Atlas of Medical Bacteriology

Atlas of Medical Bacteriology 1 Atlas of Medical Bacteriology Simplified Guide for Primary Differentiation and Diagnosis of Pathogenic Bacteria that can be isolated from patients of different diseases. University of Babylon College of Pharmacy Department of Clinical and laboratory Sciences Second Class 2014-2015 1st. Edition 2 Preface: To help students in the comprehension of Practical Part of Medical Microbiology especially the Bacteriology which is the concept of this Atlas, this work may introduce some help . Some bacteria are rare, the others very dangerous or may be obligate intracellular parasite, all of the mentioned cases need to study these bacteria by pictures in addition to the theoretical information, therefore I dedicate this work to everyone can get any benefit from it. Msc. Teacher Assistant Halah Dawood Salman 2014-2015 3 Subject No. LIST OF CONTENTS Subject Page 4 List of Contests Gram Positive Bacteria Staphylococcus 1.2 Streptococcus 1.3 Bacillus 1.4 Closteridium 1.5 Corynebacterium 1.6 Listeria 1.7 Actinomycetes 1.8 Propionebacterium 2-Gram Negative Bacteria 1- 1.1 5 10 15 20 25 27 29 31 2.1 33 Escherichia coli 2.2 Klebsiella 2.3 Enterobacter 2.4 Proteus 2.5 Salmonella 2.6 Shigella 2.7 Serratia 2.8 Pseudomonas 2.9 Vibrio 2.10 Campylobacter 2.11 Helicobacter 2.12 Haemophilus 2.13 Brucella 2.14 Bordetella 2.15 Yersinia 2.16 Neisseria 3-Acid Fast Stain Bacteria 3.1 Mycobacterium tuberculosis 3.2 Mycobacterium leprae 35 37 39 41 43 45 47 50 54 57 59 62 63 65 68 72 75 76 79 83 84 99 4- Cell Wall-Defective Bacteria (Mycoplasma) 5- Chlamydia List of abbreviations Appendix References 4 1-GRAM POSITIVE BACTERIA 1-1-Staphyllococcus spp. The staphylococci are gram-positive spherical cells, non-sporing, non-motile, usually noncapsulate, true facultatively anaerobic organisms. (1micrometer in diameter) usually arranged in grapelike irregular clusters. They grow readily on many types of media and are active metabolically, fermenting carbohydrates and producing pigments that vary from white to deep yellow. Colonies on solid media are round, smooth, raised, and glistening S. aureus usually forms gray to deep golden yellow colonies. S. epidermidis colonies usually are gray to white on primary isolation; many colonies develop pigment only upon prolonged incubation. No pigment is produced anaerobically or in broth. Various degrees of hemolysis are produced by S aureus and occasionally by other species. Staphylococcus saprophyticus is a frequent cause of cystitis in women, probably related to its occurrence as part of normal vaginal flora. S. saprophyticus can be distinguished from S. epidermidis and most other coagulase-negative staphylococci by its natural resistance to novobiocin . [Note: Urinary coagulase-negative staphylococcus is often presumed to be S. saprophyticus; novobiocin resistance can be used for confirmation.]. Light Microscope Electron Microscope 5 Scanning electron micrograph (SEM) depicting numerous clumps of methicillin-resistant Staphylococcus aureus bacteria, commonly referred to by the acronym, MRSA; Magnified 9560x Source: Centers for Disease Control and Prevention S. aureus on Nutrient Agar S. epidermidis on Nutrient Agar 6 S. aureus mannitol salt agar S. aureus on Luria Agar (LA) Media blood agar S.aureus on Trypticase soy agar(TSA) 7 On blood agar: S.auerus (ß-hemolysis) S. epidermidis (No hemolysis) S. saprophyticus (No hemolysis) S. epidermidis Growth with no mannitol fermentation S. saprophyticus On Mannitol Salt agar: S. aureus Growth with + mannitol fermentation 8 On DNase agar: S. aureus S. aureus White colonies with clear halo around colonies. S. epidermidis White colonies with NO clear halo around colonies MRSA: Methicillin-Resistant Staphylococcus aureus, MRSA are a major cause of nosocomial and life threatening infections. Infections with MRSA have been associated with a significantly higher morbidity, mortality and costs than methicillin-susceptible S. aureus (MSSA). Selection of these organisms has been greatest in the healthcare setting; however, MRSA have also become more prevalent in the community. 9 1-CHROMagar MRSA 2-Spectra MRSA 3- Brilliance MRSA 2 Agar 1- CHROMagar MRSA: is a selective and differential medium for the qualitative direct detection of nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) to aid in the prevention and control of MRSA infections in healthcare settings. 2-Spectra MRSA medium plated with methicillin-resistant Staphylococcus aureus. The ability of Spectra MRSA to identify methicillin-resistant S. aureus based on the color of the colony, in which colonies of methicillin-resistant S. aureus appear denim blue. 3- Newly Improved Chromogenic Medium for Rapid and Reliable MRSA Screening in Just 18 Hours. 1-2 Streptococcus spp. The streptococci are gram-positive spherical bacteria that characteristically form pairs or chains during growth. They are widely distributed in nature. Streptococci elaborate a variety of extracellular substances and enzymes. The streptococci are a large and heterogeneous group of bacteria, and no one system suffices to classify them. Many species of streptococci, including Streptococcus pyogenes (group A), S agalactiae (group B), and the enterococci (group D), are characterized by combinations of features, including colony growth characteristics, hemolysis patterns on blood agar (α-hemolysis, β-hemolysis, or no hemolysis). Light Microscope Micrograph 10 Gram stain of S. pneumoniae with WBC Scanning Electron Microscope Micrograph Electron Microscope Micrograph 11 S. pneumoniae colonies on a BAP(Blood Agar Plate) S. pneumoniae colonies have a flattened and depressed greenishcenter after 24-48 hours of growth on a BAP, whereas the viridans streptococci retain a raised center. . 