn out to a tip at one end and with a mouthpiece forming the other end. They are calibrated to deliver different volumes depending on requirements.
Pipettes may be sterilized in bulk inside canisters, or they may be wrapped individually in brown paper and sterilized in an autoclave or dry-heat oven. The proper procedure for the use of pipettes will be demonstrated by your instructor. Pipetting by mouth is not permissible! Pipetting is to be performed with the aid of mechanical pipette aspirators. However, a prime requirement for the cultivation of microorganisms is that they be grown at their optimum temperature.
An incubator is used to maintain optimum temperature during the necessary growth period. It resembles an oven and is thermostatically controlled so that temperature can be varied depending on the requirements of specific microorganisms. Most incubators use dry heat. Moisture is supplied by placing a beaker of water in the incubator during the growth period. A moist environment retards dehydration of the medium and thereby avoids misleading experimental results.
Its advantage is that it provides a rapid and uniform transfer of heat to the culture vessel, and its agitation provides increased aeration, resulting in acceleration of growth.
The single disadvantage of this instrument is that it can be used only for cultivation of organisms in a broth medium. It is also used as a repository for thermolabile solutions, antibiotics, serums, and biochemical reagents. The difference between the residential flora and transient flora found on skin surfaces.
The effect of hand washing on the reduction of organisms on the skin. The effectiveness of using soap alone or soap accompanied by surgical brushing. Principle Each day our hands come in contact with numerous objects and surfaces that are contaminated with microorganisms. These may include door handles, light switches, shopping carts, sinks, toilet seats, books, or even things like compost piles or body fluids, to name a few. The lack of adequate hand washing is a major vehicle in the transmission of microbial infection and disease.
The skin of a human being is sterile while in utero and first becomes colonized by a normal microbial flora at birth as it is passed through the birth canal. By the time you reach adulthood, your skin is calculated to contain 1,,,, , or one trillion, bacteria, most of which are found in the superficial layers of the epidermis and upper hair follicles. This normal flora of microorganisms is called the resident flora, the presence of which does not cause negative effects in healthy individuals.
In fact, it forms a symbiotic relationship with your skin, which is vital to your health. This beneficial relationship can change in patients who are immunocompromised, or when residential flora accidently gains entrance to the host via inoculating needles, indwelling catheters, lacerations, and the like. Microorganisms that are less permanent and present for only short periods are termed transient flora. This latter 1 flora can be removed with good hand washing techniques.
The resident flora is more difficult to remove because they are found in the hair follicles and covered by hair, oil, and dead skin cells that obstruct their removal by simple hand washing with soap. Surgical scrubbing is the best means for removal of these organisms from the skin. Surgical hand washing was introduced into medical practice in the mid-nineteenth century by the Hungarian physician Ignatz Semmelweis while working at an obstetric hospital in Vienna. He further observed that medical students examining patients and assisting in deliveries came directly from cadaver autopsy laboratories without stopping to wash their hands.
Upon his insistence, medical students and all medical personnel were required to wash their hands in a chloride of lime bleach solution before and after all patient contact. The cornerstone for the prevention of nosocomial infections is the meticulous hand washing and scrubbing of health care personnel. In the laboratory setting, your normal flora may contaminate patient samples and skew your result, leading to a misdiagnosis.
It is important for everyone in the lab to correctly wash their hands before and after handling biological materials. Media 4 nutrient agar plates per student pair Equipment Liquid antibacterial soap, 8 sterile cotton swabs, 2 test tubes of sterile saline, Bunsen burner, glass marking pencil, surgical hand brush, Quebec colony counter, stopwatch.
One student will become the washer and the other student the assistant. The washer must not wash hands before coming to the lab. The assistant will use the glass marking pencil to label the bottoms of the nutrient agar plates.
See Figure 1. The assistant will aseptically dip a sterile cotton swab into the first test tube of sterile saline. To do this: a. First light the Bunsen burner. Uncap the test tube; after removing the cap, keep the cap in your hand with the inner aspect of the cap pointed away from your palm. The cap must never be placed on the laboratory bench because doing so would compromise the aseptic procedure.
Flame the neck of the tube by briefly passing it through the flame of the Bunsen burner. Remove the tube from the flame and dip the swab in the tube, soaking it with saline. Avoid touching the sides of the tube with the swab. The assistant will then aseptically inoculate the half of the nutrient agar plate labeled R1 by streaking the far edge of the plate several times then making a zig zag streak only on the half labeled R1.
Caution: Do not gouge the surface of the agar plate. The assistant will turn on the tap on the lab sink, so that the washer can wash the right hand under warm running water, without soap, concentrating on the thumb rubbing the thumb over the right index and middle finger for one minute. The assistant will turn off the tap. The washer will shake off the excess water from the hand, but not blot dry. The assistant, using a new, dry not moistened with saline sterile cotton swab, will obtain a sample from the right thumb pad and inoculate the section of the nutrient agar plate labeled R2 in the same way that R1 was inoculated.
