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BASIC BATERIOLOGICAL TECHNIQUES A) THE PATIENT The first consideration that should be in the mind of the MLS/MT practitioner when performing basic bacteriological techniques is the patient. Remember that the intention of using these techniques is to be ab...

BASIC BATERIOLOGICAL TECHNIQUES A) THE PATIENT The first consideration that should be in the mind of the MLS/MT practitioner when performing basic bacteriological techniques is the patient. Remember that the intention of using these techniques is to be able to produce accurate results in the fastest time possible so that the results can be used by the physician in making accurate diagnosis, treatment and patient management. The patient is the source of the specimen and related information that guides the MLS/MT practitioner on the procedures/techniques that will be adopted in specimen collection and the actual work in the bacteriology laboratory. Specimen collection depends on the request of the physician. Specimens would include the following: a.) Swabs = these are collected from a.1.) skin lesions with active disease such as boils, carbuncles, furuncles, pimples; pus is mainly the specimen that will be collected. Fungal diseases which can be dry lesions will require skin scrapings instead of a swab. a.2.) infected moist sites such as eyes, ears, nasopharynx, throat (not mouth or buccal area), male urethra, vaginal and cervix, rectum. The swab is placed inside a sterile tube with cap. Usually 2 swabs are collected, one for Gram’s stained smear and the other for culture. b.) Blood – the usual specimen is a venipunctured blood. Some physicians will request blood collected from the left and right arms for blood culture and antibiotic sensitivity test. c.) CSF – this is collected by the physician through a lumbar tap. The MLS/MT prepares the sterile syringe with needle, 3 sterile tubes without anticoagulant. In Clinical Microscopy, you will be told to prepare also 3 tubes. The first is for the bloody sample, the second is for the sample with trace blood and the third is for the clear CSF. d.) Urine – a mid-stream catch is collected. If the patient cannot urinate, a suprapubic aspirate maybe collected by the physician or a catheterized urine will be collected by a trained nurse or physician. The urine is placed in a sterile container. e.) Stool – this is collected first by the patient into a clean bed pan; from there, a sample about thumb size is collected with a clean wooden spatula to a sterile plastic container. f.) Sputum – collected by the patient after making deep coughing into a sterile plastic container. A correct sputum specimen should be sticky, no bubbles, does not flow in the container, yellow to red (due to blood) and sometimes with bits of lung tissues. A sample that is watery, with bubbles, flowing inside the container, and white in color is saliva and should be discarded. The patient has to repeat the collection in a new sterile container. g.) Aspirates – these can come as purulent, sometimes bloody fluids collected by the physician in the clinic or operating room. The specimen is usually in a sealed (sterile) tube. h.) Long tubes – nasopharyngeal tube, catheter, tube used for draining incisions, etc. The tube is usually brought to the bacteriology lab by the nurse wrapped in the plastic wrapper of the tube i.) Fluid drained from the operation site (head, neck, chest, abdomen, etc. contained in a bottle or plastic bag; may have large volume like a 500 ml container. j.) Operated tissue – usually incised by the surgeon and placed in a sterile Petri dish or sterile plastic container with cap. k.) Diaper containing stool –usually from a baby having diarrhea or sometimes from an old patient l.) Seminal fluid – usually upon request from the OB-GYN department to check if there is infection in the male reproductive system. Specimen is collected by the male patient by masturbation into a sterile plastic container with cap. In clinical microscopy, there are other ways considered for collection such as coitus interruptus or the husband and wife do sex but before ejaculation, the man withdraws his sex 1|Page  organ and collect the semen into a sterile container. This is usually done due to religious restrictions or socio-cultural beliefs. B) TRANSPORT OF SPECIMEN a.) Within the general laboratory Specimens collected in the general area of the laboratory should be labeled and without delay, brought to the Bacteriology Department for processing together with the physician’s request form. b.) Within the hospital Specimens collected from in-patients should be brought immediately to the laboratory for bar coding and transported to the Bacteriology Department as soon as possible. c.) From outside the hospital It is best that the Bacteriology Laboratory is informed in advance regarding specimen collection and transport so that the necessary materials for collection can be prepared and proper instruction can be given to the MLS/MT of the requesting hospital. C) LABELLING This is a very important part of the whole procedure. The patient should verbally say his/her complete name if the patient is conscious. If not, the guard should clearly say who the patient is and say the complete name. The MLS/MT should also write the age, sex (only Male or Female), Out-patient (OP) or In-Patient (IP) with Room Number or even Bed Number for a General Ward, and Date of collection. Bar coding is often used now so after all the data have been entered into the computer, a bar code is printed and pasted on the container, NOT on the cap of the container, because the caps can be mistakenly exchanged in the process of work. Bar coding is done when the specimen is already in the bacteriology laboratory or in the receiving area of the laboratory. Once the specimen is processed in the bacteriology laboratory, the bar code is removed from the specimen container and immediately attached or pasted on to the bacteriology worksheet each time, not later, so that there is no mistake of exchanging the specimen and the bar code. D.) USE OF PRESERVATIVES 1. Boric acid = for accurate colony counts in urine. 2. Phosphate buffered saline (PBS) = fecal suspension to prevent some bacteria like Shigella from dying; other broth media may be used depending on the bacteria. 3. Formalin, polyvinyl alcohol, Schaudinns’ fluid = preserve trophozoites and cysts in stool, including helminthic eggs and larvae E.) USE OF ANTICOAGULANT Important for samples that might clot such as blood at the same time not killing the microorganism. 1. Sodium polyanethol sulfonate at 0.025% w/v = preferably in vacutainer tubes. 2. Heparin – natural anticoagulant can be used but inhibits Gram-positive Bacteria 3. Citrate and EDTA – alternative for heparin. F.) REFRIGERATION VERSUS FREEZING Refrigeration is better. The growth of microorgnisms is stopped or slowed down. Freezing can kill bacteria. G.) FRAGILE VERSUS HARDY ORGANISMS 1. Fragile = Streptococcus pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonella, Shigella, Haemophilus incluenzae, anaerobes, Mycoplasma, Viruses, Chlamydia, parasites. 