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  • 1. UNIT IIChapter-03.indd 21 7/11/2012 5:05:44 PM
  • 2. Bacteria Gram stain Gram-positive bacteria Gram-negative bacteria Cocci Bacilli Cocci Bacilli – Staphylococcus – Listeria – Neisseria – Escherichia – Streptococcus – Corynebacterium – Moraxella – Klebsiella – Enterococcus – Nocardia – Salmonella – Actinomyces – Shigella Refer Algorithm for – Enterobacter – Clostridium Gram-negative cocci Refer Algorithm for – Citrobacter Gram-positive cocci – Proteus – Yersinia Refer Algorithm for Gram-positive bacilli – Pseudomonas – Acinetobacter – Campylobacter Gram-negative – Helicobacter curved bacilli – Vibrio Refer Algorithm for Gram-negative bacilliChapter-03.indd 22 7/11/2012 5:05:46 PM
  • 3. 3 General Bacteriology BACTERIAL PHYSIOLOGY Classification on the basis of nutrition Phototrophs: Bacteria using sunlight as a source of energy Chemotrophs: Bacteria using chemical reactions as a source of energy Classification on the basis of source of compounds needed for survival Autotrophs: Bacteria which can synthesize all organic compounds on their own Heterotrophs: Bacteria which are unable to synthesize their organic metabolites and rely on preformed compounds Classification on the basis of bacteria’s relationship with oxygen and carbon dioxide Category Requirement Examples Obligate aerobe 15% to 21% O2 Mycobacterium tuberculosis Pseudomonas Micrococcus Microaerophilic 5% O2 Campylobacter Helicobacter Brucella abortus Facultative anaerobe Multiplies equally well in the presence or Enterobacteriaceae absence of O2 Staphylococcus Aerotolerant anaerobe Reduced concentration of O2 Most strains of Propionibacterium, Lactobacillus, some Clostridium species (C. histolyticum) Obligate anaerobe Strict anaerobic environment (0% O2) Most bacteroid spp., many Clostridium spp. (C. novyi), Eubacterium, Fusobacterium spp., Peptostreptococcus spp. Capnophilic 5% to 10% CO2 Neisseria, Haemophilus spp. Reference: 1. Textbook of Diagnostic Microbiology, Mahon, Lehman and Manuselis, 4th Ed., Pg. 504. Classification on the basis of temperature requirements Mesophilic: Bacteria growing best at temperatures of 25° to 40°C Psychrophilic: Bacteria growing best at temperatures below 20°C Thermophilic: Bacteria growing best at higher temperatures of 55°C to 80°C, e.g., Bacillus stearothermophilus BACTERIAL GROWTH CURVE Four phases: Lag phase→Logarithmic or exponential phase→Stationary phase→Phase of decline Change in bacterial morphology Corresponding phase Maximum cell size End of lag phase Uniform staining Log phaseChapter-03.indd 23 7/11/2012 5:05:46 PM
  • 4. 24 SMART STUDY SERIES: MICROBIOLOGY Variable Gram staining Stationary phase Irregular staining Stationary phase Presence of spores Stationary phase Presence of granules Stationary phase Production of exotoxins and antibiotics Stationary phase Involution forms Phase of decline BACTERIAL STAINS Positive stains: The dye used in reality stains the specimen for which it is intended (e.g., Gram stain, acid-fast stain) Negative stain: The dye does not adhere itself to the specimen but stains its outer boundary (e.g., India ink stain) Name of the Stain Importance Gram stain • Stains bacterial cell wall (peptidoglycan) • Distinguishes bacteria into Gram positive and Gram negative (Ziehl–Neelsen) acid-fast stain • Stains bacterial cell wall (mycolic acids) • Distinguishes bacteria into acid-fast and nonacid-fast Kinyoun stain Modification of acid-fast stain (no use of heat) Auramine O stain Fluorescent stain for Mycobacterium tuberculosis Schaeffer–Fulton stain Stains bacterial endospores Albert stain Stains metachromatic granules of Corynebacterium diphtheriae Ponder stain Neisser stain Pugh stain India Ink stain Stains bacterial capsules Nigrosin Methylene blue with anticapsular sera Stains capsules of Streptococcus pneumoniae (Quellung Reaction) Silver impregnation method Stains bacterial flagella Leifson stain Giemsa stain Stains intracellular pathogens (e.g., elementary bodies of Chlamydia, Rickettsia) Fontana stain Stains treponemes Levaditi stain Stains treponemes in tissue sections Diene’s stain Stains Mycoplasma Macchiavello stain Stains Rickettsia Warthin–Starry stain Stains spirochetes Dieterle stain BACTERIAL APPENDAGES Flagella Organs of locomotion and motility Type Arrangement Examples Monotrichous Bacteria which have one polar flagellum Vibrio cholerae Lophotrichous Bacteria with a tuft of several polar flagella Spirilla Amphitrichous Bacteria with flagella at both the ends Campylobacter (some species) Peritrichous Bacteria with flagella distributed all over the surface Members of Enterobacteriaceae, of the bacterium Bacillus sp., Listeria monocytogenesChapter-03.indd 24 7/11/2012 5:05:46 PM
