Microbiology a laboratory manual 10th edition pdf download

Download PDF. Download 1 , Download 2. These are never placed on the bench top. The pour-plate method should be demonstrated with emphasis on the gentle swirling of the molten agar so as to prevent the agar from sloshing onto the cover of the plate. Dilution refers to varying the concentration of a substance.

The dilution factor is expressed mathematically as the reciprocal of the dilution. The advantage of the serial dilution—agar plating procedure is that the cell count represents only viable cells. The disadvantage of this method is that it requires an incubation period that precludes the ability to obtain immediate results.

A variety of parameters can be used to measure cell growth chemically: for example, by an increase in protein and DNA concentrations or by determination of dry cell mass. Metabolic parameters can also be used: for example, the measurement of oxygen uptake by aerobic cells or the amount of carbon dioxide used by anaerobic organisms.

Dilution plates showing spreading colonial growth should be eliminated from the experimental data because they may be obscuring the presence of small colonial forms, and they represent the growth of more than a single colony. The spread-plate technique Experiment 3 may be substituted for the pour-plate preparation. The advantage of this is twofold: First, this procedure can be more rapidly performed, and second, only surface colonies will be present for counting.

Sanderson, M. Sensitivity of direct plating for detection of high levels of E. Current Microbiology, 55 2 — The primary purpose of this experiment is to help students to visualize the exponential growth dynamics of microorganisms in culture. Toward this end, students will use two parameters to measure growth.

The indirect method employs spectrophotometric absorbance A readings, while the direct method uses the serial dilution—agar plate method for the determination of viable cell counts. The procedure for harvesting aliquots for A determination and serial dilution for cell counts requires strict adherence to the prescribed time schedule, as outlined in the procedure section of the exercise.

To facilitate the efficient performance of these procedures within the designated time frames, a review of the use of the spectrophotometer and the serial dilution—pour-plate technique will be helpful. A demonstration and an explanation of the proper way to plot a graph using semilog paper and determination of the generation time by extrapolation from the plotted data should be presented.

In multicellular organisms, the term growth generally implies an increase in cell mass, whereas in bacteria, it implies an increase in cell number. Variations in generation time among different species of organisms have a genetic basis that governs the enzymatic utilization of substrates. Variations in generation time within a single species are dependent upon differences in the environmental and nutritional conditions. Optional Procedural Additions or Modifications To reduce the number of experimental manipulations, it is suggested that the spread-plate preparation Experiment 3 be substituted for the pour-plate procedure.

The inoculum must be composed of cells in midlog phase of growth 10—12 hours of incubation. These cultures may be placed into an ice-water bath to maintain this phase of growth for a short period of time. Peleg, M. Microbial growth curves: what the models tell us and what they cannot.

Critical Reviews in Food Science and Nutrition, 51 10 — Antibiotics would not be effective unless the abscess was incised and allowed to drain. It is only under these conditions that the antibiotic could penetrate the infected area and act on the exponentially growing cells. Generation time is a useful parameter to indicate the most suitable medium to support the growth of a specific organism. The medium in which the culture exhibited the shortest generation time would support the most abundant growth.

The identification of microorganisms in both the academic and clinical settings is dependent upon the detection of specific enzymes that produce biochemical transformations of substrates exogenously or endogenously. In this experiment, extracellular enzymatic activities will be studied. These exoenzymes hydrolyze environmental macromolecules into their basic building blocks for their transport into the cell.

Within the cell, these serve as metabolites for endoenzymatic activities. The presence of a specific exoenzyme is determined by the absence of its macromolecular substrate in the environment medium following the growth of the culture.

The use of dehydrated starch agar is less satisfactory. Milk agar is best prepared by using doublestrength nutrient agar autoclaved for 15 minutes at STP, and skim milk autoclaved for 10 minutes at 12 PSI. Juarez, Z. An extracellular protease of Streptococcus gordonii hydrolyzes type IV collagen and collagen analogues. Infection and Immunity, 67 1 —8. All biochemical reactions that occur in living cells are regulated by enzymes. Without the action of enzymes many of these reactions would not take place at perceptible rates.

All aspects of cellular metabolism are catalyzed by enzymes. This includes the digestion of large nutrient molecules such as proteins, carbohydrates, and lipids that are broken down into smaller molecules; the production and transformation of energy; and the synthesis of larger macromolecules essential for the viability of the cell.

Although milk is a sterile body fluid, microorganisms gain entry during the milking process. Many of these microorganisms contain enzymes that degrade milk carbohydrates, proteins, and lipids with the production of acid end products.