2. Hardy Organisms = Enteric bacteria, Pseudomonas, Enterococcus, other Streptococci, Staphylococcus, Yeasts, Molds, Mycobacteria, Legionella, Clostridium difficile H.) MAILING MICROORGANISMS 2|Page  Use the three tubes container/pack by CDC. 1. Innermost tube = tube with culture medium and bacteria; tube sealed with waterproof tape; tube surrounded with absorbent packing material 2. Secondary tube/container = records of the specimen 3. Tertiary/outermost tube = etiologic agent label with symbol of biohazard; address of the laboratory or institution that will receive the Specimen. The outermost tube is also called Shipping container. I.)PROTECTION OF THE PERSONNEL/MLS/MT 1. Wearing of face mask, face shield and gloves – primary importance for the health care worker. All specimens must be considered as having potential infectious microorganisms. Work is done in a BSL 2 laminar flow. Full PPE is required for highly contagious microorganism. 2. Source of the specimen should be indicated in the record. 3. Specimens must be properly submitted. The following are inappropriate: >Leaking tubes >Syringe with needle attached >Stool contaiminated with urine, barium >Anaerobes on inappropriate sources >Unpreserved specimens beyond 2 hours >Refrigerated blood cultures >Dried – up specimens >Specimens in formalin (toxic) 4. Note that specimens are collected with the assumption that the microorganisms to be cultured are aerobes and facultative anaerobes (aerobes growing anaerobically). Strict anaerobes are collected as Aspirates. Aspirate first into the syringe about 0.5 mL sterile mineral oil, then, aspirate the specimen from the infected site into the syringe. This will prevent exposure of the microorganisms to oxygen which will kill them. The specimen can be inoculated into anaerobic medium and incubated in a CO2 incubator. LEVELS OF SPECIMEN PRIORITIZATION LEVEL DESCRIPTION SPECIMENS 1 Critical/Invasive CSF, Blood, Amniotic fluid, Pericardial fluid, Bronchoalveolar lavage, Vitreous/Aqueous fluids; others like brain, heart valves 2 Unpreserved, may Sputum, stool. Body fluids not listed in No. 1, degrade or drainage from wound, pus, bone overgrow 3 Accuracy of Urine, quantitative tissue, catheter tip quantitation affected 4 Protected/preserved Swabs in holding medium(aerobic and anaerobic) 3|Page Direct examination techniques purposes: 1. Determine the quality of the specimen. 2. Aid in the diagnosis of infectious disease. 3. Guide routine culture interpretation. 4. Dictate the need for nonroutine processing. Smear preparation (in the laminar flow) 1. Swab = roll smear. Flame sterilize one side of a clean glass slide. Hold the frosted end of the slide with the left hand. See to it that the flamed side is facing above. Get the swab and position it near your left fingers by holding the stick/handle of the swab with your right hand. Then, slowly roll the swab to the right end of the glass slide. This is called “roll smear”. This will transfer the microorganisms to the slide without altering their arrangement. This technique is better than simply swabbing the slide back and forth which will destroy the cellular arrangement of the bacteria. Once done, place the slide on the floor of the laminar flow to dry. You can discard the swab by placing it in the glass jar for autoclaving. Label the frosted end of the slide with pencil. Once the smear is dry (around 5-10 minutes, hold the frosted end of the slide and pass it three times over the flame of the alcohol lamp of Bunsen burner. This is to “heat fix” the smear. It will kill the microorganisms and stick the bacterial cells, pus cells and other host cells to the slide. Once done, the smear is ready for staining. 4|Page 5|Page  TOPIC 2. STAINING OF SMEARS A. Simple staining – only one type of stain or dye is used. A.1. Positive staining – the stain colors the bacteria. Example: methylene blue staining Procedure. 1. After heat fixing the smear, place it on the staining 2. Add 2-3 drops of methylene blue to the smear using a medicine dropper. Allow the stain to react for 2 min. 3. Place tap water in a flask then, flood the smear to wash away the stain. 4. With a slide holder, tilt the smear to drain the water. Air dry the smear by placing it upright and tilted on a tissue paper. A.2. Negative or Relief staining – this technique stains the background but not the bacterial cells; used to see if there is capsule in the organism. Since the capsule is unstained, the background appears black while the capsule is a circle of light around the bacterial cell. Example: India Ink staining of CSF 1. Flame-sterilizer one side of the glass slide. Cool it. 2. With a medicine dropper, place 2 drops of fresh India ink or Nigrosine stain on the glass slide. 3. Using a flame-sterilized inoculating loop, take one loopful of the CSF and mix it with the stain in a circular manner to the size of a 25 centavo coin. 4. Flame-sterilize again the inoculating loop. 5. Get a cover slip using a forcep. Flame-starilize one size, allow it to cool, the gently place it over the CSF- stain mixture to avoid bubble formation. The smear can now be examined microscopically. 6|Page B. Differential Staining – the bacterial smear is stained with 2 dyes of different colors. B.1.) Gram’s staining – four reagents are used, namely: a.) Crystal violet = initial stain for 1 min b.) Gram’s Iodine = mordant, allows the crystal violet to bind well to the bacterial cell c.) Acetone-alcohol = decolorizer ( or 95% ethanol) d.) Safranin (red in color) = counterstain (can be substituted with carbol fuchsin dye; also red dye) 7|Page When crystal violet (CV) is added to the smear, it stains ALL bacterial cells violet. Upon addition of Gram’s Iodine as mordant, this enhances the attachment of CV to the bacteria. When Acetone-alcohol or 95% Ethanol is added as decolorizer, it dissolves the lipid part of the Gram-negative cell wall, removing the CV from the cells and rendering them colorless. Addition of safranin stains the colorless cells pink or red. Thus, Gram-positive bacterial cells are colored VIOLET while Gram-negative bacterial cells are colored PINK or RED (Principle). In summary: a.) Crystal violet: Initial stain: Gram(+) violet: Gram(-) violet b.) Gram’s iodine: Mordant: Gram(+) violet: Gram(-) violet c.) Acetone-alco: Decolori: Gram(+) violet: Gram(-) colorless d.) Safranin: Counterstain: Gram(+) violet: Gram(-) pink / red Prepare a bacterial smear. 1.1. Swab – roll smear, air dry, then, heat fix. 1.2. From a broth culture – use 1 loop of the growth on to a flame-sterilized glass slide. Air dry, then, heatfix 1.3. From a bacteria colony in tube or plate culture: Put 1 drop of sterile distilled water on to a flame-sterilized glass slide, then, pick with an inoculating needle a portion of the colony and homogenize the bacteria in the distilled water forming a circle of 25 cents. Air dry, then, heat fix. Gram’s Staining: 1. Place the smear on the staining rack. Add 5 drops of Crystal violet using a medicine dropper. React for 1 min then, wash with tap H2O. 2. Add 5 drops of Gram’s Iodine. React for 1 min then wash with H2O. 3. Decolorize briefly with Acetone-alcohol*. Wash with H2O. 4. Counterstain with 5 drops of Safranin. React for 1 min then, was with H2O. Air dry. Stained smear is ready for microscopic exam. *Over-decolorization will render Gram-positive bacteria Gram-negative. Fresh bacterial culture is required. Old cells grown beyond 48 hrs may also render Gram-positive bacteria Gram-negative or Gram-variable. 