Repeat step 5 two more times, washing the thumb for 2 minutes and then 3 minutes, respectively. The assistant will use a new, dry sterile cotton swab each time, and will aseptically inoculate R3 and R4, respectively. See Table 1.
The assistant and washer will now move to the left hand. The assistant will apply one or two drops of liquid soap to the thumb and index finger and the washer will wash for 1 minute by rubbing the thumb over the index finger.
Rinse well. Shake off water from the hand but do not blot dry. The assistant will then use a dry, sterile cotton swab to obtain a sample from the washed thumb pad and inoculate L2. Repeat step 8 two more times, not only using soap but also scrubbing the thumb with a surgical brush, for 2 minutes and then 3 minutes, respectively. The washer will obtain the surgical brush and the assistant will add saline to the brush to dampen it, and then add one or two drops of soap to the thumb and also the brush.
Caution: Place the brush bristles up on a dry paper towel between washings. The assistant will use a new, dry sterile cotton swab each time, and will aseptically inoculate L3 and L4, respectively.
Refer back to Table 1. Procedure Lab Two Examine and record the amount of growth found on each nutrient agar plate. Results may be determined by two methods. Visually observe the presence of growth on the surface of each agar plate in each section. Percent Growth Reduction. Count the colonies that appear in each section of the agar plates using a Quebec colony counter.
Record the macroscopic observations in the chart below. Record the percent growth reduction in the chart below. Compare the effectiveness of hand washing with water, with soap, and with soap and surgical scrubbing. Experiment 1: Lab Report 11 2. How does the presence of residential flora influence the infectious process? How does hand washing affect residential versus transient flora? Carry out the technique for aseptic removal and transfer of microorganisms for subculturing.
Correctly sterilize inoculating instruments in the flame of a Bunsen burner. Correctly manipulate your fingers to remove and replace the test tube closure. Principle Microorganisms are transferred from one medium to another by subculturing. This technique is of basic importance and is used routinely in preparing and maintaining stock cultures, as well as in microbiological test procedures.
Microorganisms are always present in the air and on laboratory surfaces, benches, and equipment. They can serve as a source of external contamination and thus interfere with experimental results unless proper aseptic techniques are used during subculturing.
Described below are essential steps that you must follow for aseptic transfer of microorganisms. The complete procedure is illustrated in Figure 2. Label the tube to be inoculated with the name of the organism and your initials. Hold the stock culture tube and the tube to be inoculated in the palm of your hand, secure with your thumb, and separate the two tubes to form a V in your hand.
Sterilize an inoculating needle or loop by holding it in the hottest portion of the Bunsen burner flame, until the wire becomes red hot. Then, rapidly pass the upper portion of the handle through the flame. Once flamed, the loop is never put down but is held in the hand and allowed to cool for 10 to 20 seconds. Uncap the tubes by grasping the first cap with your little finger and the second cap with your next finger and lifting the closure upward.
Note: Once removed, these caps must be kept in the hand that holds the sterile inoculating loop or needle; thus, the inner aspects of the caps point away from the palm of the hand.
They must never be placed on the laboratory bench because doing so would compromise the aseptic procedure. After removing the closures, flame the necks and mouths of the tubes by briefly passing them through the flame two—three times rapidly. The sterile transfer instrument is further cooled by touching it to the sterile inside wall of the culture tube before removing a small sample of the inoculum.
Depending on the culture medium, a loop or needle is used for removal of the inoculum. Loops are commonly used to obtain a sample from a broth culture.
Either instrument can be used to obtain the inoculum from an agar slant culture by carefully touching the surface of the solid medium in an area exhibiting growth so as not to gouge the agar. A straight needle is always used when transferring microorganisms to an agar deep tube from both solid and liquid cultures. For a slant-to-broth transfer, obtain inoculum from the slant and lightly shake the loop or needle in the broth culture to dislodge the microorganisms.
For a broth-to-slant transfer, obtain a loopful of broth and place at the base of an agar slant medium. Lightly draw the loop over the hardened surface in a straight or zigzag line, from the base of the agar slant to the top. For a slant-to-agar deep transfer, obtain the inoculum from the agar slant. Insert a straight needle to the bottom of the tube in a straight line and rapidly withdraw along the line of insertion.
This is called a stab inoculation. Following inoculation, remove the instrument and reflame the necks of the tubes. Figure 2. Broth-to-slant transfer: Obtain a loopful of broth and place at base of slant. Withdraw the loop in a zigzag motion. Slant-to-agar deep transfer: Obtain inoculum from slant. Insert the needle to the bottom of the tube and withdraw along the line of insertion.