12 The small grey, flat colonies surrounded by a zone of alpha-hemolysis are S. pneumoniae and the white colonies with no hemolytic activity are S. epidermidis (black arrow). Streptococcus agalactiae colonies growing on GBS Detect™ showing beta- hemolytic colonies. This strain is not hemolytic on a regular blood agar plate. Incubated aerobically for 24 hours at 35 deg. C. ( GBS Detect™ is recommended for the isolation and detection of gamma-hemolytic (non-hemolytic) Group B Streptococcus by inducing beta-hemolysis on sheep blood agar upon subculture from enrichment broth procedures; such as Strep B Carrot Broth™ and LIM Broth).GBS*: Group B Streptococci. Optochin test for S. pneumoniae using optochin disks. The strain on the left is resistant to optochin with no zone of inhibition, and therefore is not a pneumococcus. The strain on the right is susceptible to optochin and is S. pneumoniae. 13 CAMP-test GAS*: Group A Streptococci. GBS*: Group B Streptococci. 14 S. pneumoniae in spinal fluid FA: Florescent Antibody )digitally colored). Streptococcus pneumonia Quellung (capsular swelling) reaction can be use to demonstrate the presence of a specific capsular type of the bacterium. Encapsulated Streptococcus pneumonia 15 1-3-Bacillus Bacillus are gram-positive spore-forming bacilli. These bacilli are ubiquitous, and because they form spores, they can survive in the environment for many years. Bacillus species are aerobes. Colonies of B. anthracis are round and have a ―cut glass ‖appearance in transmitted light. Hemolysis is uncommon with B .anthracis but common with B. cereus and the saprophytic bacilli. Bacillus anthracis, commonly known as anthrax. These rod-shaped, bacteria can infect the skin (cutaneous anthrax), causing raised itchy lesions, the lungs (pulmonary anthrax), which is fatal unless treated quickly, and the digestive system (gastrointestinal anthrax), causing vomiting of blood and severe diarrhoea. All forms can be fatal if left untreated -Light Microscope- Bacillus spp. Malachite Green spore stain Bacillus Anthracis (Anthrax)Scanning electron Electron Microscope image micrograph of Bacillus anthracis spores 16 Image of Bacillus spp. as a result of capsule stain. Bacillus anthracis spores seen under phase contrast microscopy. Bacillus anthracis endospores are seen under phase contrast microscopy as lighter areas, i.e. "points of light", due to the fact that they are dehydrated, and therefore, more refractile. 17 Bacillus anthracis on chocolate agar Colonies of B. subtilis grown on a culture dish Bacillus spp. On nutrient agar B.cereus on Luria Agar (LA) 18 -1- -2- 1- Mucoid colonies of Bacillus anthracis. This culture was probably incubated at an increased CO2 tension (5% CO2) which greatly enhances production of the poly-D-glutamyl capsule and accounts for the mucoid colony type 2-Bacillus sp. on Mueller-Hinton agar. Cultivation in aerobic atmosphere, 28°C, 48 hours. Environmental isolate -1- -2- 1- Lysis of Bacillus anthracis by the lytic phage gamma. The plaque (clear area) in the region of confluent growth is where the gamma phage was applied. The plaque results from the phage's ability to lyse the bacterial cells. Since the gamma phage is specific for B. anthracis, and will not lyse B. thuringiensis or B. cereus. 2- Colonies of Bacillus cereus on the left; colonies of Bacillus anthracis on the right. B. cereus colonies are larger, more mucoid, and this strain exhibits a slight zone of hemolysis on blood agar. 19 B.cereus: Large, irregular colonies surrounded by a wide haemolytic zone. Bacillus cereus colonies on blood agar -1- -2- 1-Oxoid Brilliance Bacillus cereus Agar: (formerly Chromogenic Bacillus cereus Agar) is a chromogenic medium for the isolation and differentiation of Bacillus cereus from food samples. 2-A Bacillus subtilis bacterial colony showing signs of stationary growth after 48 hours of incubation at 37 °C (98.6 °F; magnified about 9 times). 20 1-4- Clostridium spp. The clostridia are large anaerobic, gram-positive, motile rods. Many decompose proteins or form toxins, and some do both.Their natural habitat is the soil or the intestinal tract of animals and humans, where they live as saprophytes. In general, the clostridia grow well on the blood-enriched media or other media used to grow anaerobes. Some clostridia produce large raised colonies (eg, C. perfringens); others produce smaller colonies form colonies that spread on the agar surface. Many clostridia produce a zone of hemolysis on blood agar. C. perfringens characteristically produces a double zone of -hemolysis around colonies. C. perfringens Light Microscope Clostridium tetani Light Microscope 21 This Gram-stained micrograph of Clostridium botulinum Type-A in thioglycollate broth was incubated for 48hrs at 35°C A photomicrograph of Clostridium botulinum type A viewed using a Gram stain technique. Light Microscope Clostridium difficile bacteria, light micrograph. 22 "Clostridium botulinum". This picture shows the rod-shaped bacteria, under a (SEM) scanning electron microscope. Courtesy of Science Photo Library.com Scanning Electron Microscope C.deficile Electron Microscope C.tetani C.tetani -Light Microscope 23 Botulism (Clostridium botulinum). These are Clostridium botulinum Type E colonies displaying an opaque zone grown on a 48hr egg yolk agar. -1- -2- 1- Clostridium difficile Cefoxitin Cycloserine Egg-Yolk (CCEY) AGAR. 2-Clostridium perfringens colonies on tryptose sulphite cycloserine agar (TSC agar). Ultraviolet irradiation shows yellow-green fluorescence of Clostridium difficile in agar medium. The CDC lists drug-resistant C diff as a pathogen of urgent concern. 