  • 5. GENERAL BACTERIOLOGY 25 Some noteworthy points Gram-negative bacilli Motile • Escherichia coli • Enterobacter • Proteus • Salmonella sp. Nonmotile • Klebsiella • Shigella • Salmonella Gallinarum-Pullorum • Yersinia pestis Gram-positive cocci Motile • Enterococcus casseliflavus • Enterococcus gallinarum Gram-positive bacilli Nonmotile • Bacillus anthracis • Clostridium perfringens • Clostridium tetani type 6 Bacteria which are motile at 25°C and nonmotile at 37°C (most bacteria are • Listeria monocytogenes motile at 37°C) • Yersinia enterocolitica • Yersinia pseudotuberculosis • Sphingomonas paucimobilis • Tatumella • Some species of Vibrio Fimbria Organs of attachment and adhesion Demonstrated by electron microscopy or agglutination by fimbriae-specific sera Toxins Exotoxin Endotoxin Excreted by organisms Integral part of cell wall Both by Gram-positive and Gram-negative bacteria Found mostly in Gram-negative bacteria Polypeptide Lipopolysaccharide complex Relatively unstable, heat labile (60°C) Relatively stable, heat tolerant Highly antigenic, neutralized by antitoxin Weakly immunogenic, antibodies are antitoxic Can be toxoided Cannot be toxoided Highly toxic, fatal in μg quantities Moderately toxic Usually binds to specific receptors Specific receptors not found Not pyrogenic usually Fever by induction of interleukin 1 (IL-1) production Located on extrachromosomal genes (e.g., plasmids) Located on chromosomal genes CULTURE MEDIA Classification Based on Consistency Liquid Semisolid Solid Biphasic: Castaneda system for blood culture suspected for brucellosis Agar agar (simply called agar) is the most commonly used solidifying agent Physical properties of agar melts at 95°C (sol) and solidifies at 42°C (gel), does not contribute or has any nutritive property, is not hydrolyzed by most bacteria, and is usually free from growth-promoting or growth-retarding substances. Most commonly, it is used at concentrations of 1% to 3% to make a solid agar medium. Semisolid agar: Reducing the amount of agar to 0.2% to 0.5% renders a medium semisolid.Chapter-03.indd 25 7/11/2012 5:05:46 PM
  • 6. 26 SMART STUDY SERIES: MICROBIOLOGY List of bacteria that can swarm on the semisolid media Proteus mirabilis Proteus vulgaris Vibrio parahaemolyticus V. alginolyticus Clostridium tetani Classification Based on Nutritional Component Simple: e.g., nutrient broth Complex: e.g., blood agar Synthetic (or defined): e.g., peptone water Classification Based on Functional use or Application BASAL MEDIA are basically simple media that supports most • Peptone water nonfastidious bacteria • Nutrient broth • Nutrient agar ENRICHED MEDIA: Addition of extra nutrients to grow • Blood agar nutritionally exacting (fastidious) bacteria • Chocolate agar • Loeffler’s serum slope SELECTIVE and ENRICHMENT MEDIA are designed to • Selenite F broth inhibit unwanted commensal or contaminating bacteria and • Tetrathionate broth help to recover pathogen from a mixture of bacteria. • Alkaline peptone water While selective media are agar based, enrichment media • Thiosulphate-citrate-bile salts-sucrose (TCBS) agar are liquid in consistencyQ. • Xylose lysine deoxycholate (XLD) agar DIFFERENTIAL MEDIA or INDICATOR MEDIA: Different • MacConkey’s agar bacteria can be recognized on the basis of their colony • Cystein lactose electrolyte deficient (CLED) agar • Thiosulphate-citrate-bile salts-sucrose (TCBS) agar • Xylose lysine deoxycholate (XLD) agar TRANSPORT MEDIA • Stuart’s and Amie’s: Addition of charcoal serves to neutralize inhibitory factors • Cary Blair medium and Venkatraman Ramakrishnan medium: To transport feces from suspected cholera patients • Sach’s buffered glycerol saline: to transport feces from patients suspected to be suffering from bacillary dysentery ANAEROBIC