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Bacteriology exam prep topics 4-9
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Bacteriology exam prep topics 4-9

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Questions and Answers

What is the minimum temperature required to destroy bacterial spores?

  • 70 °C
  • > 100 °C (correct)
  • 60 °C
  • 80 °C

    • Which method of sterilization uses hot air ovens?

  • Dry heat (correct)
  • Flaming
  • Moist heat
  • Pasteurization

    • What is the purpose of pasteurization?

  • To destroy all microorganisms
  • To sterilize metallic instruments
  • To flame objects
  • To destroy pathogens and spoilage-causing organisms in thermolabile products (correct)

    • What is the temperature and time required for dry heat sterilization in hot air ovens?

    <p>150 °C for 90 minutes</p> Signup and view all the answers

    Which method of sterilization uses a flame to destroy microorganisms?

    <p>Flaming</p> Signup and view all the answers

    What is the temperature and time required for moist heat sterilization?

    <p>All of the above</p> Signup and view all the answers

    What is the purpose of flaming in sterilization?

    <p>To expose metallic objects to a flame</p> Signup and view all the answers

    Why does moist heat sterilization require a lower temperature than dry heat?

    <p>Because it kills microorganisms more rapidly</p> Signup and view all the answers

    What is the primary purpose of rapidly cooling a substance after pasteurization?

    <p>To discourage the growth of any remaining viable organisms</p> Signup and view all the answers

    At what temperature are vaccines with nonsporing bacteria typically pasteurized?

    <p>60 °C</p> Signup and view all the answers

    What is the minimum temperature required to kill vegetative bacterial cells and viruses in 30 minutes?

    <p>100 °C</p> Signup and view all the answers

    What is the purpose of the incubation period in tyndallization?

    <p>To allow spores to germinate and then be killed by steam</p> Signup and view all the answers

    What is the minimum pressure required to achieve a temperature of 121 °C in an autoclave?

    <p>1.5 atm</p> Signup and view all the answers

    What type of sterilization is used for materials that are composed of thermolabile chemicals?

    <p>Tyndallization</p> Signup and view all the answers

    What is the primary advantage of autoclaving over boiling?

    <p>Autoclaving is more effective at killing spores</p> Signup and view all the answers

    What is the purpose of using free-flowing steam in tyndallization and autoclaving?

    <p>To kill vegetative cells and spores</p> Signup and view all the answers

    What is the primary purpose of evacuating all air from the inner chamber before releasing steam?

    <p>To prevent air from interfering with the sterilization process</p> Signup and view all the answers

    Which type of radiation has a poor penetrative power and is typically used for surface disinfection?

    <p>Non-ionizing radiation, such as UV light</p> Signup and view all the answers

    What is the primary mechanism by which UV light damages bacterial cells?

    <p>By forming bonds between adjacent pyrimidine bases in DNA chains</p> Signup and view all the answers

    Which type of material is typically sterilized using ionizing radiation?

    <p>Pharmaceuticals</p> Signup and view all the answers

    What is the advantage of using ionizing radiation for sterilization?

    <p>It is a fast process, achieving sterilization in a few seconds</p> Signup and view all the answers

    What is the primary disadvantage of using UV light for sterilization?

    <p>It has poor penetrative power, requiring direct exposure to the UV light</p> Signup and view all the answers

    What is the temperature that can be achieved by steam under pressure in a vacuum inner chamber?

    <p>100°C</p> Signup and view all the answers

    Which types of materials are typically sterilized using autoclaving?

    <p>Catheters and surgical instruments</p> Signup and view all the answers

    What is the primary mechanism of action of ethylene oxide?

    <p>Alkylation of proteins and nucleic acids</p> Signup and view all the answers

    What is the primary advantage of using ethylene oxide sterilization?

    <p>It can be used for heat-sensitive instruments</p> Signup and view all the answers

    What is the main limitation of using hydrogen peroxide as an antiseptic?

    <p>It is rapidly broken down by enzyme catalase</p> Signup and view all the answers

    What is the primary mechanism of action of glutaraldehyde?

    <p>Alkylation of proteins and nucleic acids</p> Signup and view all the answers

    What is the primary advantage of using glutaraldehyde disinfection?

    <p>It is effective against a wide range of microorganisms</p> Signup and view all the answers

    What is the primary difference between ethylene oxide and glutaraldehyde?

    <p>Ethylene oxide is a gas, while glutaraldehyde is a liquid</p> Signup and view all the answers

    What is the primary disadvantage of using ethylene oxide sterilization?

    <p>It is toxic and explosive in its pure form</p> Signup and view all the answers

    What is the primary application of hydrogen peroxide?

    <p>Disinfection of inanimate objects</p> Signup and view all the answers

    What is the primary reason why Gram-negative bacteria are more resistant to disinfectants and antiseptics compared to Gram-positive bacteria?

    <p>The external lipopolysaccharide layer that inhibits the penetration of biocides</p> Signup and view all the answers

    Which of the following types of microorganisms is most resistant to disinfectants and antiseptics due to their structure?

    <p>Nonenveloped viruses</p> Signup and view all the answers

    What is the recommended method for inactivating prions according to the WHO and CDC?

    <p>Combining sodium hydroxide and autoclaving at 121 °C for 1 hour</p> Signup and view all the answers

    Why are Pseudomonas and Burkholderia resistant to many antibiotics?

    <p>Their porin structure that limits the entry of antibiotics</p> Signup and view all the answers

    What is the primary mechanism by which high concentrations of H2O2 inactivate aerobic bacteria and facultative anaerobes?

    <p>Generation of reactive oxygen species</p> Signup and view all the answers

    Which of the following microorganisms is sensitive to lipid-soluble antimicrobials?

    <p>Enveloped viruses</p> Signup and view all the answers

    What is the primary reason why bacterial endospores are resistant to disinfectants and antiseptics?

    <p>Their highly resistant spore coat</p> Signup and view all the answers

    Why are Mycobacteria resistant to disinfectants and antiseptics?

    <p>Their waxy, lipid-rich cell wall</p> Signup and view all the answers

    What is the primary function of a buffering agent in a bacterial culture medium?

    <p>To regulate the pH of the medium</p> Signup and view all the answers

    What is the primary difference between liquid and semi-solid bacterial culture media?

    <p>The presence or absence of agar</p> Signup and view all the answers

    What is the primary purpose of using a selective agent, such as antibiotics, in a bacterial culture medium?

    <p>To inhibit the growth of contaminating bacteria</p> Signup and view all the answers

    What is the primary characteristic of a suitable bacterial culture medium?

    <p>It meets the nutritional requirements of the bacteria and is sterile</p> Signup and view all the answers

    What is the main purpose of transport media in microbiology?

    <p>To preserve the viability of potential pathogens during transportation</p> Signup and view all the answers

    What type of haemolysis is characterized by the complete lysis of RBCs under and around the colonies?

    <p>Beta haemolysis</p> Signup and view all the answers

    What is the primary purpose of subculturing in microbiology?

    <p>To preserve bacteria for short-term storage</p> Signup and view all the answers

    What is the main characteristic of unculturable bacteria?

    <p>They are unable to proliferate in laboratory media</p> Signup and view all the answers

    What is the primary purpose of sterilization?

    <p>To destroy or eliminate all viable microorganisms</p> Signup and view all the answers

    Which of the following factors increases the exposition time for sterilization with heat?

    <p>Presence of organic material</p> Signup and view all the answers

    What is the primary reason why moist heat is more effective at killing microorganisms than dry heat?

    <p>Moist heat causes protein denaturation more efficiently</p> Signup and view all the answers

    What is the primary difference between moist heat and dry heat sterilization?

    <p>Moist heat uses steam, while dry heat uses hot air</p> Signup and view all the answers

    Which of the following microorganisms is most resistant to heat?

    <p>Bacterial spores</p> Signup and view all the answers

    Which of the following characteristics is true about phenolics?

    <p>They remain active in the presence of organic compounds</p> Signup and view all the answers

    What is the primary mechanism of action of biguanides against microorganisms?

    <p>Affecting bacterial cell membranes</p> Signup and view all the answers

    What is the primary limitation of using alcohols as antiseptics?

    <p>They cause coagulation of a layer of protein under which bacteria can continue to grow</p> Signup and view all the answers

    Which of the following is a characteristic of chloramine?

    <p>It releases chlorine more slowly</p> Signup and view all the answers

    What is the primary reason why H2O2 is not an effective antiseptic for open wounds?