24 -1- -2- 1-Clostridium difficile On blood agar 2-This photograph depicts a colony of Clostridium sp., which had been grown on a 4% blood agar plate (BAP) over a 48 hour time period. -1- -2- 1-Clostridium Perfringens on blood agar 2-Clostridium botulinum which produces the botulism toxins growing on egg yolk agar showing the lipase reaction after 72 hours of incubation. CDC PHIL (1/3/2008). 25 1-5- Corynebacterium spp. Corynebacteria are non–spore-forming gram-positive bacilli , characteristically, they possess irregular swellings at one end that give them the ―club-shaped‖ appearance. Irregularly distributed within the rod (often near the poles) are granules staining deeply with aniline dyes (metachromatic granules) that give the rod a beaded appearance. A simple stain with methy lene blue is often used to stain these granules. On blood agar, the C. diphtheriae colonies are small, granular, and gray with irregular edges and may have small zones of hemolysis . On agar containing potassium tellurite, the colonies are brown to black with a brown-black halo because the tellurite is reduced intracellularly (staphylococci and streptococci can also produce black colonies). C. diphtheriae and other corynebacteria grow aerobically on most ordinary laboratory media. On Loeffler serum medium, corynebacteria grow much more readily than other respiratory organisms, Corynebacteria tend to pleomorphism in microscopic and colonial morphology. Light Microscope -1- Scanning Electron Micrograph -2- -3- 1-Corynebacterium diphtheriae cultivated on Tinsdale agar. Cultivation 72 hours, 37 °C in an aerobic atmosphere. 2 -Corynebacterium diptheriae on Tinsdale agar. 3- Corynebacterium diphtheriae on Tellurite agar. 26 Blood agar Hoyle's tellurite agar 1-6- Listeria monocytogenes L. monocytogenes is a short, gram-positive, non–spore-forming rod . It has a tumbling endover-end motility at 22–28°C but not at 37°C; the motility test rapidly differentiates Listeria from diphtheroids that are members of the normal microbiota of the skin. Listeria grows well on media such as 5% sheep blood agar on which it exhibits the characteristic small zone of hemolysis around and under colonies. The organism is a facultative anaerobes. The motility at room temperature and hemolysin production are primary findings that help differentiate Listeria from coryneform bacteria. -Light Microscope -Coloured Transmission Electron Microscope Image Listeria monocytogenes 27 -1- -2- -3- 1-Colorex Listeria (ISO):It is a selective medium for the isolation and presumptive identification of Listeria monocytogenes from clinical and food samples. 2-CHROMagar Listeria : It is a chromogenic media for isolation of specimins allowing direct differenciation of Listeria monocytogenes by blue colony colour with a white halo. This chromogenic culture media is based on phospholipases specific of Listeria monocytogenes. 3-Blood Agar: Listeria monocytogenes, removing colonies to see the subtle hemolysis directly beneath the colonies. (Rebecca Buxton, University of Utah). -1- -2- 1- Listeria monocytogenes residing within white blood cells. 2- Listeria bacteria. Coloured Transmission Electron Micrograph (TEM) of a human cell seen with a group of Listeria monocytogenes bacteria. The human cell is coloured green; the rod-shaped bacteria are yellow. 28 Actin Tail Bacterium -1- -2- 1- Listeria moving through cell by actin-based motility, Green = actin filament stain, Red =Listeria stain. Note the long actin filament tails behind the moving bacteria. 2-Scanning Electron Microscopy (SEM). Adhesion of L. monocytogenes on a cell wall. Experiment performed at Pasteur Institute. *Listeria monocytogenes )Facultative, Intracellular Parasite( 1-7-Actinomycetes Actinomycetes are a group of filamentous, branching, gram-positive organisms that easily fragment into slender rods . Although they superficially resemble fungi on morphologic grounds, they are prokaryotes of bacterial size. They are free-living, mostly soil organisms that are related to Corynebacteria and Mycobacteria, as well as to the Streptomycetes that are sources of important antibiotics. Actinomycosis is an opportunistic infection . The infection is probably initiated by accidental introduction of organisms into the underlying soft tissue during conditions of sufficient anaerobiasis to support their growth. They are related to the corynebacteria and include multiple genera of clinical significance such as Mycobacteria and saprophytic organisms such as Streptomyces. Members of the aerobic Actinomycetes can be categorized on the basis of the acid fast stain. Mycobacteria are truly positive acid fast organisms; weakly positive genera include Nocardia, Rhodococcus, and a few others of clinical significance. Streptomyces and Actinomadura, two agents that cause actinomycotic mycetomas, are acid fast stain negative. - Actinomyces israelii, and Arachnia propionica: A. israelii and A. propionica are part of the normal oral and intestinal flora in humans, they are strict anaerobes. Most strains of A.israelii and the other agents of actinomycosis are facultative anaerobes that grow best in an atmosphere with increased carbon dioxide. On enriched medium, such as brain-heart infusion agar, young colonies (24–48 hours) produce gram-positive substrate filaments that fragment into short chains, diphtheroids, and 29 coccobacilli. After a week, these "spider" colonies develop into white, heaped-up "molar tooth" colonies. In thioglycolate broth, A. israelii grows below the surface in compact colonies. Species are identified based on cell wall chemotype and biochemical reactions. Laboratory identification: The most typical and diagnostic finding in Actinomycosis is the presence of sulfur granules• in the draining pus. These are small, firm, usually yellowish particles, which in fact do not contain sulfur. When examined under the microscope, these appear as microcolonies composed of filaments of the organism embedded in an amorphous, eosinophilic material thought to be antigen-antibody complexes. The organism can be grown anaerobically on enriched media such as thioglycollate broth or blood agar. Growth is slow, often requiring ten to fourteen days for visible colonies. 