MEDIA: Anaerobic bacteria need special media • Robertson Cooked Meat Broth (RCMB) for growth because they need low oxygen content, reduced oxidation–reduction potential, and extra nutrients QUESTIONS 1. Mesophilic organisms are those that grow best at [Kerala 2000] a. –20°C to –7°C b. –7°C to +20°C c. 25°C to 40°C d. 55°C to 80°C e. 90°C to 20°C 2. Obligatory anaerobes cannot withstand oxygen because of absence of [APPGE 2005] a. Superoxide dismutase b. Catalase c. Peroxidase d. Cytochrome oxidase 3. Obligatory anaerobes are all except [PGI 1999] a. Clostridium botulinum b. Eikenella corrodens c. Bacteroides d. Helicobacter pylori 4. All of the following organisms are known to survive intracellularly except [AIPG 2005] a. Neisseria meningitidis b. Salmonella typhi c. Streptococcus pyogenes d. Legionella pneumophilaChapter-03.indd 26 7/11/2012 5:05:46 PM
  • 7. GENERAL BACTERIOLOGY 27 5. Cell wall structure is found in all except [UP 2007, 2006] a. Staphylococcus aureus b. Pseudomonas aeruginosa c. Mycoplasma pneumoniae d. Corynebacterium diphtheriae 6. Peptidoglycans are present in [UP 2007, 2006] a. Gram-negative bacteria b. Gram-positive bacteria c. Fungi d. Protozoa 7. Which of the following can be stained by the acid-fast method? [PGI 1988] a. Mycobacteria b. Nocardia c. Actinomyces d. Spermatic head e. All 8. Acid-fast organisms are [PGI Jun 2008] a. Spores b. Nocardia c. Legionella d. Rhodococcus e. Anaplasma 9. Dark ground microscopy is used for detection of [AIPG 1989] a. Spirochetes b. Chlamydia c. Fungi d. Virus 10. Which of the following staining methods is an example of negative staining? [Karnataka 1994] a. Gram staining b. Fontana staining c. India ink preparation d. Ziehl–Neelsen staining 11. All are capsulated bacteria except [AP 1996] a. Streptococcus pneumoniae b. Klebsiella pneumoniae c. Vibrio cholerae d. Haemophilus influenzae 12. Safety pin appearance is shown by [AI 1989] a. Mycobacterium leprae b. Mycobacterium tuberculosis c. Vibrio cholerae d. Staphylococcus aureus e. Yersinia pestis 13. Organ of adhesion of bacteria is [AP 1988] a. Capsule b. Slime c. Flagella d. Fimbriae 14. Bacteria with tuft of flagellae at one end are called [Assam 1995] a. Monotrichate b. Peritrichate c. Bipolar d. Lophotrichate 15. Lipopolysaccharide structure is a characteristic of [CUPGEE 1995] a. Exotoxin b. Endotoxin c. Both d. None 16. Which of the following is true about exotoxin? [PGI 1997] a. Heat stable b. Produced both by Gram-negative and Gram-positive organisms c. No enzymatic action d. Produced by Gram-positive organism onlyChapter-03.indd 27 7/11/2012 5:05:46 PM
  • 8. 28 SMART STUDY SERIES: MICROBIOLOGY 17. The endotoxin which leads to endotoxic shock is actually [AIIMS 2000] a. Lipoprotein b. Lipopolysaccharide c. Polysaccharide d. Polyamide 18. Which of the following toxins acts by inhibiting protein synthesis? [AI 2004] a. Cholera toxin b. Shiga toxin c. Pertussis toxin d. Heat-labile toxin of enterotoxigenic Escherichia coli 19. The usual concentration of agar used for agar medium is [Delhi 1983, 1985] a. 10% b. 20% c. 40% d. 80% 20. Which one of the following is true? [AI 2007] a. Agar has nutrient properties b. Chocolate medium is a selective medium c. Addition of selective substances in a solid medium is called enrichment media d. Nutrient broth is a basal medium 21. Heating and subsequent plating is a method used for isolating [PGI 1983] a. Corynebacterium b. Vibrio c. Salmonella d. Clostridium 22. Selenite-F broth is an enrichment media for [AIIMS 1980] a. Salmonella b. Shigella c. Escherichia coli d. Campylobactor 23. Smith Noguchi’s media is used for [AIIMS 1979, 1985; JIPMER 1981; AMU 1986] a. Salmonella b. Kiebsiella c. Spirochetes d. Bacillus 24. Dorset’s egg medium is used for the cultivation of [JIPMER 1980, AMC 1983] a. Staphylococcus b. Streptococcus c. Gonococcus d. Mycobacterium 25. Culture medium for Corynebacterium diphtheriae [JIPMER 1995, 2005] a. Loeffler’s serum slope b. MacConkey agar c. Sabouraud agar d. Lowenstein–Jensen medium ANSWERS Answer 1: c (25°C to 40°C) Temperature plays a very significant role in the growth and metabolism of bacteria. Most bacteria of medical importance are able to grow within a temperature range of 25°C to 40°C or an average incubation temperature of 37°C. The lowest temperature that kills a bacterium under standard conditions in a given time is known as thermal death point. Reference: 1. Ananthanarayan, 8th Ed., Pg. 27. Answer 2: a (Superoxide dismutase), b (Catalase), and c (Peroxidase) Obligate anaerobes are those bacteria that grow in the absence of free oxygen but fail to multiply in the presence of oxygen on the surface of nutritionally adequate solid media incubated in room air or in a CO2 incubator (containing 5% to 10% CO2).Chapter-03.indd 28 7/11/2012 5:05:46 PM