Replace the caps on the same tubes from which they were removed. Reflame the loop or needle to destroy any remaining organisms. In this experiment you will master the manipulations required for aseptic transfer of microorganisms in broth-to-slant, slant-to-broth, and slant-to-agar deep transfers.
The technique for transfer to and from agar plates is discussed in Experiment 3. A sterile inoculating needle or loop is the basic instrument of transfer. It is important that you keep in mind that transferring bacterial cultures requires aseptic or sterile techniques at all times, especially if you are working with pathogens. In short, do not contaminate what you are working with and do not contaminate yourself.
Media Per designated student group: one nutrient broth, one nutrient agar slant, and one nutrient agar deep tube. Equipment Bunsen burner, inoculating loop and needle, and glassware marking pencil. Procedure Lab One 1. Label all tubes of sterile media as described in the Laboratory Protocol section on page xv.
Following the procedure outlined and illustrated previously Figure 2. Examine all cultures for the appearance of growth, which is indicated by turbidity in the broth culture and the appearance of an orange-red growth on the surface of the slant and along the line of inoculation in the agar deep tube.
Record your observations in the chart provided in the Lab Report. Review Questions 1. Explain why the following steps are essential during subculturing: a.
Flaming the inoculating instrument prior to and after each inoculation. Holding the test tube caps in the hand as illustrated in Figure 2. Experiment 2: Lab Report 17 c. Cooling the inoculating instrument prior to obtaining the inoculum. Flaming the neck of the tubes immediately after uncapping and before recapping. Describe the purposes of the subculturing procedure.
Explain why a straight inoculating needle is used to inoculate an agar deep tube. There is a lack of orange-red pigmentation in some of the growth on your agar slant labeled S. Does this necessarily indicate the presence of a contaminant? Upon observation of the nutrient agar slant culture, you strongly suspect that the culture is contaminated. Outline the method you would follow to ascertain whether your suspicion is justified.
In the laboratory, these populations can be separated into pure cultures. These cultures contain only one type of organism and are suitable for the study of their cultural, morphological, and biochemical properties.
In this experiment, you will first use one of the techniques designed to produce discrete colonies. Colonies are individual, macroscopically visible masses of microbial growth on a solid medium surface, each representing the multiplication of a single organism. Once you have obtained these discrete colonies, you will make an aseptic transfer onto nutrient agar slants for the isolation of pure cultures.
Flame loop. The resulting diminution of the population size ensures that, following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to separate the different species.
The following are techniques that can be used to accomplish this necessary dilution: 1. The streak-plate method is a rapid qualitative isolation method.
It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate. Although many types of procedures are performed, the four-way, or quadrant, streak is described. Refer to Figure 3. Place a loopful of culture on the agar 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, and then drag it rapidly several times across the surface of Area 1.
Then touch the loop to a corner of the culture in Area 1 and drag it several times across the agar in Area 2. The loop should never enter Area 1 again. Streak Area 3 in the same manner as Area 2. The flaming of the loop at the points indicated is to dilute the culture so that fewer organisms are streaked in each area, resulting in the final desired separation.
A photograph of a streak-plate inoculation is shown in Figure 3. The spread-plate technique requires that a previously diluted mixture of microorganisms be used. The step-by-step procedure for this technique is as follows: a. 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 with the loop and replace the cover. Remove the glass rod from the beaker, and pass it through the Bunsen burner flame 20 Experiment 3 Figure 3. Allow the alcohol to burn off the rod completely. Cool the rod for 10 to 15 seconds. Remove the Petri dish cover and spin the turntable.
While the turntable is spinning, lightly touch the sterile bent rod to the surface of the agar and move it back and forth. This will spread the culture over the agar surface.
When the turntable comes to a stop, replace the cover. Immerse the rod in alcohol and reflame. In the absence of a turntable, turn the Petri dish manually and spread the culture with the sterile bent glass rod. The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette. The diluted inoculum is then added to a molten agar medium in a Petri dish, mixed, and allowed to solidify.
The serial dilution and pour-plate procedures are outlined in Experiment Before any biochemical or molecular techniques may be used to identify or characterize the causative organism, an individual bacterial colony must be isolated for testing. The isolation of Staphylococcus aureus from cultures taken from abscesses or Streptococcus pyogenes from a throat culture are two examples of clinical applications of this technique.
Prepare an environmental mixed culture. Dampen a sterile cotton swab with sterile water. Wring out the excess water by pressing the wet swab against the walls of the tube. With the moistened cotton swab, obtain your mixed-culture specimen from one of the selected environmental sources listed in the section on cultures. Place the contaminated swab back into the tube of sterile water.