30 A B -Nocardia asteroids in seputum:(A-Acid fast stain) ,(B-Gram,s stain) 1-8- Propionibacterium On Gram stain, they are highly pleomorphic, showing curved, clubbed, or pointed ends; long forms with beaded uneven staining; and occasionally coccoid or spherical forms Propionibacterium species a members of the normal microbiota of the skin, oral cavity, large intestine, conjunctiva, and external ear canal. Their metabolic products include propionic acid, from which the genus name derives. P.cnes sometimes contaminates blood or 31 cerebrospinal fluid cultures that are obtained by penetrating the skin. It is therefore important (but often difficult) to differentiate a contaminated culture from one that is positive and indicates infection. Light Microscope Electron Microscope Electron Microscope Colonies of Propionibacterium acnes on Blood agar. 32 2-Gram Negative Bacteria: 2-1-E.coli short gram-negative rods , motile , E.coli are facultative anaerobes and form circular, convex, smooth, flat, nonviscous colonies with distinct edges. Lactose fermented rapidly, some strains of E .coli produce hemolysis on blood agar , have metallic sheen on differential media. Light Microscope Electron Microscope -1- -2- 1- MacConkey agar. 2-Hektoen - Escherichia coli. Note: Orange color indicates acid production as a result of lactose fermentation. 33 -1-2-3(1) & (2)- E.coli on MacConkey agar. 3-CLED Agar . A: Proteus vulgaris (blue colour due to pH-shift to >7.6). B: Eschericha coli (colour greenish-yellow due to acid production caused by lactose fermentation). Eosin Methylene blue agar(EMB) E.coli-Green metallic sheen colonies Trypticase soy agar E.coli on Endo agar 34 E.coli on Chrom agar 2-2- Klebsiella short gram-negative rods , motile and facultative anaerobes .Capsules are large and regular in Klebsiella species, Klebsiella colonies are large and very mucoid and tend to coalesce with prolonged incubation and Lactose fermented rapidly . Klebsiella pneumonia are very viscous and have mucoid growth. The organism grows with difficulty on media containing egg yolk. Light Microscope Electron Microscope Electron Microscope Image 35 MacConkey agar EMB agar 36 Blood agar -11-Cystine -Lactose Electrolyte - Deficient agar (CLED). -2- 2- Eosin Methylene Blue Agar - Klebsiella pneumonia .Note: Mucoid colonies with dark centers due to capsule production and lactose fermentation respectively. 2-3- Enterobacter short gram-negative rods , facultative anaerobes, most Enterobacter species give positive test results for motility, produce mucoid colonies, Enterobacter aerogenes has small capsules. raised colonies, no metallic sheen; often motile; more viscous growth E nterobacter cloacae has similar to Enterobacter aerogenes . 37 Light Microscope MacConkey agar Electron Microscope EMB XLD Luria Agar (LA) Blood agar 38 Chromagar CLED 2-4- PROTEUS short gram-negative rods , facultative anaerobes , Proteus species move very actively by means of peritrichous flagella, resulting in ―swarming‖ on solid media unless the swarming is inhibited by chemicals, such as phenylethyl alcohol or CLED (cystine-lactose-electrolytedeficient) medium.; Proteus species are urease positive (smell of ammonia) . The Proteus ferments lactose very slowly or not at all. Proteus mirabilis is more susceptible to antimicrobial drugs, including penicillins, than other members of the group. Light microscope 39 Coloured TEM of the bacterium Proteus mirabilis MacConkey agar Chrom agar Swarming of Proteus mirabilis on an agar plate showing the typical growth rings 40 -1- -2- 1- CLED Agar inhibits swarming of Proteus. A: Proteus vulgaris (blue colour due to pH-shift to >7.6) B: Eschericha coli (colour greenish-yellow due to acid production caused by lactose fermentation). 2-proteus mirabilis growing on XLD agar. 2-5- Salmonella Salmonellae are short gram-negative rods but vary in length, facultative anaerobes most isolates of Salmonellae are motile with peritrichous flagella that characteristically ferment glucose and mannose without producing gas, from dextrose they produce acid and usually gas but they almost never ferment lactose or sucrose. Salmonellae grow readily on simple media, most salmonellae produce H2S. They are often pathogenic for humans or animals when ingested. They survive freezing in water for long periods. Salmonellae are resistant to certain chemicals(eg, brilliant green, sodium tetrathionate, sodium deoxycholate) that inhibit other enteric bacteria; therefore useful for inclusion in media to isolate salmonellae from feces. Light Microscope Electron Microscope 41 MAcConkey agar EMB agar XLD agar Hektoen agar SS agar 42 Bood agar Salmonella on bismuth-sulphite agar 2-6- Shigella Short gram-negative rods , Shigella species are nonmotile, not produce gas from dextrose . Shigellae are facultative anaerobes but grow best aerobically. Convex, circular, transparent colonies with intact edges reach a diameter of about 2 mm in 24 hours. Shigellae are usually do not ferment lactose but do ferment other carbohydrates, producing acid but not gas. They do not produce H2S. The four Shigella species are closely related to E. coli. Many share common antigens with one another and with other enteric bacteria (eg, Hafnia alvei and Plesiomonas shigelloides). 