  • 9. GENERAL BACTERIOLOGY 29 On exposure to molecular oxygen, a series of reactions is initiated within the bacterial cells (mediated by flavoproteins), and they result in the production of negatively charged superoxide radicals hydrogen peroxide other toxic oxygen reduction products Catalase, superoxide dismutase, and possibly peroxidases ameliorate the damaging effects of hydrogen peroxide and superoxide by converting these compounds into oxygen and water. Superoxide dismutase removes the superoxide free radical in the following reaction: 2H+ + 2 O2– → H2O2 + O2 Catalase removes hydrogen peroxide in the following reaction: 2 H2O2– → 2 H2O + O2 Option d Cytochrome oxidase enzyme helps in respiratory metabolism by catalyzing the reoxidation of the last cytochrome molecule in the electron transport chain by molecular oxygen. This final process results in the reduction of molecular oxygen to water and results in the conversion of glucose into energy in the form of ATP. EXTRA EDGE Catalase-Positive Organisms Oxidase-Positive Organisms Staphylococcus Micrococcus Enterobacteriaceae (Escherichia coli, Shigella, Salmonella, Klebsiella, Proteus) Pseudomonas Neisseria meningitidis Vibrio Pseudomonas Aeromonas Nocardia Plesiomonas Atypical Mycobacteria Helicobacter Legionella Campylobacter Reference: 1. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, 6th Ed., Pg. 879. Answer 3: a (Clostridium botulinum) and c (Bacteroides) [Please refer to the text for the classification on the basis of bacteria’s relationship with oxygen and carbon dioxide] EXTRA EDGE Classification of Anaerobes Bacilli Cocci Gram Positive SPORING Peptococcus Clostridium Peptostreptococcus Filifactor Finegoldia NONSPORING Actinomyces Bifidobacterium Eubacterium Lactobacillus Mobiluncus Propionibacterium Gram Negative Bacteroides Veillonella Prevotella Megasphaera Porphyromonas Fusobacterium Leptotrichia References: 1. Ananthanarayan, 8th Ed., Pg. 265. 2. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, 6th Ed. Pg. 881.Chapter-03.indd 29 7/11/2012 5:05:46 PM
  • 10. 30 SMART STUDY SERIES: MICROBIOLOGY Answer 4: c (S. pyogenes) Some organisms prefer to remain intracellular to achieve protection from humoral immune system protection from complement system abundance of nutrients inside the cells Types of intracellular bacteria Obligate intracellular bacteria: They are compulsorily adapted to survive in host cells by losing their ability to live an extracellular lifestyle. Facultative intracellular bacteria: Reside mainly in macrophages but certain other host cells may also get infected Obligate Intracellular Facultative Intracellular Rickettsia rickettsiae Legionella pneumophila Chlamydia trachomatis Brucella melitensis Chlamydophila pneumoniae Bordetella Salmonella typhi, S. typhimurium Mycobacterium tuberculosis, M. leprae Shigella flexneri Listeria monocytogenes Leishmania Plasmodium Trypanosoma cruzi Toxoplasma gondii Microsporidia Histoplasma capsulatum Reference: 1. Primer to the Immune Response, Academic Cell Update Edition, Pg. 210. Answer 5: c (M. pneumoniae) Mycoplasmas Cell wall–lacking bacteria containing no peptidoglycan Not attacked by cell wall–inhibiting antimicrobial agents (e.g., penicillins and cephalosporins) Contain sterols in their membranes Effect of removal of cell wall on bacteria If the bacterial wall is removed in osmotically protective media either by hydrolysis with lysozyme or by blocking peptidoglycan synthesis with an antibiotic such as penicillin, such treatments liberate protoplasts from Gram- positive cells and spheroplasts (which retain outer membrane and entrapped peptidoglycan) from Gram-negative cells. If such cells are able to grow and divide, they are called L forms. L forms are difficult to cultivate and usually require a medium that is solidified with agar as well as having the suitable osmotic strength. The difference between L forms and mycoplasmas is that when the murein is allowed to reform, L forms revert to their original bacterial shape, but mycoplasmas never do. References: 1. Ananthanarayan, 8th Ed., Pg. 387. 