8|Page 9|Page 10 | P a g e B.2. Acid fast Staining This staining technique is used for Mycobacterium spp. because the cells are difficult to stain with Gram’s staining although they are called Gram-positive. The bacterial cell wall contains mycolic acid, a waxy substance. Only when heat or phenol-containing dye can the cells be stained by the initial stain, carbol fuchsin. They are called acid fast because they are now difficult to decolorize with acid, thus, they retain the initial stain (red color). All other bacteria in the specimen (e.g., sputum) will be decolorized and will take in the counterstain, malachite green or methylene blue, and are called non-acid fast. This staining technique is routinely used in the clinical laboratory. Procedures for Acid Fast Staining: A. Ziehl-Neelsen Method 1. Place the sputum smear (or other smear prepared as requested by the physician) on the staining rack. 2. Flood the smear with the initial stain, Carbol-fuchsin. 3. Using the Bunsen burner or alcohol lamp, flame the underside of the smear until the carbol fuchsin produces steam. This will take some seconds depending on the intensity of the flame. You add some more dye to avoid drying and precipitate formation. Allow the stain to react for 5 min. 4. Using a flask with tap water, flush the smear to wash the carbol fuchsin. (it is a wrong technique to lift the smear to drain the dye because precipitates will stick to the smear. Drain only later when some dye had already been washed away.) 5. Decolorize with several drops of 3% HCl until the red color is no longer seen in the draining liquid. Then, wash the smear with tap water. 3. Counterstain with either malachite green or methylene blue. Allow the dye to react for 2 min. 4. Flush the smear with tap water to wash the dye. 5. Air dry the smear. It is best to look for a place in the lab where you can incline the smear so that water can drain to the bottom of the slide. It is also good to place a sheet of tissue paper at the bottom of the slide to hasten the absorption of water. B. Kinyoun Method 1. Place the smear on the staining rack. Then, flood the smear with carbol fuchsin containing phenol. Allow to react for 5 min. 2. Flush the smear with tap water to wash. 3. Decolorize using 5% sulfuric acid. Then, wash with tap water. 4. Counterstain with malachite green or methylene blue for 2 min. 5. Air dry the smear. NOTES: 1. The smear becomes positive for Acid Fast Bacilli (AFB, e.g. Mycobacterium) when the specimen contains 5000-10,000 bacilli per mL, thus it is important that sputum and not saliva is collected. Sputum can be redish due to blood, sticky, and has no bubbles. Saliva is watery, it flows, whitish, and has bubbles. Repeat collection is needed if the specimen is not sputum. 2. Other genera such as Nocardia spp, Rhodococcus spp. Tsukamuralla spp. and Gordonia spp. may turn out partly acid fast and should not be read as AFB. 3. If urine sediments are requested for AFB staining (suspicion of TB of the kidneys), the sex must be known. Males who are uncircumcized may produce false positive AFB because their smegma that collect under the prepuce may have Mycobacterium smegmatis. 11 | P a g e  TOPIC 3. CLASSIFICATION OF BACTERIA There are many ways by which bacteria can be classified: A. Gram’s staining 1. Gram-positive – violet or purple in color 2. Gram-negative – red or pink in color B. Acid fast staining 1. Acid fast – red in color 2. Non-acid fast – green if malachite green was used as counterstain - blue if methylene blue was used as counterstain C. Morphology 1. Cocci – round or spherical in shape 1.1. Singly – coccus, cocci 1.2. Pairs – diplococci e.g., S. pneumoniae, Neisseria spp. 1.3. Chain – Streptococcus pyogenes, Streptococcus viridans 1.4. Tetrads – Gaffkya tetragena 1.5. Packets of 8 – Sarcina lutea or Micrococcus luteus 2. Bacilli – rod-shape bacteria 2.1. Cocco-bacillus - short or oval-shaped bacilli – Bordetella spp. 2.2. Bacillus – regularly shaped rods – Bacillus spp. 2.3. Fusiform – Fusobacterium spp. 2.4. Curved or comma-shaped – Vibrio spp. 2.5. Large – Lactobacillus spp. 3. Spiral or spirochetes 3.1. Loose or irregular – Spirillum sp.; Borellia spp. 3.2. Tight – Treponema spp. 3.3. With hooked ends – Leptospira spp. 12 | P a g e D. Spore formation 1. Spore formers – Bacillus spp., Clostridium spp. 2. Non-spore formers – Other species of bacteria 13 | P a g e  E. Flagellar structure 1. Atrichous – no flagella E.g., Staphylococcus, Streptococcus, Shigella spp., All non-motile bacteria 2. Monotrichous – only one polar flagellum E.g., Vibrio cholerae 3. Lophotrichous – tuft of flagella on one end of the bacillus E.g., Spirillum volutans , Bartonella bacilliformis 4. Amphitrichous – flagellum on both ends of the bacillus E.g., Alcaligenes faecalis, Rhodospirillum rubrum 5. Amphilophotrichous – tuft of flagella on both ends of the bacillus; some authors introduced this derivative. 6. Peritrichous – flagella are found surrounding the bacillus E.g., Salmonella spp., Bacillus subtillis, Escherichia coli Tests for Motility 1. Place 1 loopful of liquid culture on a clean glass slide. Add a coverslip. Examine under subdued light using HPO. See the unidirectional movement of bacteria. Brownian movement is seen as vibratory, non- directional movement of the cells. 2. Stab a semi-solid tube butt medium. After incubation, see the type of growth from the stab. 3. Prepare a suspension of the bacteria in sterile distilled water or Trypticase Soy Broth (TSB). Then, drop on a plate culture medium at least 20 uL of the suspended bacteria. Incubate the plate at 37oC overnight. Observe the spread of the bacteria from the drop off point to indicate motility. 14 | P a g e 15 | P a g e H. Oxygen requirement 1. Obligate aerobe – always require oxygen for growth: e.g., Mycobacterium tuberculosis, Micrococcus luteus 2. Facultative anaerobe – aerobes growing anaerobically; an organism that makes ATP by aerobic respiration if oxygen is present, but is capable of switching to fermentation if oxygen is absent. e.g., Streptococcus pyogenes, Enteric bacilli 3. Obligate anaerobe - they grow in the absence of oxygen. e.g., Clostridium tetani, C. perfringens, Bacteroides spp. 4. Aerotolerant anaerobe - do not use oxygen since they usually have a fermentative metabolism, but they are not harmed by the presence of oxygen. e.g., Lactobacillus spp 5. Microaerophilic – requires only 1%-10% oxygen for growth e.g., Campylobacter spp. 16 | P a g e I. Nutritional Requirement* 1. Autotrophs – free-living, uses carbon dioxide as carbon source. 1.1. Protoautotrophs - energy is obtained from photochemical rxn. 1.2. Chemoautotrophs – energy is obtained from chemical rxn through oxidation of compounds 2. Heterotrophs – obtain their energy from more complex compounds such as sugars from their host; parasitic microorganisms *NOTE: Microbial physiologists may have more complex way of classifying microorganisms. J. Ability to Cause Disease 1. Non-pathogenic/commensal – they do not cause disease in the host (human or animal). The current view is that commensal bacteria act on the host's immune system to induce protective responses that prevent colonization and invasion by pathogens. On the other hand, these bacteria can directly inhibit the growth of respiratory pathogens by producing antimicrobial products/signals and competing for nutrients and adhesion sites. E.g., diphtheroids 2. Opportunistic – do not normally cause disease in the host unless conditions allow them to do so such us depressed immune status, dedpletion of commensals due to antibiotic use, cancer, severe burns, etc. E.g., Pseudomonas aeruginosa 3. Pathogenic – can cause disease in the host. E.g. C. tetani = pathogenicity = ability of a microorganism to cause disease = virulence = the extent of pathogenicity of a microorganism. NOTES: 1. The ability to cause disease can be due to one or more factors present in the bacteria. It can be a toxin, a capsule, or a combinbation of several factors present in the bacteria as well as conditions in the host and environment. 