43 Electron Microscope Light Microscope MAcConkey agar Blood agar 44 SS agar XLD agar Hektoen agar ENDO agar 2-7- Serratia Short gram - negative rods , facultative anaerobes, slowly fermented Lactose Serratia species produces DNase, lipase, and gelatinase. Serratia marcescens is a common opportunistic pathogen in hospitalized patients . Serratia (usually nonpigmented) causes pneumonia, bacteremia, and endocarditis. Only about 10% of isolates form the red pigment (prodigiosin) that has long characterized S. marcescens. Light Microscope Electron Microscope 45 Pigment Production by Serratia marcescens Serratia marcesens on MacConkey Agar Serratia marcesens on Blood Agar S. marcesens appear as smooth, round, medium sized colonies on blood agar. Some strains a pinkish-orange pigment. 46 produce Serratia marcescens growing on XLD agar Serratia marcescens growing on Nutrient agar 2-8-Pseudomonas The pseudomonads are gram-negative rods occurs as single bacteria, in pairs, and occasionally in short chains, motile, obligate aerobic, some of which produce water - soluble pigments. The pseudomonads occur widely in soil, water, plants, and animals. P.aeruginosa is frequently present in small numbers in the normal intestinal fl ora and on the skin of humans and is the major pathogen of the group. Other pseudomonads infrequently cause disease. P. aeruginosa grows readily on many types of culture media, sometimes producing a sweet or grapelike or corn taco–like odor. Some strains hemolyze blood. P. aeruginosa forms smooth round colonies with pigments which diffuses into the agar. Other Pseudomonas species do not produce the same pigments. P. aeruginosa in a culture can produce multiple colony types. Electron Microscope Light Microscope 47 Electron Microscope EMB- P. aeruginosa (lactose nonfermentor) MacConkey agar XLD agar P.aeroginosa Trypticase soy agar Blood Agar 48 Mullar Hinton Agar CHROMagar Pseudomonas -TSA agarPseudomonas aeruginosa on Trypic Soy Agar (TSA). 2007 Environmental Microbiology Laboratory, Inc. 49 Pseudomonas isolation agar -1- -2- 1- Pseudomonas aeruginosa on HiFluoro Pseudomonas Agar 2- Pseudomonas Agar: Pseudomonas aeruginosa on HiFluoro Pseudomonas Agar under UV light. 2-9- Vibrio spp. Vibrios are among the most common bacteria in surface waters worldwide. They are Gram negative , curved aerobic rods and are motile, possessing a polar flagellum. V. cholerae serogroups O1 and O139 cause cholera in humans, and other vibrios may cause sepsis or enteritis. Upon first isolation typical Organisms of V cholerae is a comma-shaped, curved rod 2–4 μm long . It is actively motile by means of a polar flagellum. On prolonged cultivation, vibrios may become straight rods that resemble the gram-negative enteric bacteria. V cholerae produces convex, smooth, round colonies that are opaque and granular in transmitted light. V cholera and most other vibrios grow well at 37°C on many kinds of media, including defined media containing mineral salts and asparagine as sources of carbon and nitrogen. V. cholera grows well on thiosulfate-citrate-bile-sucrose (TCBS) colonies (sucrose fermented) agar, a media selective for vibrios, on which it produces yellow that are readily visible against the dark-green background of the agar. 50 Light Microscope Vibrio cholerae. Leifson flagella stain (digitally colorized). CDC/Dr. William A. Clark 51 Electron Microscope V. cholreae V. parahaem. TCBS Agar Thiosulphate Citrate Bile Salts Sucrose Agar 52 Vibrio cholerae on Columbia Horse Blood Agar Vibrio cholerae on C.L.E.D. Vibrio species On MacConkey Vibrio species On Chrom agar 53 2-10- Campylobacter C. jejuni and Campylobacter coli have emerged as common human pathogens, C jejuni and the other campylobacters are gram-negative rods with comma, S, or ―gull wing‖ shapes. They are motile, with a single polar flagellum, and do not form spores. Selective media are needed, and incubation must be in an atmosphere with reduced O2 (5% O2) with added CO2(10% CO2). Incubation of primary plates for isolation of C jejuni should be at 42°C. Several selective media are in widespread use . Skirrow’s medium .The colonies tend to be colorless or gray. They may be watery and spreading or round and convex, and both colony types may appear on one agar plate. Light Microscope 54 Electron Microscope Electron Microscope 55 Blood Agar Campylobacter selective agar 56 2-11- Helicobacter pylori H pylori is a spiral-shaped gram-negative rod. H pylori has many characteristics in common with campylobacters. It has multiple flagella at one pole and is actively motile. H. pylori grows in 3–6 days when incubated at 37°C in a microaerophilic environment, as for C.jejuni. The media for primary isolation include Skirrow’s medium , chocolate medium, and other selective media with antibiotics. The colonies are translucent and 1–2 mm in diameter. H. pylori is a strong producer of urease. Helicobacter pylori Gram-negative, spiral to pleomorphic, spiral rod prokaryote. It can move by means of tiny flagella at the end of the cell. Light Microscope Helicobacter pylori -SEM (Scanning Electron Microscope) 57 Helicobacter pylori electron micrographs; fastidious microaerophile; typical helical shape shown in EM; causative agent of chronic gastritis, peptic ulcers and gastric cancer. Helicobacter pylori (yellow) coloured SEM (Scanning Electron Microscope) 58 H. pylori culture on sheep blood agar H. pylori culture on selective HP agar Helicobacter Pylori Agar Base-HP-Selective Medium 2-12-Haemophillus small, gram-negative, pleomorphic bacteria that require enriched media, usually containing blood or its derivatives, for isolation. Haemophilus infl uenzae type b is an important human pathogen; In specimens from acute infections, Haemophilus influenzae are short (1.5 μm) coccoid bacilli, sometimes occurring in pairs or short chains. In cultures, the morphology depends both on the length of incubation and on the medium. At 6–8 hours in rich medium, the small coccobacillary forms predominate. Later there are longer rods, lysed bacteria, and very pleomorphic forms. Organisms in young cultures (6–18 hours) on enriched medium have a definite capsule. On chocolate agar, flat, grayish brown colonies with diameters of 1–2 mm are present after 24 hours of incubation. IsoVitaleX in media enhances growth. H influenzae does not grow on sheep blood agar except around colonies of staphylococci (―satellite phenomenon‖). 