2. Jawetz, Melnick and Adelberg’s Medical Microbiology, 25th Ed., Pg. 28. Answer 6: a (Gram-negative bacteria) and b (Gram-positive bacteria) Peptidoglycan, a component of cell wall is present in both Gram-positive and Gram-negative bacteria. It is the arrangement and presence of other components that differentiate between the cell wall of Gram- positive and Gram-negative bacteria. Differences between a Gram-positive and Gram-negative cell wall Property Gram positive Gram negative Thickness of wall Thick (20 to 80 nm) Thin (10 nm) Number of layers 1 2Chapter-03.indd 30 7/11/2012 5:05:46 PM
  • 11. GENERAL BACTERIOLOGY 31 Property Gram positive Gram negative Peptidoglycan (murein) content >50% 10% to 20% Teichoic acids in wall Present Absent Lipid and lipoprotein content 0 to 3% 58% Protein content 0 9% Lipopolysaccharide content 0 13% References: 1. Ananthanarayan, 8th Ed., Pg. 17. 2. Todar’s Online Textbook of Bacteriology. Answer 7: a (Mycobacteria), b (Nocardia) and d (Spermatic head) Acid-fast structure % of H2SO4 used for decolorization M. tuberculosis 20 M. leprae 5 Nocardia 1 Bacterial spores 0.5 Cysts of Isospora, Cyclospora, Cryptosporidium 0.25 to 0.5 Kinyoun Stain It is a method of staining acid-fast microorganisms which do not involve heating the slides being stained. The Kinyoun staining method uses carbol fuchsin as a primary stain, followed by decolorization with an acid- alcohol solution and methylene blue as a counterstain. Kinyoun carbol fuschsin has a greater concentration of phenol and basic fuchsin and does not require heating in order to stain properly. Advantage: As heating is not needed, it facilitates examination in large numbers for epidemiological studies. References: 1. Mackie and McCartney Practical Medical Microbiology, 14th Ed., Pg. 793. 2. Manual of Basic Techniques for a Health Laboratory By WHO, Pg. 257. Answer 8: a (Spores), b (Nocardia), c (Legionella) and d (Rhodococcus) Acid-fast stain More popularly known as Ziehl–Neelsen staining Acid-fast bacteria are those that retain primary stain, carbol fuchsin (basic fuchsin dissolved in a phenol-alcohol- water mixture) and resist decolorization with acid-alcohol (hydrochloric acid in alcohol) or only acid (sulphuric acid) Examples of acid-fast structures: Mycobacterium tuberculosis M. leprae Bacterial endospores Clubs formed by Nocardia Cysts of Coccidian parasites (Cryptosporidium, Isospora, Cyclospora) Brucella abortus (in infected tissue or exudates) Legionella micdadei Spermatic head Reference: 1. Mackie and McCartney, Practical Medical Microbiology, 14th Ed., Pg. 802. Answer 9: a (Spirochetes) DARK GROUND MICROSCOPE Technique for improving the contrast of unstained, transparent specimens Principle: Use of reflected light instead of transmitted lightChapter-03.indd 31 7/11/2012 5:05:46 PM
  • 12. 32 SMART STUDY SERIES: MICROBIOLOGY Advantages: It enhances the visual effect produced by the material being examined, and the material need not be stained. The increased visual impact means that lesser magnification is required for searching for objects. Use: For slender organisms like spirochetes from direct urethral discharge and in serological tests like Treponema pallidum immobilization assay Reference: 1. Ananthanarayan, 8th Ed., Pg. 13. Answer 10: c (India ink preparation) Negative stain stains the background of an individual cell, rather than staining the cell itself. It is also called Relief staining or Background staining. Use: Staining of polysaccharide capsule Principle: Acidic dyes like India ink, nigrosin, or eosin have a negatively charged chromophore and do not combine readily with the negatively charged bacterial cytoplasm. Instead, they form a deposit around the bacterium, leaving the organism colorless. Reference: 1. Essentials of Biotechnology, By Ulhas K. Patil, Kalyani Muskan, Pg. 359. Answer 11: c (V. cholerae) Glycocalyx is defined as the polysaccharide-containing