    <p>It is broken down by the enzyme catalase present in animal cells</p> Signup and view all the answers

    What is the primary mechanism by which high concentrations of H2O2 inactivate aerobic bacteria and facultative anaerobes on non-living surfaces?

    <p>Generation of reactive oxygen species</p> Signup and view all the answers

    What is the primary reason why Pseudomonas and Burkholderia are resistant to many antibiotics?

    <p>Characteristics of their porins</p> Signup and view all the answers

    Which component in radiation sterilization creates highly reactive hydroxyl radicals?

    <p>Ionization of water</p> Signup and view all the answers

    What is a key characteristic of chemically defined media?

    <p>The exact amounts of purified inorganic and organic ingredients are known</p> Signup and view all the answers

    What is the primary function of differential media in bacterial culture?

    <p>Provide a presumptive identification of bacterial colonies through biochemical reactions</p> Signup and view all the answers

    Which example is appropriately classified as an enrichment media?

    <p>Selenite broth</p> Signup and view all the answers

    What distinguishes selective-differential media from other types of media?

    <p>Contains both selective agents and indicators for differentiation</p> Signup and view all the answers

    What differentiates solid media from other types of media in terms of bacteria growth?

    <p>Bacteria form visible colonies that aid in identification</p> Signup and view all the answers

    What is the primary reason UV light has poor penetrating abilities?

    <p>It is absorbed by the outer layer of biological materials.</p> Signup and view all the answers

    Which disinfectant principle is crucial for its effective use?

    <p>Ensuring the disinfectant is used at the correct concentration as indicated on the label.</p> Signup and view all the answers

    What is the recommended method for inactivating prions according to the WHO and CDC?

    <p>Combined use of sodium hydroxide and autoclaving</p> Signup and view all the answers

    What is the primary effect of UV light on bacterial cells?

    <p>Formation of bonds between adjacent pyrimidine bases in DNA</p> Signup and view all the answers

    Which method is most suitable for sterilizing heat-sensitive materials like vaccines and antibiotic solutions?

    <p>Filtration</p> Signup and view all the answers

    What is the primary chemical group involved in the action of quaternary ammonium compounds (quats)?

    <p>Cationic portion of the molecule</p> Signup and view all the answers

    Why is glutaraldehyde preferred over formaldehyde for disinfecting medical instruments?

    <p>It is less irritating and more effective</p> Signup and view all the answers

    What makes formaldehyde a less favored choice despite its effectiveness as a disinfectant?

    <p>It is banned in the EU due to its carcinogenic properties</p> Signup and view all the answers

    What characteristic of ethylene oxide makes it suitable for sterilizing materials sensitive to heat and moisture?

    <p>It carries out sterilization at ambient temperatures</p> Signup and view all the answers

    How does soap contribute to the removal of microbes during scrubbing?

    <p>By emulsifying oils and lifting debris</p> Signup and view all the answers

    What is the function of phenol red in Kligler's iron agar and Tripple sugar iron agar?

    <p>To detect the production of acid or alkaline end products</p> Signup and view all the answers

    What is the purpose of the glucose oxidation/fermentation test?

    <p>To differentiate between oxidative and fermentative bacteria</p> Signup and view all the answers

    What is the role of decarboxylase enzymes in bacterial metabolism?

    <p>To deaminate amino acids</p> Signup and view all the answers

    What is the purpose of the urease test?

    <p>To identify Proteus spp.</p> Signup and view all the answers

    What is the effect of mineral oil on bacterial growth in the glucose oxidation/fermentation test?

    <p>It creates anaerobic conditions</p> Signup and view all the answers

    What is the role of bromathymol blue in the glucose oxidation/fermentation test?

    <p>It is a pH indicator</p> Signup and view all the answers

    What is the effect of fermentation on the pH of the medium in the glucose oxidation/fermentation test?

    <p>It decreases the pH</p> Signup and view all the answers

    What is the significance of alkaline end products in the glucose oxidation/fermentation test?

    <p>They indicate the presence of oxidative bacteria</p> Signup and view all the answers

    Which system is specifically designed for the identification and differentiation of bacteria belonging to the family Enterobacteriaceae?

    <p>API® 20E</p> Signup and view all the answers

    Which molecular technique is NOT typically utilized in genotypic methods for bacterial identification?

    <p>Metabolic activity assays</p> Signup and view all the answers

    What is the main disadvantage of commercial biochemical tests compared to traditional tests?

    <p>They generally require metabolic activity for interpretation.</p> Signup and view all the answers

    Which of the following commercial systems is fully automated and also performs antibiotic susceptibility testing?

    <p>VITEK® 2</p> Signup and view all the answers

    In traditional biochemical tests, what is generally the maximum incubation period to observe a result?

    <p>24 hours</p> Signup and view all the answers

    Which system is specifically designed for the identification and differentiation of streptococcus and related genera?

    <p>RapIDTM STR System</p> Signup and view all the answers

    Why do commercial biochemical systems often take longer for result interpretation?

    <p>They rely on metabolic activity which varies among different bacteria.</p> Signup and view all the answers

    Which approach uses nucleic acid probes for bacterial identification?

    <p>Genotypic methods</p> Signup and view all the answers

    What does the haemolytic activity of bacteria indicate?

    <p>The pathogenicity of the bacteria</p> Signup and view all the answers

    Which traditional biochemical test can help identify a bacterial species after pure culture isolation?

    <p>Oxidase test</p> Signup and view all the answers

    Which bacterial enzyme is inhibited by Trimethoprim, leading to the cessation of bacterial growth?

    <p>Dihydrofolate reductase</p> Signup and view all the answers

    Why can't bacteria be identified solely based on structural differences?

    <p>Similar size, shape, and arrangement among many species</p> Signup and view all the answers

    What is the function of folic acid (vitamin B9) in bacteria?

    <p>Protein and nucleic acid synthesis</p> Signup and view all the answers

    Which bacterial species-dependant trait can be identified using biochemical tests besides haemolytic activity?

    <p>Proteloytic activity</p> Signup and view all the answers

    What is the purpose of using bacterial enzymes in the food industry?

    <p>To ferment carbohydrates for making products like dairy, wine, and beer</p> Signup and view all the answers

    What distinguishes traditional biochemical tests from genotypic biochemical tests in bacterial identification?

    <p>Traditional tests focus on biochemical activity, while genotypic tests focus on genetic material</p> Signup and view all the answers

    What is the primary responsibility of the clinician in the context of microbial identification?

    <p>Submitting and collecting appropriate samples with specific requests or adequate history</p> Signup and view all the answers

    What is one reason a sample might test negative by isolation but positive by PCR?

    <p>The sensitivity of different assays</p> Signup and view all the answers

    Which of the following is an example of an exoenzyme?

    <p>Collagenase</p> Signup and view all the answers

    Which type of enzyme is continuously produced and specific to a species?

    <p>Constitutive enzyme</p> Signup and view all the answers

    Which microorganism might require a repeat sample following a negative examination report due to intermittent shedding?

    <p>Leptospira</p> Signup and view all the answers

    What role do virulence factors play in microorganisms?

    <p>They contribute to the pathogenicity of microorganisms</p> Signup and view all the answers

    What is the role of a microbiologist in the interpretation of microbial test results?

    <p>Interpreting results in relation to the supplied information</p> Signup and view all the answers

    Which enzyme classification is based on the location within the cell?

    <p>Endoenzymes and exoenzymes</p> Signup and view all the answers

    What factors can impact the duration of a day-by-day bacteriological examination?

    <p>The time needed to obtain a pure culture and the use of antibiograms or bioassays</p> Signup and view all the answers

    Why might a negative diagnostic report not indicate the absence of the suspected microorganism?

    <p>The animal might have stopped excreting the microorganism before the sample was taken</p> Signup and view all the answers

    What key element does laboratory diagnostic quality depend on?

    <p>The clinical and epidemiological information supplied by the veterinary clinician</p> Signup and view all the answers

    Which statement is true about interpreting bacteria detection results?

    <p>Results must be interpreted with clinical signs and vaccination history</p> Signup and view all the answers

    What is one reason traditional or complex biochemical tests are performed?

    <p>To identify the bacterial species after a presumptive genus identification</p> Signup and view all the answers

    Why is detection of bacteria alone insufficient in establishing a diagnosis?

    <p>Bacteria presence needs to be supported by clinical context</p> Signup and view all the answers

    Which of the following is not a reason for a laboratory's failure to identify a pathogen?

    <p>The laboratory used the wrong type of culture medium</p> Signup and view all the answers

    Which aspect is crucial for interpreting diagnostic reports from a microbiology laboratory?