Mix gently and let stand for 5 minutes. Materials Cultures Procedure Lab Two to hour nutrient broth cultures of a mixture of one part Serratia marcescens and three parts Micrococcus luteus and a mixture of one part Escherichia coli and ten parts Micrococcus luteus. For the spread-plate procedure, adjust the cultures to an absorbance A of 0. Sources of mixed cultures from the environment could include cultures from a table top, bathroom sink, water fountain, or inside of an incubator.
Each student should obtain a mixed culture from one of the environmental sources listed above. Examine all agar plate cultures to identify the distribution of colonies. In the charts provided in Part A of the Lab Report, complete the following: a.
Draw the distribution of colonies appearing on each of the agar plate cultures. On each of the agar plate cultures, select two discrete colonies that differ in appearance. Using Figure 4.
Elevation: Flat, slightly raised, or markedly raised. Size: Pinpoint, small, medium, or large. Equipment 2. Retain the mixed-culture plates to perform Part B of this experiment. Media Four Trypticase soy agar slants per designated student group. Principle Equipment Once discrete, well-separated colonies develop on the surface of a nutrient agar plate culture, each may be picked up with a sterile needle and transferred to separate nutrient agar slants.
Each of these new slant cultures represents the growth of a single bacterial species and is designated as a pure or stock culture. This new culture will consist of daughter cells that are genetic and metabolic clones of the original bacterial cells that were transferred to the plate. This will allow for identification of the unknown bacterial species through its biochemical and molecular characteristics. Aseptically transfer, from visibly discrete colonies, the yellow M.
Procedure Lab Two 1. In the chart provided in Part B of the Lab Report, complete the following: a. Draw and indicate the type of growth of each pure-culture isolate, using Figure 4. Observe the color of the growth and record its pigmentation. Indicate the name of the isolated organisms. Do not dig into the agar. Figure 3. Can a pure culture be prepared from a mixed-broth or a mixed-agar-slant culture?
Observation of a streak-plate culture shows more growth in Quadrant 4 than in Quadrant 3. Account for this observation. Experiment 3: Lab Report 27 3. Why is a needle used to isolate individual colonies from a spread plate or streak plate? How can you determine if the colony that you chose to isolate is a pure culture? Determine the cultural characteristics of microorganisms as an aid in identifying and classifying organisms into taxonomic groups.
Principle When grown on a variety of media, microorganisms will exhibit differences in the macroscopic appearance of their growth. These differences, called cultural characteristics, are used as a basis for separating microorganisms into taxonomic groups.
They are determined by culturing the organisms on nutrient agar slants and plates, in nutrient broth, and in nutrient gelatin. The patterns of growth to be considered in each of these media are described below, and some are illustrated in Figure 4.
Nutrient Agar Slants These have a single straight line of inoculation on the surface and are evaluated in the following manner: 1. Abundance of growth: The amount of growth is designated as none, slight, moderate, or large.
Pigmentation: Chromogenic microorganisms may produce intracellular pigments that are responsible for the coloration of the organisms as seen in surface colonies. Other organisms produce extracellular soluble pigments that are excreted into the medium and that also produce a color.
Most organisms, however, are nonchromogenic and will appear white to gray. Optical characteristics: Optical characteristics may be evaluated on the basis of the amount of light transmitted through the growth. These characteristics are described as opaque no light transmission , translucent partial transmission , or transparent full transmission. Form: The appearance of the single-line streak of growth on the agar surface is designated as a.
Filiform: Continuous, threadlike growth with smooth edges. Echinulate: Continuous, threadlike growth with irregular edges.
Beaded: Nonconfluent to semiconfluent colonies. Effuse: Thin, spreading growth. Arborescent: Treelike growth. Rhizoid: Rootlike growth. Consistency: a. Dry: Free from moisture. Buttery: Moist and shiny.
Mucoid: Slimy and glistening. Nutrient Agar Plates These demonstrate well-isolated colonies and are evaluated in the following manner: 1. Size: Pinpoint, small, moderate, or large. Pigmentation: Color of colony.
Form: The shape of the colony is described as follows: a. Circular: Unbroken, peripheral edge. Irregular: Indented, peripheral edge. Rhizoid: Rootlike, spreading growth. Margin: The appearance of the outer edge of the colony is described as follows: a. Entire: Sharply defined, even.
Lobate: Marked indentations. Undulate: Wavy indentations. Serrate: Toothlike appearance. Filamentous: Threadlike, spreading edge. Elevation: The degree to which colony growth is raised on the agar surface is described as follows: a. Flat: Elevation not discernible. Raised: Slightly elevated. Convex: Dome-shaped elevation.