material lying outside the cell. Capsule A condensed, well-defined layer closely surrounding the cell that excludes particles, such as India ink, is referred to as a capsule. Slime layer If the glycocalyx is loosely associated with the cell and does not exclude particles, it is referred to as a slime layer. EXTRA EDGE List of capsulated organisms Streptococcus pneumoniae Klebsiella pneumoniae Hemophilus influenzae Pseudomonas aeruginosa Neisseria meningitides Cryptococcus neoformans Bacillus anthracis Escherichia coli Enterobacter aerogenes All organisms have a polysaccharide capsule except Bacillus antharcis, which possesses a polypeptide capsule. Reference: 1. Jawetz, Melnick and Adelberg’s Medical Microbiology, 25th Ed., Pg. 30. Answer 12: e (Yersinia pestis) Bipolar staining/safety pin appearance on staining with methylene blue is conspicuous in many bacteria, e.g., Yersinia pestis Vibrio parahaemolyticus Burkholderia mallei Burkholderia pseudomallei Campylobacter granulomatis (now known as Klebsiella granulomatis) Reference: 1. Ananthanarayan, 8th Ed., Pg. 320. Answer 13: d (Fimbriae) Reference: 1. Ananthanarayan, 8th Ed., Pg. 21. Answer 14: d (Lophotrichate) Reference: 1. Ananthanarayan, 8th Ed., Pg. 20.Chapter-03.indd 32 7/11/2012 5:05:46 PM
  • 13. GENERAL BACTERIOLOGY 33 Answer 15: b (Endotoxin) Reference: 1. Ananthanarayan, 8th Ed., Pg. 78. Answer 16: b (Produced by Gram-negative and Gram-positive organisms) Reference: 1. Ananthanarayan, 8th Ed., Pg. 78. Answer 17: b (Lipopolysaccharide) The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS (lipopolysaccharide). LPS consists of three components or regions: Lipid A, an R polysaccharide, and an O polysaccharide. LPS elicits a variety of inflammatory responses in an animal, and it activates complement by the alternative (properdin) pathway, so it may be a part of the pathology of Gram-negative bacterial infections. Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria. The biological activity of endotoxin is associated with the LPS. Toxicity is associated with the lipid component (lipid A), and immunogenicity is associated with the polysaccharide components. References: 1. Ananthanarayan, 8th Ed., Pg. 283. 2. Todar Online Textbook of Bacteriology, 2011. Answer 18: b (Shiga toxin) MECHANISM OF ACTION OF BACTERIAL TOXINS Acting by inhibition of protein synthesis ADP-ribosylating activity by inactivation of elongation factor 2 (EF-2) Diphtheria toxin of Corynebacterium diphtheriae Exotoxin A of Pseudomonas aeruginosa Interfering with 60S ribosomal subunit Shiga toxin of Shigella dysenteriae Verotoxin (Shiga-like toxin) of enterohemorrhagic Escherichia coli (EHEC) Acting on the nervous system Clostridium tetani: Tetanospasmin acts by inhibiting the release of inhibitory neurotransmitters (glycine and GABA) in the central nervous system and leading to spastic paralysis of respiratory muscles Clostridium botulinum: Botulinum toxin acts by inhibiting release of acetylcholine at neuromuscular junction in peripheral nervous system and leading to flaccid paralysis of the muscles involved. Inducing the levels of cAMP in the cells Heat-labile toxin (LT) of enterotoxigenic E. coli (ETEC) Cholera toxin (CT) of Vibrio cholerae Anthrax toxin of Bacillus anthracis Pertussis toxin of Bordetella pertussis Inducing the levels of cGMP in the cells Heat-stable toxin (ST) of ETEC Superantigens Toxic shock syndrome toxin-1 (TSST-1) of Staphylococcus aureus Pyrogenic toxin of Streptococcus pyogenes Cytolytic toxins α-Toxin of Clostridium perfringens (having lecithinase activity) α-Shemolysin of S. aureus (pore-forming activity)Chapter-03.indd 33 7/11/2012 5:05:46 PM