2. In some cases, the same species of bacteria can be pathogenic because of the factor (e.g. capsule) or non-pathogenic because of its disappearance due to mutation. E.g., Streptococcus pneumoniae Bacteria: lack membrane -bound organelles and can function and reproduce as individual cells, but often aggregate in multicellular colonies. Their genome is usually a single loop of DNA, although they can also harbor small pieces of DNA called plasmids. These plasmids can be transferred between cells through bacterial conjugation. Bacteria are surrounded by a cell wall, which provides strength and rigidity to their cells. Archaea: In the past, the differences between bacteria and archaea were not recognized and archaea were classified with bacteria as part of the kingdom Monera. Archaea are also single-celled organisms that lack nuclei. Archaea in fact differ from bacteria in both their genetics and biochemistry. While bacterial cell membranes are made from phosphoglycerides with ester bonds, archaean membranes are made of ether lipids. Eukaryotes: Unlike bacteria and archaea, eukaryotes contain organelles such as the cell nucleus, the Golgi apparatus, and mitochondria in their cells. Like bacteria, plant cells have cell walls and contain organelles such as chloroplasts in addition to the organelles in other eukaryotes. 17 | P a g e  TOPIC 4. BACTERIAL CULTURE MEDIA INTRODUCTION One of the most important reasons for culturing bacteria in vitro is its utility in diagnosing infectious diseases. Isolating a bacterium from sites in body normally known to be sterile is an indication of its role in the disease process. Culturing bacteria has the following purposes: 1.) serve as an initial step in studying its morphology and identification. 2.) obtain antigens for developing /doing serological tests or vaccines. 3.) provide cells for genetic and molecular studies. 4.) provide a reliable way to estimate their numbers (viable count). 5.) separate mixture of microorganisms on solid media. HISTORY Louis Pasteur used simple broths made up of urine or meat extracts. Robert Koch realized the importance of solid media and used potato pieces to grow bacteria. It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media. Before the use of agar, attempts were made to use gelatin as solidifying agent. Gelatin had some inherent problems; it existed as liquid at normal incubation temperatures (35-37o C) and was digested by certain bacteria. COMPOSITION OF CULTURE MEDIA Bacteria infecting humans (commensals or pathogens) are chemoorganoheterotrophs. Thus, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat. An artificial culture medium must provide all the nutritional components that a bacterium gets in its natural habitat. Most often, a culture medium contains water, a carbon and energy source , nitrogen, trace elements & growth factors. Besides these, the pH of the medium must be set accordingly. Some of the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract and malt extract. CLASSIFICATION Bacterial culture media can be classified in at least three ways: 1.)Consistency 2.)Nutritional component 3.)Functional use or purpose. 1) Classification based on consistency: 1.1.) Liquid – nutrients are present except agar (0 %), available for use in test-tubes, bottles or flasks; sometimes referred as “broths” (e.g., Nutrient Broth); bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on the surface of undisturbed broth. Bacillus anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen in pneumococci or S. pneumoniae. (See the figure of bacterial growth in Topic 3: Oxygen requirement.) Long chains of Streptococcus when grown in liquid media tend to entangle and settle to the bottom forming granular deposits. Liquid media tend to be used when a large number of bacteria have to be grown. These are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can be used to obtain viable count (dilution methods). Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria cannot be detected. 18 | P a g e  1.2.) Solid – contains 1.5%-3% agar., an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium (Ceylon moss); composed of two long-chain polysaccharides (70% agarose and 30% agarapectin); melts at 95o C (sol) and solidifies at 42o C (gel), doesn’t contribute any nutritive property, not hydrolyzed by most bacteria; free from growth promoting or growth retarding substances; may be a source of calcium & organic ions. New Zealand agar has more gelling capacity than the Japanese agar. Agar is available as fibers (shreds) or as powders. Prepared as plates, slants, slant-butt, & butt (agar deep). 1.3.) Semi-solid - agar content is 0.2-0.5% ; renders media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains (U-tube and Cragie’s tube). Certain transport media such as Stuart’s and Amies media are semi-solid in consistency. Hugh & Leifson’s oxidation fermentation test medium as well as mannitol motility medium are also semi-solid. 1.4.) Biphasic - a culture system comprises of both liquid and solid medium in the same bottle (Castaneda system for blood culture). The inoculum is added to the liquid medium and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid medium. This obviates the need for frequent opening of the culture bottle to subculture. Besides agar, egg yolk and serum too can be used to solidify culture media. While serum and egg yolk are normally liquid, they can be rendered solid by coagulation using heat. Serum containing medium such as Loeffler’s serum slope and egg containing media such as Lowenstein Jensen medium and Dorset Egg Medium are solidified as well as disinfected by a process of inspissation (thickening by coagulation using moist heat). 19 | P a g e 2. Classification based on nutritional component 2.1.) Non-synthetic – composition is not well defined 2.2.) Synthetic - composition is defined While most of the nutritional components are constant across various media, some bacteria need extra nutrients. Those bacteria that are able to grow with minimal requirements are said to non-fastidious and those that require extra nutrients are said to be fastidious. 3. Classification based on functional use or application 3.1.) Simple - Peptone water, Nutrient agar can support most non-fastidious bacteria. 3.2.) Enriched – contains additional nutrients. E.g., Blood Agar Plate (contains 5%-10% sheep blood; sometimes human blood). Other materials include serum, ascetic fluid, albumin. 3.3.) Differential – solid medium containing a dye or pH indicator to differentiate at least 2 types of bacteria. Gelatin Medium is a differential medium used to select bacteria that produces gelatinase and thereby hydrolyze gelatin (Kohn modified gelatin method), differentiating the isolate from gelatinase non- producer. 3.4.) Selective – to isolate a specific group of bacteria; medium contains certain inhibitory substance such as bile salts, dyes, alcohol, etc. that prevent the growth of certain groups of bacteria. E.g., Phenylethanol Agar (PEA) allows only Gram-positive bacteria to grow while Gram-negatives are inhibited by the alcohol. 