Organisms such as Lactobacillus and Streptococcus spp. They may produce enough acid to curdle the milk protein, forming a curd. Large, complex, highly branched polysaccharides, such as starch and cellulose, are macromolecules that cannot pass through the cell membrane and therefore must be degraded outside the cell by exoenzymes excreted by the cell.

Once degraded, these smaller molecules, such as disaccharides like lactose and sucrose, may pass through the cell membrane and be further degraded into lactose and galactose by endoenzymes of the cell. Procedural Point to Emphasize Gelatin Hydrolysis It is essential that the instructor impress upon the student the fact that the gelatin cultures must be refrigerated following their incubation and prior to their observation.

At this point in the semester, aseptic techniques should be routine for the students. Long and short versions are presented in all the experiments in this section of the manual. The long version provides the students with a comparative overview of the enzyme-dependent microbial activities on a variety of substrates.

Most of the test procedures are basic; therefore, several experiments can be readily performed during a single laboratory session by groups of four students. Carbohydrates serve as the major intracellular substrates for the production of cellular energy. Thus, one group of endoenzymes, the carbohydrases, is responsible for sustaining the bioenergetic activities in most microbes.

First, it helps students ascertain the ability of a microorganism to utilize carbohydrates as a principal energy source. Second, it enables them to determine which specific carbohydrates can be metabolized by a microorganism; this is dependent upon the presence of complementary intracellular carbohydrases.

Experimentally, carbohydrate utilization can be used as a parameter for the identification of microorganisms. This is accomplished by the action of a specific carbohydrase on the carbohydrate substrate with the production of a metabolic waste product that is then detectable in the medium.

A brief discussion of the fermentative processes with an emphasis on end-product formation would be helpful to the students. An explanation of the role of the pH indicator and the Durham tube in the carbohydrate broth medium for end-product detection should be presented.

Students should be cautioned that the presence of an acid in the medium is only discernible by the development of a canary yellow coloration throughout the culture medium.

Students need to know that, in some cases, they may obtain variable results, and this can be expected because of strain variations, incubation times, size of inoculum, or color reversion caused by refrigeration following incubation. Students should set the experimental carbohydrate media in a definite order in the test tube rack and label them carefully to prevent any mix-up, especially considering that all phenol red broths look alike.

Students should be reminded to flame the loop before and after each carbohydrate broth is inoculated. Prior to autoclaving the carbohydrates, an inverted Durham tube should be placed into each of the carbohydrate tubes.

Do not attempt to get the medium into the inverted tube; usually this tube floats on top of the medium. However, upon autoclaving, the medium will be forced up into the inverted Durham tube. The presence of a gas bubble in the Durham tube must be definite. Occasionally tiny bubbles in the gas vials may represent the loss of oxygen as the temperature changes in the medium from the refrigerator to the incubator. Zoetendal, E. The human small intestinal microbiota is driven by rapid uptake and conversion of simple carbohydrates.

Cellular respiration is a biooxidative process that occurs aerobically, with molecular oxygen serving as the final electron acceptor, or anaerobically, with an inorganic ion acting as a final electron acceptor. Fermentation is a biooxidation that utilizes an organic compound as the final electron acceptor.

Microorganisms differ in their use of pyruvic acid. Some organisms use the pyruvic acid as a final electron acceptor, resulting in the formation of acids, alcohols, and solvents. Other organisms use this compound as a stepping stone into the Krebs cycle for further ATP production. The strict anaerobes utilize the EmbdenMeyerhof glycolytic pathway to pyruvic acid with limited ATP production. The pyruvic acid is then further metabolized through fermentative pathways.

Pseudomonas species hydrolyze proteins to amino acids that then enter the Krebs cycle for generation of ATP. Clostridium perfringens is a saccharolytic organism and can utilize carbohydrates anaerobically; therefore, there is no evolution of carbon dioxide.

However, the proteolytic enzymes of its exotoxin degrade proteins with the evolution of hydrogen gas, which destroys body tissues with the further release of carbohydrates for the continued generation of ATP. The purpose of this experiment is to illustrate a rapid diagnostic procedure for the separation and presumptive identification of the enteric bacilli on the basis of differences in their carbohydrate fermentative patterns.

It is essential to adhere to the designated incubation period of 18—24 hours to prevent the complete degradation of the carbohydrate substrates, thereby inhibiting the catabolism of proteins.

Students should be reminded not to fail to inoculate the butt of the TSI agar. Failure to do so will produce invalid results. Students should be told that they cannot use an inoculating loop for this experiment. The loop will split the butt, giving the medium the false appearance of gas production. A prolific H2S producer may produce so much black precipitate ferrous sulfide that acidity produced in the butt is completely masked.