  • 14. 34 SMART STUDY SERIES: MICROBIOLOGY LOCATION OF VARIOUS BACTERIAL TOXINS (AND OTHER ENZYMES) Plasmid borne Tetanospasmin of C. tetani Anthrax toxin of B. anthracis Pertussis toxin of B. pertussis Heat-labile toxin (LT) of ETEC Penicillinase enzyme of S. aureus Chromosomal Cholera toxin (CT) of V. cholerae Pertussis toxin of B. pertussis Shiga toxin of S. dysenteriae Exotoxin A of P. aeruginosa Pyrogenic toxin B of S. pyogenes Phage encoded Botulinum toxin of C. botulinum Diphtheria toxin of C. diphtheriae Verotoxin (Shiga-like toxin) of EHEC Pyrogenic toxin A, C of S. pyogenes Reference: 1. Ananthanarayan, 8th Ed., Pg. 283. Answer 19: (None of the above) The usual concentration of agar used is 1% to 2%. Answer 20: d (Nutrient broth is a basal medium) Nutrient broth serves as a basal media for preparation of media like sugar fermentation, etc. Answer 21: d (Clostridium) C. perfringens needs a heat treatment for the spores to get activated. “Mild heating is of value in recovering heat-sensitive spores from faces as heat shock activates the spores and increases the rate of germination.” Reference: 1. Mackie and McCartney Practical Medical Microbiology, 14th Ed., Pg. 537. Answer 22: a (Salmonella) Reference: 1. Ananthanarayan, 8th Ed., Pg. 288. Answer 23: c (Spirochetes) Reference: 1. Ananthanarayan, 8th Ed., Pg. 379. Answer 24: d (Mycobacterium) Reference: 1. Ananthanarayan, 8th Ed., Pg. 364. Answer 25: a (Loeffler’s serum slope) EXTRA EDGE A COMPREHENSIVE LIST OF CULTURE MEDIA FOR BACTERIOLOGY • Alkaline peptone water Enrichment media for Vibrio cholerae • Alkaline salt transport medium Transport media for diarrheal diseases suspected of being caused • Taurocholate peptone transport medium by V. cholera • Anaerobic media Liquid media by addition of • glucose (0.5% to 1%) • ascorbic acid (0.1%) • cysteine (0.1%) • sodium merceptoacetate (0.1%) • thioglycollate (0.1%) • particles of cooked meat brothChapter-03.indd 34 7/11/2012 5:05:46 PM
  • 15. GENERAL BACTERIOLOGY 35 • Bile salt agar Selective media for V. cholerae • Thiosulphate citrate bile salts-sucrose agar (TCBS) • Monsur’s tellurite taurocholate gelatin agar • Bile esculin agar (contains 40% bile) Selective media for Enterococcus species (black coloration of the medium) • Blood agar Enriched media (supports the growth of fastidious organisms, e.g., Streptococccus) Indicator media to show hemolytic properties of certain organisms Staphylococcus aureus: ␤-hemolytic; Streptococcus pneumonia and S. viridans-␣-hemolytic; Enterococcus: nonhemolytic) • Bordet-Gengou agar Isolation of Bordetella pertussis • Charcoal blood agar • Regan-Lowe medium (charcoal agar with blood, cephalexin, and amphotericin B) • Brain heart infusion broth Used in blood culture bottles (both adult and pediatric patients) • Buffered charcoal yeast agar (BCYA) Specialized media for isolation of Legionella • Feeley Gorman agar • Campylobacter thioglycollate broth Selective holding media for recovery of Campylobacter species • Castaneda medium Biphasic medium for the isolation of Brucella • Cefoxitin cycloserine fructose agar (CCFA) Selective media for isolation of Clostridium difficile form suspected • Cefoxitin cycloserine egg-yolk agar (CCEY) cases of pseudomembranous colitis/antibiotic-associated diarrhea • Cefsulodin-Irgasan-Novobiocin medium (CIN Selective media for Yersinia (and may be used for Aeromonas also) medium) • Columbia-colistin nalidixic acid agar (CNA agar) Selective media for isolation of Gram-positive cocci • Cooked meat broth In general, used for preservation and storage of bacterial cultures • Nutrient agar slopes • Semisolid nutrient agar stabs • Heated blood agar slopes • Crystal violet blood agar Selective media for Streptococcus pyogenes • Cysteine lactose electrolyte deficient media Most commonly used media for culturing urine samples (CLED media) • Egg saline medium Preservation of cultures of Gram-negative bacilli • Egg yolk agar Detection of lipase and lecithinase activity of Clostridium species • Ellner’s medium Specialized media to induce sporulation in Clostridium • Medium of Duncan and Strong • Medium of Phillips • Alkaline egg medium • Fildes blood-digest agar and broth Enriched media for recovery of Haemophilus influenzae • Levinthal’s agar • Firm agar 4% to 6% agar Prevents swarming of Proteus mirabilis, P. vulgaris, Clostridium tetani • Fletcher’s agar Solid media for isolation of Leptospira • Ellinghausen and McCullough medium • EMJH media • Glycerol saline transport medium Transport stool specimen for typhoid bacilli • Heated blood agar/chocolate agar Growth of fastidious organisms (e.g., Hemophilus influenzae, Neisseria gonorrhoeae, S. pneumoniae • Hoyle’s tellurite lysed blood agar Selective media for isolation of Corynebacterium from throat swabs • Tinsdale medium • Loeffler serum slope Stimulation of metachromatic granules in Corynebacterium diphtheriaeChapter-03.indd 35 7/11/2012 5:05:46 PM