59 Light Microscope Haemophilus influenzae : are small, pleomorphic, gram-negative bacilli or coccobacilli with random arrangements. Haemophilus influenzae In specimens from patients with pneumonia (Sputum) caused by Haemophilus influenzae, both neutrophils and bacteria are usually plentiful. However, if examination of the slide is not thorough, the coccobacilli may be inconspicuous in the background of pink-staining mucus. Other organisms, such as Eikenella corrodans or Bacteroides species, are also pleomorphic gram-negative coccobacilli; but they rarely cause pneumonia. 60 Haemophilus influenzae Bacteria TEM picture ( Transmission Electron Microscope) False-colour scanning electron micrograph (SEM) of Haemophilus influenzae (Pfeiffer's bacillus), a Gram negative, non-motile, non-sporing species of bacilli (rod-shaped bacteria) Haemophilus influenzae type b bacteria(HIB) TEM of Haemophilus influenzae TEM ( Transmission Electron Microscope)- False-colour transmission electron micrograph (TEM) of Haemophilus influenza. 61 Haemophilus influenzae (only grows on chocolate) Must strains of Haemophilus spp does not grow on 5% Sheep Blood Agar, which contains hemin (factor X) but lacks NAD (factor V). H. influenzae colonies on a CAP(Chocolate Agar Plate). 2-13- Brucella The brucellae are obligate parasites of animals and humans and are characteristically located intracellularly. They are relatively inactive metabolically. The appearance in young cultures varies from cocci to rods 1.2 μm in length, with short coccobacillary forms predominating. They are gram negative but often stain irregularly, and they are aerobic, nonmotile, and nonspore forming. Brucellae have Small, convex, smooth colonies appear on enriched media in 2–5 days. Brucellae are adapted to an intracellular habitat, and their nutritional requirements are complex. Some strains have been cultivated on defined media containing amino acids, vitamins, salts, and glucose.. 62 Light Microscope A magnified view of Brucella - Scan Microscopy TEM of Brucella abortus bacteria TEM ( Transmission Electron Microscope) Brucella colonies on agar 2-14-Bordetella minute, gram-negative coccobacilli bacteria resembling H influenzae. With toluidine blue stain, bipolar metachromatic granules can be demonstrated. A capsule is present. Primary requires enriched media. Bordet-Gengou medium (potato-blood- isolation of B. pertussis glycerol agar) that contains penicillin G, 0.5 μg/mL, can be used; however, a charcoal(Regan Lowe) is preferable because of containing medium supplemented with horse blood the longer shelf life. The plates are incubated at 35–37°C for 3–7 days aerobically in a moist environment (eg, a sealed plastic bag). 63 Light Microscope Electron Microscope Blood agar Charcoal agar 64 Charcoal Blood Agar with cephalexin *With the addition of blood this medium is used to isolate Bordetella pertussis. 2-15-Yersinia Short, pleomorphic gram-negative rods that can exhibit bipolar staining. Microaerophilic or facultatively anaerobic. Most have animals as their natural hosts, but they can produce serious disease in humans. Y pestis exhibits striking bipolar staining with special stains such as Wright, Giemsa, Wayson, or methylene blue . It is nonmotile. It grows as a facultative anaerobe on many bacteriologic media. Growth is more rapid in media containing blood or tissue fluids and fastest at 30°C. In cultures on blood agar at 37°C, colonies may be very small at 24 hours. A virulent inoculum, derived from infected tissue, produces gray and viscous colonies, but after passage in the laboratory, the colonies become irregular and rough. The organism has little biochemical activity, and this is somewhat variable. Y enterocolitica and Y. pseudotuberculosis these are nonlactose-fermenting gram-negative rods that are urease positive and oxidase negative. They grow best at 25°C and are motile at 25°C but nonmotile at 37°C. 65 Photomicrograph of Gram stain of Yersinia enterocolitica, the causative agent of yersiniosis.Credit: Centers for Yersinia pestis — Gram Stain Gram-negative bacilli (0.5 to 0.8 by 1 to 3 microns), single or Disease Control and Prevention short chained. Sometimes bipolar staining (CDC)(Image Number: 2153) (―closed safety pin‖). -Light Microscope- Electron Microscope 66 Electron Microscope Cefsulodin Irgasan Novobiocin (CIN) Agar -A differential and selective medium for the isolation of Yersinia enterocolitica. Fermentation of  mannitol in the presence of neutral red produces characteristic "bull's-eye" colonies. These are colourless with a red centre. A zone of precipitated bile may also be present.Crystal violet, sodium desoxycholate, cefsulodin, Irgasan (triclosan) and novobiocin are inhibitory agents. Typical Y. enterocolitica colonies will have deep-red centres surrounded by a transparent border giving the appearance of a "bull's-eye". Other Yersinia species and Enterobacteriaceae grow on the medium  and may look similar to Y. enterocolitica.  