3.5.) Special - for the isolation of certain genus or species of bacteria. E.g., Lowenstein-Jensen medium – for Mycobacterium spp. NOTE: An Enriched medium is different from an Enrichment Medium. The latter is used to increase the growth of an important microorganism before doing a sub-culture. 20 | P a g e 21 | P a g e  TOPIC 5. ANTIBIOTIC SENSITIVITY TEST INTRODUCTION Antibiotic sensitivity or susceptibility test is an essential step in the characterization of a bacterial isolate from a clinical specimen. The results will aid the physician in the treatment and management of the patient. As such, this has to be performed with accuracy and precision. ACTIONS OF ANTIBIOTICS O BACTERIA 1. Act against cell wall integrity: Beta-lactam antibiotics, vancomycin 2. Effect against cell membrane structure & function: Polymyxins 3. Inhibition of protein synthesis: Aminoglycosides, Tetracyclines, Macrolides, Clindamycin, Chloramphenicol 4. Inhibition of essential metabolites: Sulfonamides, Trimethoprim 5. Interference with nucleic acid metabolism: Rifampin, Quinolones, Metronidazole 22 | P a g e TYPES OF ANTIBIOTIC SENSITIVITY TEST 1. Disc diffusion - qualitative 2. Tube dilution - quantitative 3. Strip diffusion – quantitative 1. DISC DIFFUSION TEST This is based on the ability of an antibiotic in an impregnated disc to diffuse into a plate medium seeded with a lawn of the test bacterial isolate. The diameter zone of inhibition is interpreted as Sensitive, Intermediate, and Resistant, based on previous studies of the Manufacturer of the antibiotic discs. 1.1.McFarland Standard Regardless of the method used, the concentration of the bacterial inoculum must be standardized. The McFarland turbidity standards are prepared by adding specific volumes of 1% Sulfuric acid and 1.175% Barium chloride. The commonly used preparation is: >McFarland 0.5 standard: 99.5 mL 1% Sulfuric acid + 0.5 mL 1.175% Barium chloride. The precipitate of Barium sulfate produces a turbidity comparable to a bacterial suspension of 1.5 x 108 colony forming units (CFU)/mL. 1.2. Inoculum of bacteria a.) From the plate culture of the bacteria, pick individually 3-5 similar colonies with a flame-sterilized inoculating needle and inoculate a 5 mL sterile Trypticase Soy Broth or Mueller-Hinton Broth. Incubate the tube at 37 C for at least 12 hours or until the turbidity is greater than the McFarland standard. The bacterial must be in the Logarithmic or Exponential phase. b.)Get a fresh 5 mL broth and slowly add to it the bacteria to be tested using sterile 100 mL micropipet. Compare the turbidity with the McFarland standard. To do this, place the 2 tubes side by side in front of a white paper having thick and thin black lines seen against the light. The black lines behind the McFarland 23 | P a g e  standard should have the same intensity with the tube containing the bacterial suspension. The bacteria should be plated within 15 minutes, otherwise it will overgrow. 0.5 McFarland Standard Bacterial Suspension 1.3.) Mueller-Hinton Agar – this is prepared according to the Manufacturer’s Instruction (Difco, BBL or Merck Brand). A 100 mL preparation is sufficient for 4 standard Petri dishes. Mix the powdered medium in distilled water. Place a cork or cotton plug to avoid evaporation of water. Boil in a water bath until the agar is melted. Autoclave to sterilize at 121oC, 15 pounds/ square inch (PSI) pressure for 15 min. The Petri dishes top and bottom must be wrapped with bond paper or Manila paper and autoclaved together with the medium. Cotton swabs wrapped also with bond paper should be autoclaved as well. After autoclaving, allow the temperature of the autoclave to return to zero BEFORE opening the autoclave. Cool the MHA to around 40oC before dispensing in 4 Petri dishes. The hotness can be felt by feeling the arm skin on the flask. Dispensing should be done in the Laminar flow to avoid contamination. The Petri dish cover is placed halfway on top of the Petri dish bottom to allow some evaporation to take place but not to collect moisture of condensation on the underside of the Petri dish cover. After around 15 min, the MHA has already solidified. The plates can be covered with the dish top and placed upside down, then wrapped with the bond paper or Manila paper used for autoclaving. The plates can be stored in the refrigeration section at 4-8oC and should be used within 1 week for the antibiotic sensitivity test. The paper wrapper should be labelled with the name of the medium and date of preparation prior to refrigeration. 1.4.) Inoculation of the MHA plates At least 2 MHA plates are used for every bacterial isolate to be tested. Get the MHA plates from the refrigerator and allow to pre-ward at room temperature at least 15 min before inoculating the plate. The wrapper should be removed only when inoculation is about to be done. Work inside the Laminar flow. Remove the paper wrapper from the two MHA plates. Place the plates in the “down-up” position but retain the cover of each plate. Open the tube containing the standardized bacteria. With a sterile cotton swab, dip it into the broth. Wring the swab against the inner wall of the tube to remove the excess broth. Cover again the tube and set it aside. Then, open one MHA plate with your left hand and spread the bacteria using the moist cotton swab with your right hand by making cris-cross action on the surface of the MHA. Once done, rotate a bit the plate and do second streaking. Rotate a bit the plate and do third streaking. Rotate the plate a bit and do the fourth streaking. Finally, streak the inner side of the plate by making a circular motion of the swab. By doing this, you are assured that the whole surface of the MHA has been seeded to make a lawn of the bacteria. Place the inoculated MHA aside with the dish top covering half of the dish bottom to partially dry the MHA surface. Repeat the process for the second MHA plate. 24 | P a g e 1.5. Placing the antibiotic discs The antibiotic discs are purchased in small plastic canisters of 100 discs per type. It is important to have a collection of discs having different antibiotics. Each bacterial isolates can be tested with as many as 20 antibiotics of different types. Bring to the laminar flow the canisters from the refrigerator. It is best to place the canisters inside a plastic box to avoid contamination. A forcep is also needed. Inside the laminar flow, it is best to have an electric coil for sterilization of inoculating loop, inoculating needle and forceps. After about 10 min from inoculation of the MHA plates, sterilize a forcep and carefully get one disc from the canister. Once done, carefully place the disc on a pre-determined spot on the MHA plate. Once the disc is placed on the plate, it should not be removed anymore. Sterilize the forcep before taking another disc. 1.6. Reading the zones of inhibition After incubation, the plates are taken from the incubator. Plates are placed on the working table and all discs used are checked using the laboratory-made checklist. Zone of inhibition is measured with a caliper or ruler with millimeter calibration. The diameter is measured by placing the caliper or ruler on the zone of inhibition across the center of the disc. For an irregular zone, 2 readings are done. Hypothetical Example: Diameter Zone of Inhibition (Manufacturer) Reading Antibiotic X 10 ug/disc 0 – 7 mm (R) 8 –11 (I) 12 & above (S) 14 mm (S) Antibiotic Y 15 ug/disc 0 – 9 mm (R) 10-14 (I) 15 & above (S) 12 mm (I) Antibiotic Z 12 ug/disc 0 - 8 mm (R) 9-13 (I) 14 & above (S) 8 mm (R) Report: Organism: Staphylococcus aureus Antibiotic X = S (sensitive) Antibiotic Y = I (intermediate) Antibiotic Z = R (resistant) Additional antibiotics used are also reported. 