  • 16. 36 SMART STUDY SERIES: MICROBIOLOGY • Lowenstein-Jensen medium Selective media for isolation of Mycobacterium tuberculosis from • Middlebrook media sputum and other samples • MacConkey agar Differential media for Enterobacteriacaeae (i.e., lactose fermenting and nonlactose fermenting) • MacConkey bile salt lactose agar Differential and media for isolation of Salmonella and Shigella from • Brilliant MacConkey agar stool specimens • Leifson’s deoxycholate-citrate agar (DCA) • Wilson and Blair’s brilliant green bismuth- sulphite agar (BBSA) • Taylor’s xylose lysine deoxycholate agar (XLD) • Hektoen enteric agar • Salmonella-Shigella agar • Mannitol salt agar Selective and indicator media for S. aureus • Modified Barbour Stoenner Kelly medium (BSK) Specialized media for Borrelia burgdorferi • Modified Korthoff’s medium Liquid media for isolation of Leptospira • Modified New York City medium (contains colistin, Selective media for Neisseria gonorrhoeae lincomycin, trimethoprim, amphotericin B) • Mueller-Hinton agar Performing antimicrobial susceptibility for bacteria • Nutrient agar 1% to 2% agar Basal media in microbiology Supports the growth of all nonfastidious organisms • Nonnutrient agar Cultivation of parasites (e.g., Acanthamoeba) • Peptone water • Basal media for preparation for carbohydrate fermentation media • To ascertain whether a bacteria is motile or nonmotile • Basis for Indole test • Phenol-red egg yolk polymyxin agar Selective media for isolation of Bacillus cereus from food, feces, and vomitus • Pike’s media Preservation of S. pyogenes, pneumococci, and Hemophilus influenzae in nose and throat swabs • Polymyxin B-lysozyme-EDTA-thallous acetate Selective media for isolation of Bacillus anthracis from soil and other medium (PLET) materials containing numerous spore formers of other species • Polymyxin B, neomycin, fusidic acid media (PNF) Selective media for S. pyogenes (or ␤-hemolytic Streptococcus) • PPLO medium (contains sterol) Specialized media for Mycoplasma pneumoniae • Prereduced anaerobically sterilized (PRAS) media Commercially available media for anaerobic organisms • Proteose peptone-yeast extraction broth Media for carrying out biochemical tests for anaerobes • Requirements of X and V factors Isolation of Haemophilus influenzae • RPMI 1640 medium Cultivation of malarial parasites (i.e., Plasmodium) • Robertson cooked meat broth (RCMB) Growth of anaerobes (e.g., Clostridium) Maintaining stock cultures of anaerobic organisms • Salt-cooked meat broth (cooked meat broth with Enrichment media for isolation of S. aureus from heavily 10% NaCl) contaminated materials • Semisolid agar 0.05% to 0.1% agar Prevents convection current and allows the growth of anaerobic and microaerophilic organisms • Skirrow’s Campylobacter medium (contains Selective media for Campylobacter jejuni polymixin B, trimethoprim, vancomycin) • Preston Campylobacter medium (contains polymixin B, rifampicin, trimethoprim) • Campy blood agar • CVA medium (contains cefoperazone, vancomycin, amphotericin)Chapter-03.indd 36 7/11/2012 5:05:46 PM
  • 17. GENERAL BACTERIOLOGY 37 • Smith-Noguchi medium Cultivation of nonpathogenic treponemes (e.g., Reiter strain of Treponema phagedenis) • Sorbitol MacConkey agar Isolation of verocytotoxin-producing (enterohemorrhagic) E. coli of 0157 type (as it fails to ferment D-sorbitol) • Stuart transport media Maintaining the viability of gonococci on swabs during • Amies transport media transportation • Tetrathionate broth Enrichment media for isolation of Shigella and Salmonella from • Gram-negative broth stool samples • Selenite-F broth • Thayer-Martin medium (contains vancomycin, Selective media for Neisseria gonorrhoeae colistin, nystatin) • Thioglycollate broth All purpose enrichment broth for anaerobes, aerobes, • Trypticase Soy broth microaerophilic, and fastidious organisms • Todd Hewitt broth with antibiotics Selective and enrichment for Streptococccus agalactiae in female genital specimens) • Triple sugar iron agar (TSI) medium Differentiation of various members of Enterobacteriaceae • Wilkins-Chalgren agar Performing antimicrobial susceptibility of anaerobic bacteriaChapter-03.indd 37 7/11/2012 5:05:46 PM