    <p>The clinical and environmental context provided</p> Signup and view all the answers

    What is CAMP test used for?

    <p>presumptive identification of Group B beta-hemolytic streptococci (Streptococcus agalactiae).</p> Signup and view all the answers

    Between what is the basis of the CAMP test enhanced haemolytic activity with?

    <p>between beta haemolysis producing strains Staphylococcus aureus and the extracellular protein (CAMP factor) produced by S. agalactiae.</p> Signup and view all the answers

    With what bacteria is the positive result of the CAMP test seen?

    <p>All of them</p> Signup and view all the answers

    What is the primary function of the cell wall in bacteria?

    <p>To provide structural support and maintain cell shape</p> Signup and view all the answers

    What is catalase test used for?

    <p>differentiating many gram positive organisms for example staphylococci catalase positive, streptococci and enterococci catalase negative</p> Signup and view all the answers

    what does the catalase test detect?

    <p>Detects the enzyme catalase that converts H2O2 (hydrogen peroxide) to water and gaseous (molecular) oxygen.</p> Signup and view all the answers

    What to catalase positive organisms produce during the test?

    <p>Catalase positive organisms rapidly produce bubbles when exposed to a solution containing hydrogen peroxide</p> Signup and view all the answers

    Which type of coagulase is responsible for the formation of a clot when bacteria are incubated with plasma?

    <p>Free coagulase</p> Signup and view all the answers

    What is the primary function of the coagulase enzyme in Staphylococcus aureus?

    <p>To escape phagocytosis</p> Signup and view all the answers

    Which method is used to detect bound coagulase in Staphylococcus aureus?

    <p>Slide coagulase test</p> Signup and view all the answers

    What is the primary difference between coagulase positive and coagulase negative staphylococci?

    <p>Their ability to clot plasma</p> Signup and view all the answers

    Why is the tube coagulase test performed even if the slide coagulase test is negative?

    <p>To detect free coagulase</p> Signup and view all the answers

    What is the main purpose of the oxidase test in bacterial identification?

    <p>To detect the presence of cytochrome c oxidase in bacteria</p> Signup and view all the answers

    Which reagent is typically used in the oxidase test?

    <p>1% tetramethyl-p-phenylene-diamine solution</p> Signup and view all the answers

    Which of the following statements is true regarding anaerobic bacteria and the oxidase test?

    <p>Anaerobic bacteria are oxidase negative</p> Signup and view all the answers

    What distinguishes Pseudomonas aeruginosa from Escherichia coli in the oxidase test?

    <p>Pseudomonas aeruginosa is oxidase positive, while Escherichia coli is oxidase negative</p> Signup and view all the answers

    Why is the oxidase test considered a rapid test?

    <p>Results are typically obtained within a few minutes</p> Signup and view all the answers

    What are organisms that can utilize both aerobic and anaerobic pathways called?

    <p>Facultative anaerobes</p> Signup and view all the answers

    Which end product is not typically associated with pyruvate metabolism?

    <p>Citric acid</p> Signup and view all the answers

    Which of the following is necessary for detecting gas production during carbohydrate fermentation?

    <p>Durham tubes</p> Signup and view all the answers

    In carbohydrate fermentation media, what is the purpose of adding a specific carbohydrate?

    <p>To act as a substrate for fermentation</p> Signup and view all the answers

    Which pH indicator changes color to yellow at acidic pH?

    <p>Phenol red</p> Signup and view all the answers

    If an organism uses peptones instead of carbohydrates for energy, what color will phenol red turn?

    <p>Deep red</p> Signup and view all the answers

    What causes the medium to turn yellow in a positive carbohydrate fermentation test?

    <p>Production of acid</p> Signup and view all the answers

    What is the end product of glucose degradation before it enters further metabolic pathways?

    <p>Pyruvic acid</p> Signup and view all the answers

    Which of the following is not an organic acid typically produced during carbohydrate fermentation?

    <p>Sulfuric acid</p> Signup and view all the answers

    What is the primary purpose of Kligler's iron agar and Tripple sugar iron agar tests?

    <p>To differentiate among Enterobacteriaceae based on carbohydrate fermentation patterns and hydrogen sulfide production</p> Signup and view all the answers

    What is the common substrate in both Kligler's iron agar and Tripple sugar iron agar tests?

    <p>Sodium thiosulfate</p> Signup and view all the answers

    Which of the following sugars is present in Tripple sugar iron agar but not in Kligler's iron agar?

    <p>Sucrose</p> Signup and view all the answers

    What is the purpose of phenol red in both Kligler's iron agar and Tripple sugar iron agar tests?

    <p>To indicate the pH of the medium</p> Signup and view all the answers

    Which of the following is a characteristic of Enterobacteriaceae that is used in the differentiation of these bacteria using Kligler's iron agar and Tripple sugar iron agar tests?

    <p>Ability to ferment glucose</p> Signup and view all the answers

    Which condition does oxidative bacteria require to produce acids from metabolized carbohydrates?

    <p>Aerobic conditions only</p> Signup and view all the answers

    What role does the mineral oil layer play in the glucose oxidation/fermentation test?

    <p>Ensures anaerobic conditions</p> Signup and view all the answers

    What is indicated if the medium turns blue in the glucose oxidation/fermentation test?

    <p>Alkaline end products, indicating peptone usage</p> Signup and view all the answers

    What is the role of bromothymol blue in the medium?

    <p>Indicates changes in pH</p> Signup and view all the answers

    How do fermentative bacteria behave differently from oxidative bacteria in the glucose oxidation/fermentation test?

    <p>Produce acids under both aerobic and anaerobic conditions</p> Signup and view all the answers

    What is the purpose of adding a pH indicator to the nutrient medium in decarboxylase enzyme assays?

    <p>To indicate alkalinity resulting from decarboxylation</p> Signup and view all the answers

    What is the role of decarboxylases in bacterial metabolism?

    <p>To degrade amino acids and produce alkaline products</p> Signup and view all the answers

    Why are decarboxylases considered adaptive enzymes?

    <p>Because they are induced by the presence of specific amino acid substrates</p> Signup and view all the answers

    What is the purpose of cultivating bacteria on a nutrient medium containing a specific amino acid substrate?

    <p>To detect the production of decarboxylase enzymes</p> Signup and view all the answers

    What is the end-product of decarboxylation reactions?

    <p>Alkaline compounds</p> Signup and view all the answers

    What is the primary function of urease in microbiological testing?

    <p>To distinguish Proteus species from other urease-producing organisms</p> Signup and view all the answers

    What is the purpose of Christensen's agar in urease testing?

    <p>To detect the presence of urease and subsequent ammonia production</p> Signup and view all the answers

    What is the end product of urease activity on urea?

    <p>Ammonia and carbon dioxide</p> Signup and view all the answers

    Why is urease testing particularly useful in identifying Proteus species?

    <p>Because Proteus species produce urease at a faster rate than other organisms</p> Signup and view all the answers

    What is the purpose of the pH indicator (phenol red) in Christensen's agar?

    <p>To detect the production of ammonia</p> Signup and view all the answers

    Which biochemical test within the IMViC series is specifically designed to detect the production of acetoin?

    <p>Vogues-Proskauer test</p> Signup and view all the answers

    Which reagent is commonly used in the Indole test to detect the presence of indole?

    <p>Kovac's reagent</p> Signup and view all the answers

    What is the primary purpose of the Methyl-Red test?

    <p>To detect mixed acid fermentation</p> Signup and view all the answers

    Which medium is used for the Citrate test in the IMViC series?

    <p>Simmon's citrate agar</p> Signup and view all the answers

    A positive result in the Vogues-Proskauer (VP) test indicates the presence of which metabolic intermediate?

    <p>Acetoin</p> Signup and view all the answers

    What is the byproduct of tryptophan metabolism that is detected in the indole test?

    <p>Indole</p> Signup and view all the answers

    What is the purpose of adding Kovac's reagent in the tube test for indole detection?

    <p>To detect the presence of indole</p> Signup and view all the answers

    What is the color change observed when Kovac's reagent is added to a positive indole test?

    <p>Colorless to cherry red</p> Signup and view all the answers

    What is the alternative method to the tube test for detecting indole?

    <p>Spot test using filter paper</p> Signup and view all the answers

    What is the enzyme responsible for the conversion of tryptophan into indole?

    <p>Tryptophanase</p> Signup and view all the answers

    What is the primary purpose of the methyl red test?