67 2-16-Neisseria The typical Neisseria is a gram-negative, nonmotile diplococcus, approximately 0.8 μm in diameter. Individual cocci are kidney shaped; when the organisms occur in pairs, the flat or concave sides are adjacent. In 48 hours on enriched media (eg, modified Thayer-Martin, Martin-Lewis, GC-Lect, and New York City), gonococci and meningococci form convex, glistening, elevated, mucoid colonies 1–5 mm in diameter. Colonies are transparent or opaque, nonpigmented, and nonhemolytic . The neisseriae grow best under aerobic conditions, but some grow in an anaerobic environment. They have complex growth requirements. -1- -2- 1-Gram stain of Neisseria gonorrhoeae from blood culture growth. 2-Gram stain of Neisseria gonorrhoeae, the agent of the STD gonorrhea. The bacteria are seen as pairs of cocci (diplococci) in association with host pmn's(polymorphonuclear leukocytes).CDC . -1-21-Light microscopy of Neisseria meningitidis. Gram stain.Gram-negative cocci that typically appear in pairs with the opposing sides flattened. 2-Gram stain of N. meningitides. 68 Neisseria meningitidis Neisseria gonorrhoeae Scanning electron micrograph images Scanning electron micrograph images of gonococcal-lymphocyte interactions. False-colour transmission electron micrograph (TEM) of Neisseria meningitides. a species of non- motile, non-capsulate, aerobic, Gramnegative bacteria causing meningitis. Neisseria gonorrhoeae Electron microscope picture 69 Neisseria meningitides colonies on CAP (Chocolate Agar Plate) at 35-37°Cwith ~5%CO2 (or in a candlejar). Colonies of Neisseria meningitidis on Blood Agar Plate (BAP) . Some strains form pearl-like colonies on the surface of this media. Cultivation 24 hr. in an aerobic atmosphere enriched with 5% CO2, 37 °C. 70 N. meningitidis colonies on a Blood Agar plate Chocolate Agar- Gonococcal Agar N.gonorrhoeae colonies on chocolate agar plate (Chocolate Agar / Martin Lewis with Lincomycin) For the cultivation and isolation of Neisseria gonorrhoeae. Neisseria meningitidis colonies on chocolate agar (Thayer-Martin agar)with positive oxidase test as indicated by black colonies. 71 GC SELECTIVE AGAR (NEW YORK CITY) 3-Acid-fast‖ bacilli 3-1: Mycobacterium tuberculosis. The mycobacteria are rod-shaped, aerobic bacteria that do not form spores. Although they do not stain readily, after being stained, they resist decolorization by acid or alcohol and are therefore called ―acid-fast‖ bacilli. The Ziehl-Neelsen technique of staining is used for identification of acid-fast bacteria. Mycobacterium tuberculosis causes tuberculosis and is a very important pathogen of humans. Mycobacterium leprae causes leprosy. In tissue, tubercle bacilli are thin, straight rods measuring about 0.4 × 3 μm . The media for primary culture of mycobacteria should include a nonselective medium and a selective medium: Semisynthetic agar media—Inspissated egg media and Broth media. (LÖwensteinJensen medium is example for Inspissated egg media , small inocula in specimens from patients will grow on these media in 3–6 weeks. These media with added antibiotics are used as selective media) . Mycobacteria are obligate aerobes and derive energy from the oxidation of many simple carbon compounds. Increased CO2 tension enhances growth. The growth rate is much slower than that of most bacteria. The doubling time of tubercle bacilli is about18 hours. Saprophytic forms tend to grow more rapidly, to proliferate well at 22–33°C, to produce more pigment, and to be less acid fast than pathogenic forms. 72 Mycobacterium tuberculosis. Acid-fast stain. CDC. Mycobacterium tuberculosis revealed with a special stain specific for Mycobacteria,acid-fast Ziehl-Neelsen stain; Magnified 1000X. Image courtesy: CDC/Dr. George P. Kubica (1979). Mycobacteria http://www.flickr.com/photos/ajc1/510668360/ Mycobacterium tuberculosis scanning electron micrograph. Mag 15549X. CDC 73 Transmission electron micrograph of Mycobacterium tuberculosis In vitro culture of Mycobacterium tuberculosis on an agar slope. James King-Holmes 2011 . Mycobacterium tuberculosis colonies on7H 11 agar Mycobacterium tuberculosis colonies on agar. Courtesy Center for Hopkins University Tuberculosis Research, Johns 74 Mycobacterium tuberculosis Colonies on Lowenstein-Jensen medium. CDC. 3-2: Mycobacterium leprae Typical acid-fast bacilli—singly, in parallel bundles, or in globular masses—are regularly found in scrapings from skin or mucous membranes (particularly the nasal septum) in lepromatous leprosy. Since discovering of leprosy it has not been cultivated on nonliving bacteriologic media. Scrapings with a scalpel blade from skin or nasal mucosa or from a biopsy of earlobe skin are smeared on a slide and stained by the Ziehl-Neelsen technique. Biopsy of skin or of a thickened nerve gives a typical histologic picture. The bacilli are often found within the endothelial cells of blood vessels or in mononuclear cells. When bacilli from human leprosy (ground tissue nasal scrapings) are inoculated into footpads of mice, local granulomatous lesions develop with limited multiplication of bacilli. Inoculated armadillos develop extensive lepromatous leprosy. Photomicrograph depicting an acid fast stain of Mycobacterium leprae bacteria. Electron Microscope Micrograph 75 4-Cell Wall-Defective Bacteria (Mycoplasma ) - Overview Mycoplasmas are small, prokaryotic organisms with no peptidoglycan cell walls. Instead, they are enclosed in a single, trilaminar plasma membrane. They are, therefore, plastic and pleomorphic, and thus cannot be classified as either cocci or rods. Because of