25 | P a g e NOTES: 1. Antibiotic X should have a known concentration, E.g., 100 ug/mL. 2. T1 contains antibiotic (1 mL) + Standardized bacteria in TSB 1 mL. Because of the dilution, T1 has now 50 ug/mL Antibiotic X. 3. T2 up to T8, the concentration of Antibiotic X is reduced by ½. T2=25 ug/mL; T3=12.5 ug/mL; T4=6.2 ug/mL, T5=3.1 ug/mL., etc. 4. T9 is the Negative control without antibiotic. 5. To get the Minimum Inhibitory Concentration of the antibiotic, look for the highest dilution without turbidity (growth). E.g., T1=NG, T2=NG, T3=NG, T4=NG, T5=NG, T6=G, T7=G, T8=G, T9=G Thus, T5 is the MIC=3.1 ug/mL of Antibiotic X. 6. To determine the Minimum Bactericidal Concentration (MBC), get a loopful of the mixture from each tube with No Growth (NG). Streak separately on MHA plates and incubate at 35oC overnight (O/N). The plate (=tube) with No Colonies (NC) is the MBC. E.g., T1=NC, T2=NC, T3=NC, T4=NC, T5=WC Thus, the MBC is T4=6.2 ug/mL. Why is the MIC different from MBC? By experience, even if the tube appears to have no growth (no visible turbidity) there are still few cells that are alive, thus, when plated, they will form colonies. Usually, a higher concentration of the antibiotic will reveal the MBC. In this activity, the MIC is T5 but the MBC is T4. It is important for the Medical Laboratory Scientist to observe very clearly which tube has No Growth and has Growth. 26 | P a g e 3. Strip diffusion (E test) The strip (AB Biodisk, Sweden) is impregnated with varying concentration of the antibiotic starting with the highest concentration at the tip and in decreasing amount along the length of the strip. When the strip is placed on a lawn of bacteria on MHA, the antibiotic will diffuse into the medium, inhibiting/killing the bacteria on both sides of the strip. Instead of a circular zone of inhibition, this time, the inhibited area looks semi-oval fan with a stick. The area with the least inhibition of growth will indicate the least amount or concentration of the antibiotic that can inhibit the growth of the test organism. Strips of different antibiotics can be placed on one MHA plate. 27 | P a g e 28 | P a g e 29 | P a g e  BACTERIOLOGICAL TECHNIQUES FOR THE IDENTIFICATION OF CLINICALLY IMPORTANT BACTERIA IMPORTANCE OF COLONIAL MORPHOLOGY OF BACTERIA 1. Provide a presumptive diagnosis to the physician in time of critical need. 2. Enhance the quality of patient care through rapid reporting of results and by increasing this cost- effectiveness of laboratory testing. 3. Play a significant role in quality contro, especially of automated procedures and other commercially available identification systems. Notes: 1. Colonial morphology of primary cultures are noted after 18-24 hrs of incubation of plate cultures. Thus, streaking is very important to get isolated colonies. Observe that streaking is aimed to dilute the distribution of the bacterial cells on the plate. After the primary streaking, the initial of the secondary streaking overlaps the primary streaking but not all streaks. Same with the 3rd and 4th streakings 30 | P a g e 31 | P a g e Bacillus anthracis = Medusa head colonies Nocardia spp. = brittle, form splinters, bread Proteus spp.=swarming colonies crumbs Corynebacterium ssp = rough edges Staphylococcus aureus = creamy (butyrous) Strep. pneumoniae= umbilicate or check-shape Niesseria spp. = sticky colonies Corynebacterium spp (diphtheroids) = waxy Staphylococcus aureus = convex colonies Streptococcus species = pin point or dew drop F. ODOR colonies It is not safe to inhale the smell of a culture but rather, to smell as the D.PIGMENT OR COLOR odor dissipates while the Petri dish is open in the Staphylococcus epidermides = white colonies laminar flow. Enterococcus spp. = gray colonies Staphylococcus aureus = old socks/stockings Neisseria spp, Micrococcus(non-pathogenic) = unwashed for a few days yellow, off white colonies Pseudomonas aeruginosa = fruity (apples, pears, Prevotella melaninogenica = brown-black grapes combined) Pseudomonas aeruginosa = green colonies Proteus mirabilis = putrid Serratia marcescens = red colonies Haemophilus = musty basement room Kluyvera sp. = blue Nocardia = freshly plowed field, soil-like Chromobacterium violaceum = purple NOTE: You can observe that some bacterial species E. CONSISTENCY OF COLONIES have multiple Determined by touching the colony with a sterile colonial morphology inoculating loop. 32 | P a g e  TOPIC 6B. CLINICALLY SIGNIFICANT COCCI-Staphylococcus spp A. Genus Staphylococcus and Micrococcus (Family Micrococcaceae) 1.)Gram-positive cocci appearing singly, in pairs, in short chains and mostly in clusters (bunch of grapes); 0.5- 1.5 u in diameter; facultative anaerobe, except S. saccharrolyticus obligate anaerobe; non-spore former, non- motile. 2.)Colonies appear after 18-24 hrs incubation at 37C, white, cream to yellow/light gold, buttery consistency; some B-hemolytic. 3.)Common habitat of the skin & mucous membrane. 4.)Coagulase test =very important biochemical test to identify S. aureus (+), a human pathogen. S. intermedius, S. dedlphini, S. hyicus, S. schleiferi are coagulase(+) but are animal strains. Coagulase(-) human strains include S. epidermidis, S. saprophyticus. Other staphylococcus species are only “nice to know”. There are two types of coagulase based on the technique used. a.)Bound coagulase = “clumping factor”: on a glass slide, mix 1 drop of human, rabbit or pig plasma and 2 colonies of Gram-positive cocci in cluster; result is clot formation (+)=S. aureus. b.)Free coagulase =should be performed if the slide test is negative. Prepare in a tube a 1:6 diluted rabbit plasma available commercially (1 vol plasma + 5 vol 0.85% NaCl; e.g., 0.5 mL rabbit plasma + 2.5 mL NaCl) Transfer 1 mL dil rabbit plasma into another tube. From the plate culture, pick at least 3 similar colonies and imulsify in the plasma. Incubate at 35C water bath. Observe for clot formation after 1, 2, 3, and 4 hrs. PRINCIPLE: Coagulase acts as Coagulase-reacting Factor (CRF) that behave like a blood clotting factor that converts fibrinogen in plasma to fibrin to form a clot. Observation is done hourly because after 4 hours, the clot may be dissolved through fibrinolysis. 33 | P a g e 8. Methicillin Resistant Staphylococcus aureus (MRSA) This test detects S. aureus strains that are resistant to methicillin. The test does not only test for methicillin resistance but also to other B-lactam antibiotics such as oxacillin, penicillin, amoxicillin and cephalosporins. MRSA strain produces an altered penicillin binding protein (PBP2) since they have a mecA gene. An MHA plate seeded with Gram-positive cocci in clusters is added with a methicillin disc and incubated at 35-37C for 24 hours. MRSA is resistant, no zone of inhibition. 9. Cultivation: Use BAP (Gram-positive cocci, Gram-positive bacilli and Gram-negative bacilli will grow. Phenylethanol Agar only Graam positives will grow. Mannitol Salt Agar containing 7.5% NaCl will allow halophilic (salt-loving) bacteria to grow. Mannitol is fermented and in the presence of phenol red as pH indicator, colonies of S. aureus will be yellow. 