    <p>To determine stable acid end products from glucose fermentation</p> Signup and view all the answers

    During the methyl red test, which of the following acids is NOT produced by MR positive organisms during prolonged incubation?

    <p>Sulfuric acid</p> Signup and view all the answers

    Which color change indicates a positive result in the citrate utilization test?

    <p>Green to blue</p> Signup and view all the answers

    What role does $ ext{α-naphthol}$ play in the Voges-Proskauer test?

    <p>Catalyst for the conversion to diacetyl</p> Signup and view all the answers

    Which indicator is used in the Simmon’s citrate agar for the citrate utilization test?

    <p>Bromothymol blue</p> Signup and view all the answers

    What is the metabolic end product detected by the Voges-Proskauer test?

    <p>Acetylmethylcarbinol</p> Signup and view all the answers

    Why do Enterobacteriaceae usually test positive for either the methyl red test or the Voges-Proskauer test, but rarely both?

    <p>They typically follow only one metabolic pathway for glucose utilization</p> Signup and view all the answers

    How is MR-VP broth expected to change color in a positive methyl red test?

    <p>From yellow to red</p> Signup and view all the answers

    Which component is NOT required for a Voges-Proskauer test?

    <p>Methyl red</p> Signup and view all the answers

    What is the primary purpose of controlling factors such as pH and temperature during bacterial cultivation?

    <p>To ensure optimal growth and reproduction of bacteria</p> Signup and view all the answers

    What is the primary purpose of using agar-agar in bacterial culture media?

    <p>To solidify the medium</p> Signup and view all the answers

    What is the primary purpose of semi-solid media in microbial cultivation?

    <p>Determination of bacterial motility</p> Signup and view all the answers

    What is the typical concentration of agar in solid media?

    <p>1.5-2.0%</p> Signup and view all the answers

    What is the purpose of broth medium in microbiology?

    <p>To propagate many microorganisms and for fermentation studies</p> Signup and view all the answers

    What is the primary difference between liquid and semi-solid media?

    <p>Concentration of agar</p> Signup and view all the answers

    What is the advantage of using solid media in microbiology?

    <p>Microorganisms grow in a physically informative way</p> Signup and view all the answers

    What is the primary characteristic of agar-agar that limits its use as a culture medium?

    <p>It is not nutritious</p> Signup and view all the answers

    What is the main difference between complex media and defined media?

    <p>Defined media have exact composition known</p> Signup and view all the answers

    What is the primary purpose of adding beef extract and peptone to agar-agar?

    <p>To provide nutrients to support life</p> Signup and view all the answers

    What is the temperature range at which agar-agar melts and solidifies?

    <p>97-100°C and 42°C</p> Signup and view all the answers

    What is the primary characteristic of general-purpose media?

    <p>They are capable of sustaining growth of a large variety of bacteria</p> Signup and view all the answers

    What is the name of the medium that contains 1-5% of agar-agar?

    <p>The medium has 'agar' in its name</p> Signup and view all the answers

    What type of medium is used to support the growth of fastidious bacteria?

    <p>Enriched media</p> Signup and view all the answers

    What is the purpose of heating blood agar to create chocolate agar?

    <p>To lyse red blood cells and release nutrients for bacterial growth</p> Signup and view all the answers

    What is the primary purpose of using a selective medium?

    <p>To inhibit the growth of unwanted bacterial species</p> Signup and view all the answers

    What type of medium is used to detect a particular bacterium that may be present in small proportions?

    <p>Enrichment medium</p> Signup and view all the answers

    What is the primary characteristic of Blood Agar?

    <p>It is used to detect hemolysis patterns</p> Signup and view all the answers

    What is the primary purpose of using Bromocresol Purple Lactose Agar?

    <p>To detect the presence of enterobacteriaceae</p> Signup and view all the answers

    What type of medium is Lowenstein Jensen medium?

    <p>Selective medium</p> Signup and view all the answers

    Which type of media is used to promote the growth of anaerobes?

    <p>Thioglycollate broth</p> Signup and view all the answers

    What is the primary purpose of Rappaport-Vassiliadis soya peptone broth?

    <p>To select for Salmonella</p> Signup and view all the answers

    Which type of media is used to prevent the overgrowth of contaminating organisms during transportation?

    <p>Transport media</p> Signup and view all the answers

    What is the primary purpose of Mannitol salt agar?

    <p>To differentiate between fermenting and non-fermenting bacteria</p> Signup and view all the answers

    Which type of media is used to promote the growth of a specific type of bacteria and differentiate between genera and/or species?

    <p>Selective-differential media</p> Signup and view all the answers

    What is the primary purpose of subculturing in microbiology?

    <p>To obtain a pure culture of bacteria</p> Signup and view all the answers

    Which type of media is used to inhibit the growth of other bacteria except Staphylococci?

    <p>Mannitol salt agar</p> Signup and view all the answers

    What is the primary reason for immediately inoculating samples that may contain anaerobic bacteria into appropriate media?

    <p>To prevent desiccation of the sample</p> Signup and view all the answers

    What is the primary purpose of the triple packaging system for international transport of infectious substances?

    <p>To prevent leakage of the sample during transport</p> Signup and view all the answers

    What information must be included on the sample submission form supplied by the laboratory?

    <p>The identity of the sample, animal ID, date of collection, and specific details of the tests required</p> Signup and view all the answers

    Why is it essential to screw together or tape the container caps of samples to be submitted to the laboratory?

    <p>To prevent leakage of the sample during transport</p> Signup and view all the answers

    What is the primary purpose of the laboratory supplying sample submission forms?

    <p>To ensure that the laboratory receives all necessary information about the sample</p> Signup and view all the answers

    What should the veterinary practitioner do if they require tests that are not part of the routine investigation?

    <p>Contact the laboratory to discuss the required tests</p> Signup and view all the answers

    What is the primary purpose of using separate leak-proof containers for each sample?

    <p>To prevent contamination of the samples</p> Signup and view all the answers

    Why is it essential to label samples clearly and unambiguously?

    <p>To ensure that the laboratory receives the correct information about the sample</p> Signup and view all the answers

    What is the primary purpose of the international standards for the transport of infectious substances?

    <p>To ensure the safe transportation of infectious substances</p> Signup and view all the answers

    What is the primary reason for not submitting blood to the laboratory in a syringe?

    <p>It may cause hemolysis</p> Signup and view all the answers

    Why should the needle be removed before expelling a blood sample into a sterile tube?

    <p>To prevent hemolysis</p> Signup and view all the answers

    What is the recommended method for collecting a urine sample to minimize contamination?

    <p>Cystocentesis</p> Signup and view all the answers

    What is the significance of sampling pus from the edge rather than the center of an abscess?

    <p>The edge provides the best bacterial results</p> Signup and view all the answers

    What is the primary reason that anaerobic bacteria samples need to be placed in transport media?

    <p>To prevent exposure to oxygen</p> Signup and view all the answers

    What is a critical consideration when transporting specimens to avoid compromising the sample?

    <p>Avoiding drying and exposure to a noxious atmosphere</p> Signup and view all the answers

    What is a crucial step in the collection of a milk sample from a mastitic udder?

    <p>Disinfecting teats using 70% ethanol on cotton wool</p> Signup and view all the answers

    Why is it advisable to take more than one blood sample within a 24-hour period for bacterial isolation?

    <p>To account for intermittent bacteria presence</p> Signup and view all the answers

    What is the recommended method for collecting samples from intact pustules or vesicles on skin lesions?

    <p>Sterile syringe and fine needle</p> Signup and view all the answers

    Why are short cotton swabs generally unsatisfactory for obtaining nasopharyngeal specimens in large animals?

    <p>They cannot reach the nasopharyngeal area effectively</p> Signup and view all the answers

    Which of the following is essential to increase the likelihood of detecting the causal organism in swabs and discharges?

    <p>A larger sample volume</p> Signup and view all the answers

    What should be done with whole fetus and placenta samples when it is impossible to submit them entirely?

    <p>Submit fetal stomach contents and uterine discharge</p> Signup and view all the answers

    Which samples are important to rule out zoonotic pathogens in the case of abortion?

    <p>Listeria, Brucella, Leptospira</p> Signup and view all the answers

    Which method is recommended for collecting samples from raw surfaces of non-intact pustules or vesicles?

    <p>Use a swab</p> Signup and view all the answers

    Which type of samples should be prioritized for submission when evaluating for respiratory diseases?