Umbonate: Raised, with elevated convex central region. Nutrient Broth Cultures Media These are evaluated as to the distribution and appearance of the growth as follows: Per designated student group: five each of nutrient agar slants, nutrient agar plates, nutrient broth tubes, and nutrient gelatin tubes.
Uniform fine turbidity: Finely dispersed growth throughout. Flocculent: Flaky aggregates dispersed throughout. Pellicle: Thick, padlike growth on surface. Sediment: Concentration of growth at the bottom of broth culture may be granular, flaky, or flocculant. Nutrient Gelatin This solid medium may be liquefied by the enzymatic action of gelatinase.
Liquefaction occurs in a variety of patterns: 1. Crateriform: Liquefied surface area is saucer-shaped. Napiform: Bulbous-shaped liquefaction at surface. Infundibuliform: Funnel-shaped. Saccate: Elongated, tubular. Stratiform: Complete liquefaction of the upper half of the medium. While not truly a diagnostic tool, recognition of these patterns of characteristics will aid in a clinical lab setting by helping to minimize the list of potential bacterial species to test for.
Using aseptic technique, inoculate each of the appropriately labeled media listed below in the following manner: a. Nutrient agar slants: With a sterile needle, make a single-line streak of each of the cultures provided, starting at the butt and drawing the needle up the center of the slanted agar surface. Nutrient agar plates: With a sterile loop, prepare a streak-plate inoculation of each of the cultures for the isolation of discrete colonies.
Nutrient broth cultures: Using a sterile loop, inoculate each organism into a tube of nutrient broth. Shake the loop a few times to dislodge the inoculum. Nutrient gelatin: Using a sterile needle, prepare a stab inoculation of each of the cultures provided. Alright, now in this part of the article, you will be able to access the free PDF download of Microbiology: A Laboratory Manual PDF using our direct download link mentioned at the end of this article.
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If you feel that we have violated your copyrights, then please contact us immediately click here. You may send an email to madxperts [at] gmail. Save my name, email, and website in this browser for the next time I comment. Friday, February 3, Clean both with lens paper and Windex. Visualization of the single-celled bacteria in the unstained state is a challenging experience for beginning students of microbiology.
To lower the frustration level of students, the instructor should apprise them of the fact that differentiating living bacteria from microscopic debris is an arduous task. It may be helpful to the students for the instructor to prepare a demonstration to clarify the distinction between Brownian movement and bacterial motility.
As this may be the first time that students are required to transfer microorganisms from sterile cultures, it is important to stress the principles of aseptic transfer techniques. Review the steps as outlined in Experiment 2. This is a good opportunity to instruct the students in the proper procedures for the disposal of contaminated materials and equipment. The immersion of the hanging-drop slides into a container of disinfectant is recommended.
Minion, J. Comparison of LED and conventional fluorescence microscopy for detection of acid fast bacilli in a low-incidence setting. PLoS One, 6 7 :e Living microbial preparations are done to detect physiologic processes, such as motility and binary fission, and to observe their natural size and shape. Stained smears, on the other hand, distort the size, shape, and arrangement and allow you to view only dead organisms; thus, it is not possible to see motility.
True motility is a directional movement, while with Brownian movement, the microorganisms vibrate at a constant rate without progressing in any particular direction. True motility and uniformity in the shape of the particles can be used as criteria for differentiation of living organisms from debris. The distinction between viable and nonviable particles may not always be accurate, particularly when viewing the smaller life forms.
Similarities between refractive indices, size, shape, and their movement may preclude distinction between the particles. Bacteria are more difficult to observe in an unstained state because of their small size, the movement of cells caused by Brownian. The microorganisms used in this experiment have been selected to illustrate the size variations that exist in the microbial population. The approximate size of the protozoans is 1.
The prokaryotic bacterial cells are almost one-tenth the size of the erythrocytes. The above spectrum of cell types may be viewed with the light microscope. Viruses, subcellular particles, are measured in nanometers and can be seen only by means of an electron microscope.
Procedural Point to Emphasize The instructor should indicate that the critical step in this procedure is the alignment of the stage and ocular micrometers so that the lines on both coincide. A demonstration and individual assistance may be required. Cheadle, M. Sporocyst size of isolates of Sarcocystis shed by the Virginia opossum Didelphis virginiana. Veterinary Parasitology, 95 2—4 : — The same calibration factor cannot be used to determine the size of an organism under all objectives.
The stage micrometer is calibrated only for use with the oil-immersion objective. If a stage micrometer division contains 12 ocular divisions, the distance between two lines on the ocular micrometer is 0. The measurements of Bacillus subtilis in the stained and unstained state would not be the same. Heat fixation causes dehydration of the cells, and their measurements will be less than that of their native state. These initial staining exercises are designed to instruct students in the proper technique for the preparation of a bacterial smear, which is the prerequisite for all staining procedures.