10. Toxins produced by S. aureus: a.) Cytolytic = virulence factor b.) Enterotoxins = associated with food poisoning resulting in diarrhea; pancit, foods containing cream (pasta, steamed fish), toxic shock syndrome toxin-1 (TSST-1) c.) Exfoliative toxins=causes scalded skin syndrome or Ritter’s disease especially in infants and children. 11. Protein A: present in Cowan 1 strain and other isolates of S. aureus. It binds with the Fc portion of IgG thereby avoiding opsonization. Used for co-agglutination test in serology to identify other bacteria. 34 | P a g e  TOPIC 6Ca-STREPTOCOCCUS & ENTEROCOCCUS 1.) Members are Gram(+) cocci in pairs and in chain. 2.) Metabolism is fermentative; facultative anaerobic; require nutrients for growh; grows well on BAP; with one of three types of hemolysis. 3. Except the viridans group, cell wall contain Group C carbohydrate substance used for serologic typing. 4. Groupings: 4.1. Hemolytic patterns: Alpha, Beta, Gamma (non-hemolytic). An Alpha prime of small area of intact red cells and wide zone of complete hemolysis was reported. 4.2. Physiologic: type of infection in the host 4.2.1. Pyogenic type-pus forming strains, Beta-hemolytic type, constitutes most of Lancefield groups 4.2.2. Lactic acid type-mostly found in dairy produced, non-hemolytic, Lancefield group N 4.2.3. Enterococcus type – found in human intestine, now under Genus Enterococcus 4.2.4. Viridance type – do not have the group C carbohydrate substance, normal flora of human respiratory tract, alpha or gamma hemolytic, may be opportunistic 4.3. Serologic/Lancefield grouping – Rebecca Lancefield extracted the group C carbohydrate substance and used this as antigen to raise antibodies(Abs)/immunoglobulins (Igs) in animals; serotypes could be organized from Group A= S. pyogenes Group B= S. agalactiae; Group C= S. equisimilis; Group D= S. bovis, equinus, & Enterococcus faecalis, E. faecium, E. durans; Groups F, G, etc. No Group C carbohydrate substance: Strep. pneumoniae, S. anginosus, S. sanguis, S. mitis, S. mutans, etc. 35 | P a g e  4.4. Biochemical grouping NOTE: The secret of remembering tests is to see which is unique to a test. For instance, in a test, all are + except __? Or all are neg. except? Hippurate Hydrolysis: to differentiate S. agalactiae from other Beta hemolytic Streptococcus. Principle: Hippuricase from bacteria hydrolysis sodium hippurate to sodium benzoate & glycine. Addition of Ninhydrin reacts with glycine to form a purple color Inoculate a tube having 0.5 mL of 1% sodium hippurate with 3-4 colonies of B-hemolytic Strep. Incubate at 35C for 2 hrs. Add 0.2 mL Ninhydrin. See a purple color for positive test. CAMP Test. To differentiate S. agalactiae from other B-hemolytic Strep. On BAP, streak on side with B-hemolytic Staphylococcus aureus. Then, streak B-hemolytic Strep in tangent with the S. aureus streak. Incubate at 35C for 18-24 hrs. Observe an arrow-head demolysis. PYR test: Pyrolidonyl aminopeptidase test to differentiate S. pyogenes from other B-hemolytic Strep. Inoculate 1 mL PYR tube with B-hemolytic Streptococcus. Incubate for 4 hrs at 35-37C. Add 3 drops PYR reagent and see a red color. 36 | P a g e 37 | P a g e  TOPIC 6Cb-STREPTOCOCCUS CLINICAL IMPORTANCE 1. Streptococcus pyogenes 1.1. Cell wall structure 1.2. Virulence factors 1.2.1. M protein stimulates strain-specific antibodies that confer resistance to that strain. 1.2.2. Streptolysin O=oxygen labile, antigenic resulting in Anti-Streptolysin O (ASO) antibodies; associated with post streptococcal infection and disease; causes sub-surface hemolysis on BAP; streaking with cut-streaks on BAP 1.2.3.Streptolysin S =oxygen stable but not antigenic, causes surface hemolysis on BAP 1.2.4. DNAases= four types A, B, C, D; most common is B.; they cause antibody formation 1.2.5. Streptokinase =lysis of fibrin clot; enzyme is antigenic. 1.2.6. Hyaluronidase or spreading factor 1.2.7. Erythrogenic toxin =cause of scarlet fever; due to exotoxins A, B, C. 38 | P a g e 1.3. Bacterial pharyngitis or ‘strep throat” common among children 5-15 years old 1.4. Pyodermal infection =empitigo, cellulitis, erysipelas, wound infection or even gangrene 1.5. Post-streptococcal infection =caused by Anti-streptolysin O antibodies that react with the heart muscle resulting in rheumatic fever; ASO also form immune complexes with Streptolysin O which are deposited in the glomeruli causing acute glomerulonephritis. 1.6. Streptococcal toxic shock syndrome 2. Streptococcus agalactiae 2.1. mastitis in cattle 2.2. infection in newborns, early onset and late onset. 3. Group D Streptococcus 3.1. S. bovis causing bacteremia and gastrointestinal tumors 4. Enterococcus 4.1. Diseases similar to group D Streptococcus 5. Strep. pneumoniae 5.1. Contain 82 capsular serotypes; virulence factor 5.2. Associated with lobar pneumonia, bacteremia, meningitis 5.3. Colonies are described as “checker-shape”, become flat after sometime due to autolytic activity 5.4. enzymes: neuraminidase, protease, hyaluronidase 6. Viridans Streptococci 6.1. S. mutans, S. salivarius, S. sanguis, S. mitis, S. anginosus. 6.2. Oropharyngeal commensals but are opportunistic pathogens causing bacteremic, meningitis, dental caries, 7. Aerococcus 7.1. Aerococcus viridans =only species 7.2. Tend to form tetrads instead of cocci in chain 7.3. Causes different types of infection although rarely 8. Leuconostoc 8.1. Appears like cocco-bacilli in chain 8.2. L.lactis, L. mesenteroides, L. paramesenteroides 8.3. Rarely cause meningitis, endocarditis, septicemia 9. Pediococcus 9.1. Appears in pairs, tetrads, and clusters 9.2. There are 8 species, 2 isolated from humans: P. acidilactici, P. pentosaceus. 10. Gemella =very rare human pathogens (nice to know). 39 | P a g e  TOPIC 7: GRAM-NEGATIVE COCCI & COCCOBACILLI-NEISSERIACEAE The Family Neisseriaceae contains four genera: Neisseria, Moraxella, Acinetobacter, Kingella: plump cocci, coccobacilli, rods, Gram negative, non-motile, strictly or preferentially aerobic, optimum temperature for growth 32-36C. 1. Neisseria: coffee-bean diplococci, adjacent sides flat, cell division 2 planes, Catalase +, Oxidase +, G+C content 46.5-53.5% in DNA. Human isolates/pathogens: N. gonorrhoeae, N. meningitidis Nonpathogens: N. cinerea, N. elongata (rod shape), N. flavescens, N. lactamica, N. mucosa, N. polysaccharea, N.sicca, N. subflava Animal isolates: N. caviae, N. canis, N. denitrificans, N. macacae, N. ovis, N. parelongata (rod) 2. Moraxella: 2.1.Subgroup Branhamella: cocci, adsjacent sides flat, cell division 2 planes, Catalase +, Oxidase +; G+C content 40-47.5% in DNA Human isolates: M.(B.) catarrhalis Animal isolates: M.(b.) caviae, M.(B.) cuniculi, M.(B.) ovis 2.2.Subgroup Moraxella: short rods, pairs & chains, cell division 1 plane Catalase +, Oxidase +, G+C content 40-47.5% in DNA Isolates: M.(M.) atlantae, M(M.) cuniculi, M.(M.) bovis, M.(M.) lacunata, M.(M.) nonliquifaciens, M.(M.) osloensis, M.