    <p>Nasopharyngeal specimens</p> Signup and view all the answers

    Which of the following steps is necessary when obtaining a skin biopsy from ulcers?

    <p>Clean the superficial area before biopsy</p> Signup and view all the answers

    What is the first step in the 5i approach?

    <p>Inoculation</p> Signup and view all the answers

    How long does it generally take for bacteria to form colonies during incubation?

    <p>24-48 hours</p> Signup and view all the answers

    Which phenotypic method involves using light microscopy to observe the Gram status of bacteria?

    <p>Cellular morphology</p> Signup and view all the answers

    What is the third step in the bacteriological examination plan?

    <p>Isolation</p> Signup and view all the answers

    Which of the following is not a phenotypic method for bacterial identification?

    <p>Genotypic methods</p> Signup and view all the answers

    What is the purpose of the isolation step in the 5i approach?

    <p>Separating bacteria to obtain pure colonies</p> Signup and view all the answers

    Which test is not typically used for bacterial biochemical characterization?

    <p>PCR test</p> Signup and view all the answers

    What does the inspection step in the 5i approach primarily involve?

    <p>Evaluating colony morphology</p> Signup and view all the answers

    What are the conditions typically maintained during the incubation step?

    <p>Most favorable temperature and atmosphere</p> Signup and view all the answers

    Which methods of bacterial identification often require days to weeks for results?

    <p>Phenotypic methods</p> Signup and view all the answers

    What is the primary purpose of direct microscopy on the first day of bacteriological examination?

    <p>To observe the shape and staining characteristics of the bacteria</p> Signup and view all the answers

    What is the advantage of using Gram stain over Romanowsky-type stain?

    <p>Gram stain is more specific for gram-positive bacteria</p> Signup and view all the answers

    What is the purpose of using selective media in primary isolation?

    <p>To differentiate between gram-positive and gram-negative bacteria</p> Signup and view all the answers

    What is the primary advantage of using CNAA media in primary isolation?

    <p>It is more selective for gram-positive bacteria</p> Signup and view all the answers

    What is the primary purpose of streaking media plates in primary isolation?

    <p>To produce individual isolated colonies after incubation</p> Signup and view all the answers

    What might be the reason for the presence of microorganisms in a sample?

    <p>All of the above</p> Signup and view all the answers

    What should be done when a repeat sample is required after a negative examination report?

    <p>Repeat the sample following a negative examination report</p> Signup and view all the answers

    What is the primary purpose of inoculating media with a portion of the specimen in a semi-quantitative fashion?

    <p>To estimate the relative abundance of organisms in the sample</p> Signup and view all the answers

    What is the significance of seeing colonies growing on all four quadrants of the petri plate?

    <p>There are large numbers of microorganisms in the sample</p> Signup and view all the answers

    What might lead to conflicting results in microbiology?

    <p>The sensitivity of different assays</p> Signup and view all the answers

    What is the purpose of evaluating the cytology of a sample obtained from the infected site?

    <p>To aid in the determination of clinical significance of the isolated microorganisms</p> Signup and view all the answers

    What is essential for the benefit of the owner and pet in microbiology?

    <p>Cooperation between the veterinary practice and laboratory</p> Signup and view all the answers

    What is the benefit of obtaining a pure culture?

    <p>It allows for the identification of a single type of microorganism</p> Signup and view all the answers

    What is the significance of a negative diagnostic report?

    <p>The laboratory was unable to isolate the microorganism</p> Signup and view all the answers

    Study Notes

    Cultivation of Bacteria

    • Cultivation is the process of propagating organisms by providing proper environmental conditions to isolate a pure culture and identify a possible pathogenic bacteria.
    • Factors that must be controlled during growth include:
      • Nutrients
      • pH
      • Temperature
      • Aeration
      • Salt concentration
      • Ionic strength
    • Media must meet the following requirements:
      • Contain all necessary chemical components that bacteria need
      • Have a certain pH (usually neutral or weakly alkaline)
      • Be sterile

    Types of Media

    • Liquid media:
      • Contain nutrients, but no gelling agents
      • Examples: nutrient broth, tryptic soy broth, brain-heart infusion broth
      • Used for propagation of many organisms, accumulation of bacterial toxic end products, and fermentation studies
    • Semi-solid media:
      • Prepared with agar at 0.5% or less concentration
      • Used for cultivation of microaerophilic bacteria or determining bacterial motility
      • Examples: motility test medium, Stuart's and Amies transport media
    • Solid media:
      • Contain agar at a concentration of 1.5-2.0% or other solidifying agents
      • Used for growth of bacteria in a physically informative way, forming visible colonies
      • Examples: nutrient agar, blood agar, MacConkey agar, Baird-Parker agar

    Agar-Agar

    • A polysaccharide extracted from the cell walls of red algae
    • Inert and non-nutritious, so it must be impregnated with nutrients to support life
    • Melts at 97-100°C and solidifies at 42°C

    Classification of Media based on Composition

    • Chemically defined media:
      • Exact amounts of highly purified inorganic and organic ingredients are known
      • Examples: citrate broth
    • Complex media:
      • Prepared using digests of microbial, animal, or plant products
      • Highly nutritious, but impure substances
      • Examples: tryptic soy broth
    • General-purpose (basal) media:
      • Capable of sustaining growth of a large variety of bacteria
      • Examples: nutrient agar

    Classification of Media based on Functional Use

    • Enriched media:
      • Basal media with additional nutrients
      • Support growth of most bacteria, including fastidious species
      • Examples: blood agar, chocolate agar, Loeffler's medium
    • Differential (indicator) media:
      • Designed to give a presumptive identification of bacterial colonies
      • Often contain a fermentable sugar and a pH indicator
      • Examples: Bromocresol purple lactose agar, CLED agar
    • Selective media:
      • Contain inhibitory substances that prevent growth of unwanted bacteria
      • Used in diagnostic bacteriology
      • Examples: Lowenstein Jensen medium, 6.5% NaCl broth
    • Enrichment media:
      • Liquid media that permit the growth and detection of a particular bacterium
      • Used in diagnostic bacteriology
      • Examples: selenite broth, Rappaport-Vassiliadis soya peptone broth
    • Selective-differential media:
      • Contain both selective and differential agents
      • Examples: Mannitol salt agar, MacConkey agar, Baird-Parker agar
    • Transport media:
      • Ensure the viability of potential pathogens in the sample
      • Prevent drying and overgrowth of contaminating organisms
      • Examples: Cary Blair transport medium, Stuart's transport medium

    Haemolysis

    • Alpha haemolysis:
      • Incomplete and partial lysis of RBCs
      • Caused by H2O2
      • Examples: Streptococcus pneumoniae
    • Beta haemolysis:
      • Complete lysis of RBCs
      • Caused by exotoxins
      • Examples: Streptococcus pyogenes
    • Gamma haemolysis:
      • No haemolysis
      • Examples: Enterococcus faecalis

    Preservation of Bacteria

    • Subculturing:
      • A short-term preservation of bacteria
      • Limitations: death of cells, risk of contamination, and mutations
    • Long-term methods:
      • Paraffin method
      • Freeze-drying (lyophilization)
      • Freezing at -70°C
      • Ultra-freezing (cryopreservation) in liquid nitrogen at -196°C

    Unculturable Bacteria

    • Metabolically active in their native environment, but unable to proliferate in laboratory media
    • Examples: intracellular bacteria like Rickettsia, Coxiella, Chlamydia, Ehrlichia

    Physical Methods of Microbial Control

    • Sterilization:
      • All viable microorganisms are eliminated or destroyed
      • No levels of sterility
    • Disinfection:
      • Destruction or significant reduction of microorganisms on nonliving materials
      • Effective against vegetative cells
    • Antisepsis:
      • Inactivation and destruction of vegetative microbial forms on animals or living tissues
      • Chemical means (antiseptics)
    • Asepsis:
      • Absence of bacteria, viruses, and other microorganisms
      • Related to the practice of preventing the introduction of harmful microorganisms into the body

    Effects of Control Methods on Bacteria

    • Cell-wall injury
    • Cell membrane damage
    • Alteration of the colloidal state of cytoplasm
    • Inactivation of cellular enzymes
    • Interference with the structure and function of DNA

    Heat Application

    • Common way to destroy microorganisms
    • Factors affecting sterilization with heat:
      • Type of heat
      • Temperature and time
      • Number of microorganisms
      • Type of microorganisms
      • Type of material
      • Presence of organic material

    Radiation

    • Non-ionizing radiation:
      • Low energy, poor penetrative power
      • UV light
    • Ionizing radiation:
      • High energy, good penetrative power
      • X-rays, gamma rays, and high-energy electron beams