In addition, the microscopic observation of the stained smears is intended to familiarize students with cellular morphology—the size, shape, and arrangement of bacteria. The performance of these experiments will also reinforce the use of the oil-immersion lens. Materials Both broth and agar slant cultures of the selected organisms should be available in order for students to gain experience in performing aseptic transfers and bacterial smear preparations from both types of cultures.
When heat fixing a smear, students should be instructed to pass the slide through the outer portion of the flame to prevent overheating the smear. Excess heat can distort the morphology through plasmolysis of the cell wall.
In performing the staining procedure, students are frequently afraid to sufficiently wash their slides following the application of each staining reagent. It should be emphasized that if the smear is heat fixed properly prior to the start of the staining procedure, then this fear is unfounded. Furthermore, they should be instructed to wash both sides of the slide under running water to remove all residual stain, as it may interfere with the microscopic recognition of the microorganisms.
As the students are still novices in aseptic transfer techniques, a review of this procedure is recommended. Agar cultures are used in this experiment to help the student prepare smears of the correct thickness.
A good smear should allow the student to read newsprint through the smear. Broth cultures, on the other hand, allow the student to view the morphological characteristics on the smear because the cells are widely separated and do not clump on the smear. Students should be cautioned to clean slides well. A dirty or greasy slide will produce a poor smear preparation because grease may prevent the smear from adhering to the glass. Likewise, grease may cause the suspension to coalesce and not spread evenly on the slide.
Dust particles on a slide might easily be mistaken for microorganisms. These slides could be saved and used in Experiment 9. Finished slides can be wrapped in paper toweling and secured with a rubber band. Students tend to use too much inoculum when preparing their bacterial smears from agar slant cultures. It should be stressed that a sufficient number of organisms will be obtained by touching the surface of the culture with a loop or needle without digging into the agar. It should also be mentioned that broth cultures.
Weinstein, R. Factitious meningitis. Diagnostic error due to nonviable bacteria in commercial lumbar puncture trays. Journal of the American Medical Association, 8 —9. Youssef, D. Negative image of blastomyces on diff-quik stain. Acta Cytologica, 55 4 — Basic dyes are used preferentially for bacterial staining because the chromogen is cationic and has an affinity for the negatively charged DNA.
Also, the bacterial cell surface generally has a negative charge, which attracts the basic stain. Simple staining procedures cannot be used for purposes other than the determination of cell morphology. The structural bacterial components are too small to be viewed with a simple light microscope.
Failure to heat fix the E. Heat is required to cause coagulation of bacterial proteins, which then adhere to the glass slide. The sparse number of remaining cells would not be readily discernible. The coffee-discolored laboratory coat is not permanently stained, and the color will wash out.
The reason for this is that the coffee is not a stain. It is only a chromogen and lacks the auxochrome component. Therefore, ionization cannot occur, and there will be no binding to the cloth fibers. Thick smears do not allow sufficient light to pass through the preparation for good visualization of the organisms.
Also, dense smears contain tightly packed and superimposed cells that do not lend themselves to accurate determination of cell shape and arrangement. Air-drying prevents the cells from shrinkage and distortion, thereby protecting their size and shape, and allows for the visualization of the natural cellular morphology. Excessive heating may distort the morphology, causing plasmolysis of the cell wall.
On the other hand, an improperly heat-fixed smear could wash off the slide. The presence of grease from fingers or any other exogenous source may interfere with the adherence of the culture to the slide and will result in the production of an unsatisfactory smear. The presence of dirt or dust on the glass surface will produce artifacts in the stained smear and serve as a source of confusion for the student viewing the organisms in the stained smear.
Negative staining is presented as an alternative technique to the hanging-drop procedure for the observation of living cells. 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.
Solutions Manual for Microbiology A Laboratory Manual 10th Edition by Cappuccino by zed08 - Issuu.
Unlike the sterile human skin found in utero, adult human skin is colonized by about one trillion bacteria, which constitute the normal residential and transient flora of the skin. The necessity for surgical washing of hands was introduced in the mid-nineteenth century, in Vienna, by Ignatz Semmelweis.
Semmelweis showed that hand washing prior to delivery decreased the incidence of puerperal fever child birth fever resulting in maternal mortality. Routine surgical scrubbing by surgeons is an essential practice for all surgical procedures in modern medicine. Although the skin is never completely sterilized, the residential and transient flora can be significantly reduced смотрите подробнее prolonged hand washing with soap and hot water.
Since students have not yet learned aseptic techniques, the instructor should demonstrate to the class the method used to inoculate sterile agar plates with sterile cotton swabs. Proper opening and closing of saline tubes after passing the lips of the tube through the Bunsen burner flame.