their extremely small size, mycoplasmas frequently pass through bacteriologic filters. Mycoplasmas are also the smallest of known free-living, selfreplicating prokaryotic cells. The many Mycoplasma species are widely distributed in nature, and include several commensals commonly found in the mouth and genitourinary tract of humans and other mammals. For these reasons, Mycoplasmas are often recovered as contaminants or adventitious flora from biologic materials, including clinical samples. Three Mycoplasma species are definitively associated with human disease, namely Mycoplasma pneumoniae, which is the cause of a primary atypical pneumonia, and Mycoplasma hominis and Ureaplasma urealyticum, which are associated with a variety of genitourinary diseases, such as urethritis, pelvic inflammatory disease, and intrapartum infections . -Colony production Mycoplasmas produce minute colonies on specialized agar after several days of incubation. These are best visualized under 30 to 100x magnification. The central portion of the colony penetrates the agar while the periphery spreads over the adjacent surface, in some cases giving the colony a characteristic fried egg appearance. -Mycoplasma Pneumoniae causes a lower respiratory tract infection (primary atypical pneumonia, so named because the signs and symptoms are unlike typical lobar pneumonia). -Laboratory identification Direct microscopic examination of clinical material for M.pneumoniae is of limited value. Sputum is scanty and nonpurulent, and the pathogen stains poorly or not at all using standard bacteriologic stains. Sputum samples or throat swabs can be cultured on special media; however, isolation of the organism usually requires eight to fifteen days and, therefore, cannot aid in early treatment decisions. Serologic tests are the most widely used procedures -Genital Mycoplasmas Mycoplasma hominis and Ureaplasma urealyticum are common inhabitants of the genitourinary tract . They grow more rapidly than M.pneumoniae, and can be 76 distinguished by their carbon utilization patterns; U. urealyticum hydrolyses urea. M. hominis degrades arginine, - Other Mycoplasmas Several other species of Mycoplasma can be recovered from human sources, for which no pathogenic role is established to date. One such organism, AIDSassociated Mycoplasma, or M. incognitus, has been isolated in high frequency from patients with that disease, in which the organism may play a role, possibly as a secondary invader. 77 78 5-Chlamydiae I. Overview Chlamydia is a genus of small bacteria that are obligate intracellular parasites, depending on the host cell for energy in the forms of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). They grow in cytoplasmic vacuoles in a limited number of host cell types. The genus is divided into three species: Chlamydia trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae. C.trachomatis infections cause diseases of the genitourinary tract and the eye, including many cases of nongonococcal urethritis and ocular infections such as trachoma. C.psittaci and C.pneumoniae infect various levels of the respiratory tract. For example, C.psittaci causes psittacosis, and C. pneumoniae causes atypical pneumonia - General Features of Chlamydiae Chlamydiae are small, round-to-ovoid organisms that vary in size during the different stages of their replicative cycle. The chlamydial DNA genome is less than 109 daltons in size, making it among the smallest found in prokaryotic cells. Chlamydiae possess ribosomes and synthesize their own proteins and, therefore, are sensitive to antibiotics that inhibit this process, such as tetracyclines and macrolides - Laboratory identification: Useful stains: Chlamydiae are not stained using the Gram stain, but can be visualized under light microscopy by stains that preserve the host cell architecture. Direct immunofluorescence is also a common and useful procedure. In C.trachomatis only, a matrix of glycogenlike material accumulates in the inclusions, which can be shown by staining with iodine. Other species do not give this reaction. -C. trachomatis causes a range of genitourinary and eye infections -Chlamydia Psittaci Psittacosis, or more broadly, ornithosis, denotes a zoonotic disease that is transmitted to humans by inhalation of dust contaminated with respiratory secretions or feces of infected birds. The human disease usually targets the lower respiratory. 79 -Chlamydia Pneumoniae C.pneumoniae is a respiratory pathogen causing pharyngitis, followed by laryngitis, bronchitis, or interstitial pneumonia. significant cause of community-acquired respiratory infection, worldwide and without seasonal incidence. 80 sometimes It is a occurring 81 82 List of Abbreviations Abbreviations CLED EMB SS agar MRSA GBS XLD TSA TEM SEM WBC Spp. CDC CIN CAMP test CAP BAP GC STD CCEY TSC HP Meaning Cystine -Lactose Electrolyte - Deficient Eosin Methylene Blue Salmonella Shigella Agar Methicillin-Resistant Staphylococcus aureus Group B Streptococci Xylose lysine deoxycholate Tryptic Soy Agar Transmission Electron Microscope Scanning Electron Microscope White Blood Cell Species Center for Disease Control and Prevention Cefsulodin Irgasan Novobiocin Christie-Atkins, Munch-Petersen test Chocolate Agar Plate Blood Agar Plate Gonococcal Sexually Transmitted Disease Cefoxitin Cycloserine Egg-Yolk Tryptose Sulphite Cycloserine Helicobacter Pylori 83 Appendix-1 84 Appendix -2 85 Appendix -3 86 Appendix -4 87 Appendix -5 88 Appendix -6 89 Appendix -7 90 Appendix -8 91 Appendix -9 92 Appendix -10 93 Appendix -11 94 Appendix -12 95 Appendix13 96 Appendix -14 97 Appendix -15 98 REFERENCE: 1-Chen, P. et al. 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