    Filtration

    • Passage of a liquid or gas through a screen-like material with pores small enough to retain microorganisms
    • Used to sterilize:
      • Bacteriological media
      • Serum
      • Vaccines
      • Antibiotic solutions
      • Other heat-sensitive materials

    Chemical Methods of Microbial Control

    • Principles of effective disinfection:

      • Read the label
      • Use the correct concentration
      • Consider the environmental pH and temperature
      • Mechanical removal of material

    • Halogens:

      • Iodine (I2)
      • Chlorine (Cl2)
      • Used to disinfect:
        • Skin
        • Wounds
        • Eating utensils
        • Food manufacturing equipment

    • Phenols:

      • Phenol (carbolic acid)
      • Phenolics
      • Exert antimicrobial activity by injuring lipid-containing plasma membranes
      • Used to disinfect:
        • Throat lozenges
        • Eating utensils
        • Food manufacturing equipment### Phenolics

    • Stable and persist for longer periods after application

    • Suitable for disinfecting pus, saliva, and feces

    • Cresols are very good surface disinfectants

    • O-phenylphenol is the main ingredient in most formulations of Lysol

    Biguanides

    • Have a broad spectrum of activity
    • Primarily affect bacterial cell membranes
    • Especially effective against G+ bacteria
    • Chlorhexidine is a well-known biguanide
    • Frequently used for microbial control on skin and mucous membranes
    • Combining chlorhexidine with a detergent or alcohol is used for surgical hand scrubs and preoperative skin preparation

    Alcohols

    • Denature proteins, disrupt membranes, and dissolve lipids
    • Effectively kill bacteria and fungi, but not endospores and non-enveloped viruses
    • Evaporate rapidly, leaving no residue
    • Unsatisfactory antiseptics when applied to wounds due to protein coagulation
    • Ethanol and isopropanol are commonly used alcohols
    • Recommended concentration of ethanol is 70% (60-95%)

    Detergents and Surfactants

    • Detergents contain surfactants and are cleaning agents
    • Soap has little value as an antiseptic but helps in mechanical removal of microbes
    • Surfactants can be anionic, cationic, amphoteric, or nonionic
    • Cationic surfactants (quaternary ammonium compounds) are bactericidal, fungicidal, amebicidal, and virucidal
    • Quaternary ammonium compounds are inactive against endospores, mycobacteria, and certain other bacteria

    Aldehydes

    • Effective antimicrobials that inactivate proteins by forming covalent cross-links
    • Formaldehyde and glutaraldehyde are two examples
    • Glutaraldehyde is less irritating and more effective than formaldehyde
    • Used to disinfect hospital instruments, including endoscopes and respiratory therapy equipment
    • 2% solution is bactericidal, tuberculocidal, and virucidal in 10 minutes and sporicidal in 3-10 hours

    Ethylene Oxide Gas

    • Gaseous chemosterilant that requires a closed chamber similar to a steam autoclave
    • Activity depends on alkylation, replacing labile hydrogen atoms in proteins and nucleic acids
    • Kills all microbes and endospores in a few hours
    • Carries out sterilization at ambient temperatures and is highly penetrating
    • Used for instruments that cannot tolerate heat, moisture, or abrasive chemicals

    Hydrogen Peroxide

    • PEROXgens are a group of oxidizing agents that include hydrogen peroxide, peracetic acid, ozone, and others
    • Hydrogen peroxide generates reactive and cytotoxic oxygen species in cells, initiating oxidation of biomolecules
    • Effectively disinfects inanimate objects and is sporicidal at high concentrations
    • Broken down into water and oxygen by catalase enzymes in animal cells

    Microbial Characteristics and Microbial Control

    • Gram-negative bacteria are generally more resistant than Gram-positive bacteria to disinfectants and antiseptics
    • Pseudomonas and Burkholderia are unusually resistant to biocides
    • Mycobacteria, endospores, and protozoan cysts and oocysts are very resistant to disinfectants and antiseptics
    • Bacterial endospores are affected by relatively few biocides
    • Viral resistance to biocides depends on their structure, with enveloped viruses being more susceptible to lipid-soluble antimicrobials

    Coagulase Test

    • Differentiates Staphylococcus species into two groups: coagulase positive and coagulase negative staphylococci
    • Coagulase causes a clot to form when bacteria are incubated with plasma
    • Coagulase enzyme interacts with fibrinogen, converting it to fibrin, which creates a coating around bacteria to help them escape phagocytosis

    Types of Coagulase

    • Two types: free coagulase and bound coagulase
    • Bound coagulase is also known as the clumping factor
    • Bound coagulase is detected rapidly by a slide test
    • Free coagulase is detected in the test tube as a result of the formation of a clot

    Coagulase Test Results

    • Positive results: Staphylococcus aureus, pseudointermedius
    • Negative results: Staphylococcus epidermidis

    Detection Methods

    • Bound coagulase is found in the bacterial cell wall and can be detected using a slide method
    • Free coagulase is secreted outside the cell wall and can be detected when bacteria are cultivated in a tube containing plasma, resulting in a clot formation after a few hours
    • Slide test is used less frequently than the tube method, and if slide test is negative, a tube test is performed anyway

    Oxidase Test

    • Detects Cytochrome c oxidase, an enzyme involved in electron transport and nitrite metabolism in some bacteria.
    • The enzyme can accept electrons from artificial substrates, producing a dark oxidized product.
    • Used to differentiate between groups of Gram-negative bacteria.

    Oxidase Test Results

    • Pseudomonas aeruginosa: oxidase positive
    • E. coli: oxidase negative

    Electron Transport Chain

    • All aerobic (facultative anaerobic) bacteria use the electron transport chain (ETC).
    • Not all ETCs are identical among bacteria.
    • Some bacteria have Cytochrome c oxidase, the last enzyme transferring electrons to oxygen in the ETC.

    Anaerobes

    • Anaerobes are oxidase negative.

    Oxidase Test Procedure

    • A rapid test performed on a filter paper.
    • Uses a reagent, usually 1% tetramethyl-p-phenylene-diamine solution.

    Carbohydrate Fermentation

    • Most organisms generate energy through bioxidation of a substrate carbohydrate metabolism.
    • Organisms use carbohydrates differently depending on their enzymes, with some using fermentation.
    • Sugar fermentation can be either anaerobic (glucose) or aerobic.

    Anaerobic Fermentation

    • Anaerobic fermentation involves the degradation of glucose to pyruvic acid.
    • Pyruvate metabolism results in various end products, including lactic, formic, acetic acids, and hydrogen or carbon dioxide production.

    Carbohydrate Fermentation Medium

    • A typical carbohydrate fermentation medium consists of:
    • Nutrient broth ingredients (support for growth of all organisms)
    • A specific carbohydrate (e.g. glucose, sucrose, lactose, maltose, mannitol) serving as a substrate for fermentating capabilities
    • pH indicator (e.g. phenol red, which changes color from red to yellow at acidic pH)

    Detection of Fermentation

    • Fermentation that ends in acid production will cause phenol red to turn yellow (positive reaction).
    • Acid production can be accompanied by gas formation, which is visible as bubbles.
    • Durham tubes (inverted small tubes) are used to detect gas production.
    • No reaction is seen if the culture is not able to ferment the carbohydrate (tubes remain red, no gas production).

    Alternative Energy Source

    • Sometimes, the culture uses peptones as an energy source, producing alkaline end products.
    • Phenol red turns to a deep red color in this case.

    Kligler's Iron Agar and Triple Sugar Iron Agar Tests

    • Used to differentiate among Enterobacteriaceae (Gram-negative bacilli that ferment glucose and produce acids)
    • Differentiation based on carbohydrate fermentation patterns and hydrogen sulfide production

    Composition of Agars

    • Kligler's Iron Agar: contains lactose and glucose, phenol red, and sodium thiosulfate (H2S substrate)
    • Triple Sugar Iron Agar: contains lactose, glucose, and sucrose, phenol red, and sodium thiosulfate (H2S substrate)

    Glucose Oxidation/Fermentation (O/F) Test

    • Used to determine how bacteria metabolize carbohydrates, such as glucose.
    • Distinguishes between oxidative and fermentative bacteria.

    Oxidative Bacteria

    • Form acids from metabolized carbohydrates.
    • Produce carbohydrates only under aerobic conditions.

    Fermentative Bacteria

    • Produce acid under both aerobic and anaerobic conditions.