The flora of the skin microbilogy and residential is usually nonpathogenic and взято отсюда benefit the host by preventing transient pathogens from colonizing the skin surface. On the other hand, the residential flora is capable of causing skin diseases when they are able to enter the blood, especially in immunosuppressed people.
The residential and transient microorganisms mnaual the skin respond differently to hand washing. Transient flora are susceptible to antiseptics and are easily removed with hand washing. Residential organisms are more difficult to detach from the skin because of a layer of oil and entrapment in the hair follicles and dead skin cells that obstruct their removal by simple hand washing and require vigorous hand scrubbing with soap and water.
Tip This being the first laboratory session for students, the instructor should circulate through the laboratory and assist students who are having problems with dexterity and manipulation of equipment. Katz J. Hand washing and hand disinfection: More than your mother taught you. Anesthesiology Clinics of North America, 22 3 — The oil layer, dead cells, and organisms trapped in hair follicles prevent the removal of all microorganisms from the skin with water alone.
Soap helps to remove the oil and soap. Surgical gloves play a significant role in preventing cross-contamination of both the surgeon and patient. Surgeons wear gloves because it is impossible to remove all of the organisms from the skin even with the most vigorous hand washing. However, surgical gloves are not a substitute for hand washing.
Tears in gloves may also occur if fingernails, natural or artificial, are too long. Aseptic technique forms the basis for the successful manipulation of organisms in the microbiological laboratory.
The development of proper aseptic transfer manuxl can be acquired only through the repetitive performance of this task until the steps involved become second nature to the student. Microbiology a laboratory manual 10th edition pdf download accomplish this end, it is advisable to allow students to practice this technique using cultures and sterile media in various forms, microbiology a laboratory manual 10th edition pdf download.
The necessary manual dexterity required for the handling of culture tubes and closures while flaming inoculating instruments will be acquired through repetition. Beginning students in microbiology have difficulty appreciating the diminutive size of microorganisms. Thus, they have the tendency to procure excessive amounts of inoculum for transfer.
It should be stressed that the inoculating instrument needs only to touch /3154.txt growth, not to be dragged over the agar, to obtain a sufficient number of cells for the transfer. When broth cultures are used, the organisms must be suspended by vigorous tapping of the bottom of the tube. A single loopful will suffice for use as the inoculum.
It should be stressed that the transfer procedure should be performed manaul rapidly as possible. However, to ensure that viable cells are obtained from the stock culture, the hot loop or needle must be cooled by tapping it against the inner surface of the culture tube before securing the inoculum.
The students should be reminded that the entire inoculating wire must be microbiology a laboratory manual 10th edition pdf download until it turns red. Considering students are novices and lack the necessary manual dexterity at this point, it is wise for the instructor to circulate through the laboratory and assist students who are unable to manipulate the uncapping and recapping of culture tubes while holding the transfer instrument.
The purposes of the subculturing procedure are intended to establish a routine method for the transfer from one medium to another for the preparation and maintenance of stock cultures and to provide media fdition the performance продолжить microbiological test procedures.
A straight inoculating needle is used to inoculate an agar deep micrbiology in order to maintain the redox potential of the medium.
The absence of pigmentation on some S. This organism is capable of producing variants that may not produce any pigment. Thus, some colonies are red, while others are colorless. Also, the rate of pigment production may vary within one culture, producing a mixture of pigmented and nonpigmented microbiology a laboratory manual 10th edition pdf download. To determine the presence of contamination microbioology the S. Streak-plate preparations of both colonies may also be helpful for a comparison of cultural characteristics.
Lypson, M. An assessment tool for aseptic technique in resident physicians: A journey towards validation in the real world of limited supervision. The inoculating instrument is flamed prior to inoculation to prevent contamination of the stock cultures.
Flaming after inoculation prevents contamination of the laboratory table when the instrument is returned to the table. The test tube closures are held in the manner prescribed to maintain their sterility. Once removed, they must be kept between the fingers of the hand and never placed on the laboratory tabletop.
Insertion of a hot needle directly into or onto the culture laboeatory must not be done, as this will kill the cells. Flaming the neck of the test tube is a precaution intended to kill any organisms that might be present on the neck of the tube or the inner surface of the closure if the aseptic procedure has been compromised.
The purposes of this experiment are to instruct students in the preparation of pure cultures from a mixed microbial population and to compare the cultural characteristics of the resultant agar plate and agar slant cultures. Toward this end, students are first introduced to two methods that are used to separate microorganisms, namely the streakplate and spread-plate techniques.