If H2S is produced, an acid condition does exist even though it is not discernible, and the sample should be reported as positive for acid. Ma, M. Influence of pH of TSI medium on the detection of hydrogen sulfide production by Campylobacter hyointestinalis. Letters in Applied Microbiology, 44 5 —9.

The TSI test is designed for the rapid separation and presumptive identification of enteric organisms. The lower concentration of glucose in the medium allows for detection of the utilization of this substrate only.

The purpose of the phenol red pH indicator is to detect carbohydrate fermentation that is indicated by a color change in the medium from orange-red to yellow, which is caused by the presence of acidic end products. The limited length of the incubation period is important to prevent the breakdown of proteins in the medium, which would result in the formation of end products that would obscure the observation of the results.

The purpose of this battery of biochemical tests is to separate and identify the members of the family Enterobacteriaceae. Once again, the basis of these tests is predicated on the availability of specific endoenzymes whose presence is determined by the presence or absence of their metabolic end products in the medium.

This particular group of tests has clinical as well as environmental significance. From the medical perspective, members of this family of microorganisms include pathogens and organisms that constitute the normal intestinal flora.

From an environmental aspect, E. Students should be cautioned to be careful to add the correct test reagents to the appropriate cultures. In addition, the following points should be stressed for the performance of the biochemical tests. Methyl Red Test No attempt should be made to record the methyl red results prior to 48 hours of incubation. They should be checked, however, after 24 hours. If the methyl red tests are performed too early, false-positive results may occur because the organisms have not had sufficient time to completely metabolize the acidic products that have accumulated following the fermentation of the glucose.

Hydrogen Sulfide Test Students should be made aware that H2S production is most prominent with motile organisms and that they will produce a black color throughout the medium. Nonmotile organisms may exhibit blackening along the line of growth. A reversal of the order may give a weakpositive or false-negative result. Also, a to minute reaction time is required prior to the observation of the cultures. Citrate Test A positive reaction is ascertained by the presence of growth on the slant surface and not by the color change of the medium that accompanies the microbial growth.

Citrate Test It is a good idea to streak the citrate medium first when inoculating a number of biochemical tests from the same culture. Any carryover of glucose or other nutrient onto the citrate medium may produce a falsepositive result. Both E. The low pH is maintained by E. On the other hand, E. Pyruvic acid is a utilizable intracellular metabolite and therefore is not excreted into the medium. Indole is a waste product and can be detected in the medium.

The rationale for the use of Simmons citrate is to identify organisms that are enzymatically capable of metabolizing citrate as the sole carbon source for energy production. Standing at room temperature for any length of time may cause color changes and decrease sensitivity.

Store the reagent in an amber bottle with a glass stopper. Motile cultures grown in SIM media display a diffuse growth away from the line ofinoculation.

Proteus spp. Variations in the pH of positive reactions can occur because of differences in the size of the inoculum. Lee, J. Shigella flexneri infection in a newly acquired rhesus macaque Macaca mulatta. Laboratory Animal Research, 27 4 —6. Stoffels, L. Thiosulfate reduction in Salmonella enterica is driven by the proton motive force. Journal of Bacteriology, 2 — Murphy, T. Every textbook comes with a day "Any Reason" guarantee.

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Updated to reflect changes in the lab manual, this guide is a valuable teaching aid for instructors. For each experiment, this guide provides detailed lists of required materials, tables that let instructors calculate the amount of media and equipment needed for their class, procedural points to emphasize, suggestions for optional procedural additions or modifications, helpful tips for preparing or implementing each experiment, and answers to the Review Questions in the lab manual.

Information on laboratory safety protocol for instructional and technical staff is also provided. In this manual, comprehensive introductory material is given at the beginning of each major area of study, and specific explanations and detailed directions precede each experiment. This approach augments, enhances, and reinforces course lectures, enabling students to comprehend more readily the concepts and purposesof each experiment. This also provides a review aid if the laboratory and lecture sections are not taught concurrently.

The manual should also reduce the time required forexplanations at the beginning of each laboratory session and thus make more time available for performing the experiments. Undetected location. NO YES. Laboratory Exercises in Microbiology, 5th Edition. The lab manual covers most core aspects of the microbial world very well: bacteria, fungi, and protists, but does not include any experiments with the viruses.

A simple exercise with bacteriophage T4 and Escherichia coli could be added to A simple exercise with bacteriophage T4 and Escherichia coli could be added to illustrate plaque formation. The lab manual is very good in giving the students a place to record results in a format that will allow them to see how to collect data and interpret it.

Leaving a blank page for notes also is good. Key words are defined well in the introductory portion of each exercise. For the most part the content was excellent and quite accurate.