    Test Conditions

    • Anaerobic conditions are ensured by a mineral oil layer over the medium in one of the tubes.
    • Medium contains ph indicator bromothymol blue, which is:
      • Neutral (green) in normal conditions.
      • Turns yellow in acidic conditions.

    Nonsaccharolytic Bacteria

    • Use pepton as the source of energy.
    • Produce alkaline end products.
    • Turn bromothymol blue to blue color.

    Utilization of Amino Acids

    • Some bacteria can deaminate and decarboxylate amino acids using enzymes called deaminases and decarboxylases.
    • Decarboxylases are adaptive enzymes produced in the presence of specific amino acid substrates (e.g., lysine, ornithine, arginine).
    • Decarboxylase enzymes are used to differentiate members of the Enterobacteriaceae family.
    • The activity of decarboxylase enzymes is determined by cultivating nutrient mediums.
    • The nutrient medium used contains glucose, a specific amino acid substrate, and a pH indicator (bromothymol blue).
    • If the bacterium ferments glucose, decarboxylation occurs, causing the pH to become alkaline despite the fermentation of glucose.
    • The decarboxylation process creates an acidic environment, activating the decarboxylase (if produced).
    • The end products of decarboxylation are alkaline.

    Urease Test

    • Urease is an enzyme produced by certain organisms, particularly useful in identifying Proteus spp.
    • The urease test helps distinguish between lactose-fermenting and non-lactose-fermenting enteric microorganisms.

    Principle of Urease Test

    • Urease is a hydrolytic enzyme that cleaves ammic compounds, such as urea, producing an alkaline end product, ammonia.
    • Christensen's agar is used to detect urease, which contains urea and a pH indicator (phenol red).

    Detection of Urease

    • The presence of ammonia in the medium creates an alkaline environment.
    • As a result, the pH indicator phenol red turns deep pink in color, indicating urease production.

    IMViC Tests

    • IMViC tests are a set of diagnostic tools used to differentiate members of the Enterobacteriaceae family.
    • The IMViC acronym represents four individual tests:

    IMViC Components

    • Indole test: detects the presence of indole, a compound produced by certain bacteria.
    • Methyl-Red (MR) test: determines the ability of bacteria to ferment glucose and produce acid.
    • Voges-Proskauer (VP) test: detects the production of acetoin, a compound produced by certain bacteria.
    • Citrate test: assesses the ability of bacteria to utilize citrate as a sole source of carbon and energy.

    Indole Test

    • Tryptophan, an essential amino acid, can be metabolized into indole and pyruvic acid through the enzyme tryptophanase, with ammonia mediating the conversion.
    • Indole test is used for the identification of microorganisms, and not all microorganisms exhibit this characteristic.
    • Indole can be detected in two ways:

    Detection Methods

    • Tube Test:
      • A tube containing tryptose water is inoculated with the culture.
      • Kovac's reagent is added after inoculation.
      • Color change is observed at the meniscus.

    Alternative Detection Method

    • Spot Test:
      • Filter paper is used.
      • Test culture and Kovac's reagent are applied.
      • Two reagents are used:
        • Benzaldehyde produces a red color in a positive result.
        • Cinnamaldehyde produces a blue or green color in a positive result, and pink in a negative result.

    Methyl Red Test

    • Used to determine if an organism can produce stable acid end products from glucose fermentation
    • In the first 24 hours, glucose is converted to pyruvic acid, resulting in a positive MR reaction
    • During prolonged incubation (2-5 days), MR-positive organisms metabolize pyruvic acid to lactic, acetic, and formic acids, maintaining an acidic pH
    • A positive result turns MR-VP broth red after adding methyl red (a pH indicator)

    Voges-Proskauer Test

    • Used to determine if an organism produces acetylmethylcarbinol, a neutral end product of glucose metabolism
    • Acetylmethylcarbinol is converted to diacetyl in the presence of α-naphthol, 40% KOH, and atmospheric oxygen
    • A positive result turns the broth pinkish-red
    • The test requires 6 drops of α-naphthol and 2 drops of KOH

    MR-VP Test

    • Most Enterobacteriaceae demonstrate one or the other metabolic pathway, but rarely both
    • Only one of these tests will be positive

    Citrate Utilization Test

    • Used to determine if bacteria can utilize sodium citrate as their only carbon source
    • Distinguishes between coliforms, such as Klebsiella aerogenes (citrate-positive) and E. coli (citrate-negative)
    • Simmon's citrate agar is used, containing bromothymol blue as the pH indicator
    • Citrate metabolism results in alkaline end products, causing the medium to change from green to blue in a positive result

    Cultivation Process

    • Cultivation is the process of propagating organisms by providing proper environmental conditions, mainly to isolate a pure culture and identify pathogenic bacteria from clinical samples.

    Requirements for Growth

    • Organisms require all elements in their organic matter, a full complement of ions, and a source of energy for growth.
    • Factors to be controlled during growth include nutrients, pH, temperature, aeration, salt concentration, and ionic strength.

    Cultivation in Laboratory Setting

    • Cultivation in a laboratory setting is done using a suitable medium, which is a specifically prepared environment that ensures the preservation, growth, and reproduction of bacteria.

    Medium Requirements

    • Media must contain all necessary chemical components required by bacteria.
    • Media must have a neutral or weakly alkaline pH environment.
    • Media must be sterile.

    Components of Media

    • Nutrients (proteins, amino acids)
    • Energy source (carbohydrates)
    • Essential metals and minerals
    • Buffering agents (phosphate, acetate)
    • Indicators for pH change (phenolred)
    • Selective agents (antibiotics)
    • Gelling compound – agar-agar

    Classification of Bacterial Culture Media

    • Bacterial culture media can be classified based on composition, consistency, and purpose.

    Bacterial Culture Media

    • Liquid media:
    • Contains nutrients, but no gelling agents like gelatin or agar
    • Examples: nutrient broth, tryptic soy broth, brain-heart infusion broth, selenite F broth, glycose broth, alkaline peptone water
    • Used for: propagating many organisms, accumulating bacterial toxic end products, fermentation studies

    Semi-Solid Media

    • Prepared with agar at 0.5% or less concentration
    • Used for: cultivating microaerophilic bacteria, determining bacterial motility
    • Examples: motility test medium, Stuart's and Amies transport media

    Solid Media

    • Contain agar at a concentration of 1.5-2.0% or other inert solidifying agents
    • Bacteria grow in a physically informative way, forming visible colonies, aiding identification
    • Examples: nutrient agar, blood agar, MacConkey agar, Baird-Parker agar

    Agar-Agar

    • Agar-agar is a polysaccharide extracted from the cell walls of red algae.
    • It is inert and non-nutritious, requiring impregnation with nutrients (e.g., beef extract and peptone) to support life.
    • Agar-agar melts at 97-100 °C and solidifies at 42 °C.
    • Media containing 1-5% agar-agar typically have "agar" in their name.

    Bacterial Culture Media

    Classification by Composition

    • Chemically defined media: exact amounts of highly purified inorganic and organic ingredients are known (e.g., citrate broth).
    • Complex media: prepared using digests of microbial, animal, or plant products (e.g., casein, beef extract, yeast extract); exact composition is unknown.
    • General-purpose (basal) media: capable of sustaining growth of a large variety of less fastidious bacteria (e.g., nutrient agar, used for primary isolation of microorganisms).

    Bacterial Culture Media

    • Enriched media: basal media with additional nutrients (e.g., blood, serum, egg yolk) supporting growth of most bacteria, including fastidious species.
    • Examples of enriched media: blood agar, chocolate agar, Loeffler's medium.

    Blood Agar

    • 5-10% blood added to a general purpose base.
    • Important in clinical medium for differentiating and identifying bacterial pathogens based on hemolysis patterns.
    • Hemolysis patterns can be partial or complete.

    Chocolate Agar

    • Heating blood agar to rupture red blood cells, releasing nutrients for fastidious bacteria growth.
    • Not used for hemolysis testing.
    • Ruptured red blood cells give the plate a chocolate brown color.

    Differential (Indicator) Media

    • Designed to give a presumptive identification of bacterial colonies based on biochemical reactions.
    • Often contain fermentable sugars and a pH indicator for a color change in the media.
    • Examples: Bromocresol purple lactose agar for enterobacteriacea, nonselective Cysteine Lactose Electrolyte deficient (CLED) agar for urinary pathogens.