Students should be made aware that the streak-plate technique is the most frequently used procedure for the separation of organisms from a mixed culture, whereas spreadplate sownload are used preferentially for the quantitation of cell populations. Glasson, J. Evaluation of an automated instrument for inoculating and spreading samples onto microbiology a laboratory manual 10th edition pdf download plates. Journal of Clinical Microbiology, 46 4 —4. Characterization of an Enterococcus faecium small-colony variant isolated /17646.txt blood culture.
International Journal microbiology a laboratory manual 10th edition pdf download Medical Microbiology, 1 —4. Students should be apprised of the following when performing the streak-plate procedure: a. Petri dish covers should never be completely removed; this will avoid exposing the medium увидеть больше the cover to exogenous contamination.
The cover should be raised and held at the smallest angle that is sufficient for the introduction of the inoculating wire, and it should be done only for as long as it takes to inoculate each designated area of the plate. It is essential /22364.txt the inoculating instrument be flamed and cooled microbiology a laboratory manual 10th edition pdf download to the inoculation of each area of the plate.
Once the inoculum is obtained from the previously streaked area, the loop or needle should not be passed over that area again during the streaking process. As this is the first time students are performing plate inoculations, they should be reminded of the fact that agar plate cultures are always incubated in an inverted position.
In this way, the culture is distributed evenly and should produce distinct discrete colonies. Optional Procedural Additions or Modifications Because of the time constraints in the laboratory, an expanded examination of cultural characteristics, as presented in Experiment 4, is frequently.
In order to gain an awareness of differences in cultural characteristics, it is suggested that students observe their culture preparations from Experiment 3 owners download manual ford 1993 aerostar note these variations. A pure culture can be obtained from a mixed culture only by first performing a streak-plate or spread-plate inoculation for the separation of the organisms into discrete colonies.
Microbiology a laboratory manual 10th edition pdf download Quadrant 4 of a streak-plate inoculation contains more growth than Quadrant 3, microgiology the inoculating wire was repeatedly dragged through Quadrant 3 nanual, more likely, microbiology a laboratory manual 10th edition pdf download entered Quadrant 1 during its inoculation.
The inoculating needle is the manual stand desk of choice to isolate individual discrete colonies because it is thin enough to touch the microbiology a laboratory manual 10th edition pdf download of the colony. The center of the colony is the best area for isolation and transfer to an agar slant as a subculture.
An inoculating loop is too imprecise and therefore unsatisfactory. The purity of a chosen colony may be determined by the following: a. Subculturing the isolate in microbiologu broth medium or on an agar slant medium b. Gram staining the subculture following incubation to verify its purity. Cultural characteristics are determined genetically for each particular organism. As such, these characteristics remain constant and are micdobiology.
This property of colonial constancy is important because it allows the microbiologist to use rownload macroscopic growth patterns as an aid in the identification of various microbial species. A standard descriptive vocabulary has been developed to describe the cultural and colonial appearance of microorganisms grown in artificial culture media.
A single cell dividing cessna maintenance free download binary fission on agar divides z of times, producing a single round colony. Its appearance is determined by fundamental characteristics, such as pigment production, type of cell wall, presence or absence of a capsule, and motility.
Because of environmental paboratory, growth patterns may not always coincide exactly with those illustrated in the figures in the manual. Gelatin cultures: For the rapid resolidification of liquefied gelatin, cultures should be refrigerated for about 30 minutes. This process can be expedited by placing liquefied tubes in a beaker of crushed ice узнать больше a few minutes to determine if gelatin microbiology a laboratory manual 10th edition pdf download liquefied.
Sutula, J. Culture media for differential isolation of Lactobacillus casei Shirota from oral samples. The compound microscope is an indispensable tool in the study of microbiology. However, the visualization of microorganisms, particularly prokaryotes, requires that microbiolog students become adept in editino use of the oilimmersion objective.
Materials Slides Stained slides of selected microorganisms, prepared by the instructor, may be substituted for the commercial slide preparations. Following their use, the immersion oil can be removed from the slides with the application of xylol and gentle blotting with lens paper. Students should be made aware of the fact that the microscope is an expensive piece of equipment, and больше на странице, proper care is required at all times.
To prevent damage to the microscope, it is important to emphasize the proper means of transporting it to and from the laboratory bench. Also, to maintain the instrument in proper working condition, students must check the objective lenses for the presence of residual oil at the start and end of. Xylol is never to be micdobiology by students for the removal of oil from the lens system of the microscope. In addition to the instructions in the manual for the proper use of the oil-immersion objective, the following are some suggestions that may be helpful to facilitate student use of this objective.
The body tube of a microscope is never lowered while looking through the ocular lens to ensure that the objective lens and the slide are not damaged by the forceful contact between the two. The iris diaphragm adjusts the amount of light coming through the specimen. The coarse adjustment is used to bring the specimen into view.
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