    Selective Media

    • Contains inhibitory substances that prevent unwanted bacterial species growth and promote growth of a particular bacterium or group.
    • Used in diagnostic bacteriology.
    • Examples: Lowenstein Jensen medium for mycobacteria, 6.5% NaCl broth for enterococci.

    Enrichment Media

    • Liquid media that permit growth and detection of a particular bacterium, which may have been a small proportion of the original inoculum.
    • Selective, inhibiting normal flora growth, allowing the desired bacterium to grow until it reaches the desired numbers.
    • Examples: Selenite broth for salmonellae, Rappaport-Vassiliadis soya peptone broth for salmonella, Fraser broth for Listeria.

    Selective-Differential Media

    • Contains both selective and differential agents.
    • Examples: Mannitol salt agar inhibiting other bacteria except staphylococci, MacConkey agar containing crystal violet and lactose, Baird-Parker agar, XLD agar.

    Transport Media

    • Ensures viability of potential pathogens in the sample and prevents drying and overgrowth of contaminating organisms.
    • Examples: Cary Blair transport medium, Stuart's transport medium (semisolid), Amies transport medium with/without charcoal (semisolid).

    Special Media for Anaerobes

    • Requires less oxygenation concentration and extra nutrients.
    • Examples: Thioglycollate broth, Robertson's cooked meat medium.
    • Other special media: Miller Hilton agar for antibiogram and bacterial enumeration.

    Collection of Tissue Samples

    • Tissue samples should be collected as soon as possible after death (post-mortem) to ensure optimal quality.
    • Samples should be at least 1 cm³ and placed in separate sterile screw-capped jars.
    • Order of collection: outside body cavities, thorax, and abdominal tissues.

    Samples in Case of Abortion

    • Whole fetus and placenta should be submitted for examination.
    • If not possible, collect tissue samples from the fetus, affected placenta, and fetal stomach contents (abomasal for ruminants), and uterine discharge.
    • Rule out zoonotic pathogens such as Salmonella, Listeria, Leptospira, and Brucella.

    Swabs and Discharges

    • Fluids are preferred over swabs, as they provide a larger sample volume and increase the likelihood of detecting the causal organism.
    • Short cotton swabs are generally unsatisfactory for obtaining nasopharyngeal specimens in large animals.
    • Samples should be collected from the site of infection, and guarded swabs may be necessary to avoid contaminants.

    Samples from Skin Lesions

    • Intact pustules or vesicles: disinfect the surface with 70% alcohol, allow it to dry, then aspirate the material with a sterile syringe and fine needle.
    • Raw surfaces of ulcers: take a swab or biopsy of wound tissue after cleaning and breaching the superficial area.
    • Skin scrapings can be done using a blunt scalpel, scraping until blood is seen.

    Blood Samples

    • Withdraw blood into an aseptically syringe or vacutainer.
    • Prevent blood from clotting, and do not freeze whole blood prior to submission to the laboratory.
    • Take multiple samples within a 24-hour period, as bacteria can be intermittent.

    Fecal and Urine Samples

    • Feces: collect a sample the size of a thumb and forward to the laboratory without transport medium.
    • Rectal swabs should be placed in a transport medium to avoid desiccation.
    • Urine samples: collection by cystocentesis, catheter, or mid-stream urine is acceptable.

    Samples from Abscesses

    • Collect several milliliters of pus using a syringe, and/or take a swab from the wall of the abscess (not the center).
    • Shave and disinfect the area with 70% ethanol or iodine solution before collection.

    Samples from Eyes and Ears

    • Eyes: collect conjunctival swabs, corneal scrapings, or conjunctival biopsies.
    • Ears: take swabs, ensuring not to touch the ear canal.

    Collection of Mastitic Milk

    • Collect samples as soon as possible after first clinical signs are seen, before treatment with intramammary or systemic antibiotics.
    • Udder and teats must be dry and disinfected with 70% ethanol before collection.

    Specimens for Anaerobic Culture

    • Many anaerobes die within 20 minutes if exposed to oxygen, so specimens from animals that have died >4 hours ago are usually unsuitable.
    • Use anaerobic transport medium, and prioritize processing in the microbiology laboratory.

    Transport of Specimens

    • Process specimens as soon as possible to obtain useful outcomes.
    • Drying and exposure to noxious atmospheres compromise specimens.
    • Transport media do not contain nutrients, but microorganisms remain viable for at least 24-48 hours.
    • Use cold packs or wet ice during transportation, and refrigerate samples at +4°C if delayed.

    Sample Submission

    • Samples must be submitted individually in separate leak-proof containers, clearly marked, and indicating the identity of the samples, animal ID, and date of collection.
    • Use sample submission forms, which must be completed by the veterinary practitioner and contain specific details of the tests required, clinical history, differential diagnosis, vaccination, therapy, and animal age.

    Bacteriological Examination

    • Bacteriological examination involves cultivation, isolation, and identification of the causative agent.
    • A pure culture is required for successful identification.

    Bacteriological Examination Plan

    • The 5i approach is used: inoculation, incubation, isolation, inspection, and identification.
    • Inoculation: introducing bacteria into an environment where they will grow and reproduce.
    • Incubation: maintaining bacteria at the most favorable temperature and atmosphere for 24-48 hours to form colonies.
    • Isolation: separating bacteria from a natural living mix of microorganisms to obtain separate colonies on solid media.
    • Inspection: evaluating colony morphology and observing expected characteristics.
    • Identification: using different tests, such as biochemical, serological, and molecular tests.

    Bacterial Identification and Characterization

    • Phenotypic methods:
      • Cellular morphology: using light microscopy.
      • Staining characteristics: Gram status, cell structures (flagella, endospores, etc.).
      • Growth characteristics: culturing requirements (oxygen, osmotic pressure, temperature, etc.), colony morphology.
      • Biochemical characteristics: oxidase, catalase, urease, nitrate, and other tests.
    • Genotypic methods:
      • Give results that are a product of the expression of bacterial genes.
      • These approaches can be successful in bacterial identification but often require days to weeks for some pathogens.

    Timeframe

    • Bacteriological examination of a sample generally takes about 3-10 days.

    Bacteriological Day-by-Day Examination Plan

    • On the first day, the sample is inoculated on different media and direct microscopy can be done to guide the choice of therapy before culture results are available.
    • Direct microscopy (direct smear) can help identify the shape and staining characteristics of the infectious agent.

    Staining Methods

    • Gram stain: useful for observing gram-bacteria, but cellular contents of the sample are not readily discernible.
    • Romanowsky-type stain (Wright's or Giemsa): used to differentiate cells in pathological specimens, detect parasites in blood, and more commonly used in cytology.

    Primary Isolation

    • Media plates are streaked in a way that produces individual isolated colonies after incubation.
    • Media used include:
      • Blood agar
      • MacConkey agar (for Gram-negative bacteria)
      • CNAA (for Gram-positive bacteria)
      • Other selective, differential, or selective-differential media
    • Inoculation is performed in a semi-quantitative fashion to enable estimation of the relative abundance of organisms in the sample.

    Growth Assessment (2nd Day)

    • Evaluation of bacterial growth on different media
    • Determination of whether it's a pure culture or a mixed culture
    • Number of types of colonies present
    • Possible pathogen is determined by specific criteria

    Colony Morphology and Identification (2nd Day)

    • Evaluation of colony morphology on different media
    • Microscopy - Gram staining
    • Rapid biochemical tests (e.g., production of catalase, oxidase, indole, coagulase)
    • Inoculation of other biochemical tests, antimicrobial susceptibility testing, serological tests, and bioassays

    Evaluation of Test Results (3rd Day)

    • Evaluation of traditional biochemical tests, complex biochemical tests, antibiogram, and other tests
    • Interpretation of diagnostic results depends on clinical and epidemiological information supplied by the veterinary clinician
    • A negative diagnostic report does not necessarily mean the suspected microorganism is not the aetiological agent of the condition.

    Interpretation of Diagnostic Results

    • Detection of bacteria doesn't always establish a diagnosis - results must be interpreted in combination with clinical signs, vaccination history, etc.
    • Isolation of microorganisms may represent contamination of sample by feces, soil, or post-mortem invasion.
    • Some bacteria may be shed intermittently, and a repeat sample following a negative examination report might be worthwhile.
    • Conflicting results may reflect the sensitivity of different assays.
    • Teamwork between veterinary practice and laboratory is essential for the benefit of the owner and pet.

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    1. Cultivation of bacteria 5. Physical methods of microbial control 6. Chemical methods of microbial control 7. Sample collection 8. Bacteriological examination of samples 9. Biochemical tests

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