(M.) phenylpyruvica 3. Acinetobacter: Log phase (short rods), Stationary phase (cocco-bacilli), cell division 1 plane, Catalase +, Oxidase - , G+C content 38-47% in DNA Isolates: A. baumannii, A. calcoaceticus, A. haemolyticus, A. junii, A. johnsonii, A. lwoffii, etc. 4. Kingella: rods, pairs & chains, cell division 1 plane, Catalase - , Oxidase +, G+C content 47.3-53.8% in DNA NOTE: In the bacterial growth curve, there are 4 stages better seen in a broth culture because of extent of turbidity that can be measured using a photometer: a.) Lag or adaptation phase- when the bacterial cells are just introduced into the fresh culture medium, may take a few hours b.) Log or exponential phase – rapid cell division in a short period of time, cells are physiologically active, (best time to introduce antibiotics) c.) Stationary or plateau phase –the number of cells dividing is about equal to cells dying, nutrients in the medium are starting to be consumed d.) Death or decline phase – many cells are dying more than new cells growing and dividing because of depleted nutrients in the medium and toxins that are accumulating. 1. N. gonorrhoeae: 1.1. Humans are the only known host. 1.2. Gram-negative coffee-bean shaped diplococci when seen on Gram-stained smear of clinical specimens; urethral smear in males, cervico-vaginal smear in females, anal smear or throat smear in sexually active persons; eye smear in infants born of mothers with cervico-vaginal gonorrhea 40 | P a g e  Neutrophil or pus cell 1.3. Cell wall contains capsule, pili (T1-T5), outer membrane, LPS (endotoxin), Proteins I, II, III (antigens), peptidoglycan layer, cytoplasmic membrane. 1.4. Short incubation period 2 to 7 days. Males have urethritis, dysuria, prostatitis, and epididymitis. Asymptomatic infections are present. In females, the endocervis is infected with vaginal discharge and dysuria, may progress into pelvic inflammatory disease (PID, resulting to sterility, ectopic pregnancy or perihepatitis (Fitz-Hugh-Curtis syndrome) Infection of other sites: bacteremia, anorectal, oropharyngeal, ophthalmia neonatorum in newborns , requires silver nitrate application on the eyes of normally born infants (Crede’s prophylaxis). 1.5. Dacron or rayon swabs are preferred since cotton swab and calcium alginate swab can kill the bacteria 1.6. Gram-stained smear: the Gram-negative coffee-bean shaped diplococci intracellularly located in neutrophils or pus cells is indicative: Report of Gram-stained smear: Gram-negative coffee bean shaped diplococci intracellularly located characteristic of gonococci seen. 1.7. Culture medium: Thayer-Martin Agar. Swab is streaked in a Z manner. And cross-streaked with a sterile inoculating loop. When the bacteria grow, the Plate can be positioned to shown an N growth (for Neisseria). The TM medium contains VCN Vancomycin to inhibit Gram(+), Colistin to inhibit Gram(- ), and Nystatin to inhibit yeasts (fungus). Cultivation is done by putting the plates inside a candle jar to provide 3-5% carbon dioxide for growth. 41 | P a g e 1.9. Antibiotic Susceptibility test: Ceftriazone should be used since this is the antibiotic of choice. Penicillins have already shown resistance in many strains of N. gonorrhoeae. 2. Neisseria meningitidis 2.1. Found mostly in nasopharynx but may be found also in urogenital and rectal areas; CSF, blood and aspirates. 2.2. Nasopharyngeal swab and CSF are the preferred specimens. 2.3. Stains A, B, C, Y and W-135 are most often associated with epidemics. Meningococcasl pneumonia associated with serogroup Y; W-135 is associated with invasive disease. 2.4. Meningitis often occur in young adults; meningococcemia is fatal in 25% of cases; may cause disseminated intravascular coagulation (DIC), septic shock, hemorrhage of adrenal glands (Waterhause- Freiderichsen syndrome) which is fatal. 2.5. Gram-staining of CSF, aspirate are important presumptiveidentification 2.6. Culture: Chocolate agar (Blood Agar Base + Sheep Blood and boiled to release hemoglobin from red cells, becomes brown in color). 2.7. Serological test: coagglutination test 2.8. Neufeld-Quellung reaction or capsular swelling test 2.9. Fermentation test 2.10. Molecular test 3. Acinetobacter 3.1. Most important species: A. baumanni 3.2. Opportunistic infection, accounts for 1-3% of opportunistic infections 3.3. UTI, pneumonia, endocarditis, septicemia 3.4. Easily identified using Vitek 2 automated system 42 | P a g e 43 | P a g e NOTES: 1. Grouping of bacteria is not an easy task because of their different characteristics. However, a good Gram- stained smear can help in the tentative assignment of the bacteria. This will guide the MT/MLS in the next steps to be undertaken. 2. Gram-positive cocci appearing singly, pairs, chains and small clusters will have to consider Staphylococcus group and Streptococcus group. Culture on BAP will help and the source of specimen will make you decide whether to incubate without Candle jar technique or with Candle jar (throat specimen). Catalase test is done next on the colonies but have to be subcultures on TSA since rbcs on BAP can cause false (+) test. 3. Gram-negative cocci will go to Neisseria group. Specimens from urethra, cervicovaginal, anal, throat, and eyes have to consider gonococci so, Thayer Martin Agar in Candle jar has to be done. CSF or nasopharyngeal swab has to be cultured on CAP with candle jar to isolate meningococci. 4. Bacillus are large bacilli with spores located almost at the center of the cells. B. anthracis is of clinical importance seen in sputum, stool or skin swab. Growth on BAP by contaminating species is common. Clostridium have spores but located towards the end or at the end of the cells. Swab or aspirate from wound (Clostridium tetani or C. perfringens –gangrene) have to be considered. C. botulinum is considered in food samples or stool. Anaerobic cultivation is required which is not done commonly in laboratories because of the required CO2 incubator and other facilities. 5. Corynebacterium or diphtheroids has to be considered when swabs from oral lesions are taken (diphtheria patients, usually children). Diphtheroids can be seen from skin lesions. Actinomyces are seen in aspirates from the neck and other areas and sputum. 6. The Gram negative bacilli is the largest grpup. Enterics belong to Family Enterobacteriaceae (Escherichia, Shigella, Edwardsiella, Salmonella, Citrobacter, Klebsiella, Enterobacter, Hafnia, Serratia, Proteus, Providencia, Morganella, Yersinia, Erwinia); Pseudomonads (Pseudomonas, Burkholderia, Stenotrophomonas, Xanthomonas); Vibrio, (Campylobacter, Helicobacter, etc). Although stool is the most common specimen, other clinical samples can show enterics and Vibrio, enterics and pseudomonads can be isolated also in other clinical specimens. Challenge in the cultural, biochemical and other tests predominate. 7. The Gram-negative coccobacilli under the Pasteurella Group (Pasteurella, Yersinia and Francesilla), Haemophilus, Bordetella, Brucella, are also isolated from various specimens. 8. Gram-negatives of other forms such as Fusobacterium and other anaerobic Gram-negative bacilli can be isolated also if there are facilities for anaerobic cultivation, needless to say they are included. 9. The spirochetes that are difficult to Gram-stain are not included in the schematic diagram buy they are clinically important such as Spirillum, Treponema, Borrelia, and Leptospira. 10. The genus Mycobacterium, although considered Gram-positive because of similar cell wall to Gram-positive bacteria, are classified differently as Acid Fast Bacilli because of their mycolic acid containing cell wall. 11. Other microorganisms such as Chlamydia, Mycoplasma, Rickettsia, and others like Wall Deficient Microbial Forms (WDMF) are also included in Bacteriology. 44 | P a g e  BACILLUS SPECIES 1. Taxonomy This genus is one of the largest and most ubiquitous, and has gained notoriety with taxonomists for its extreme phenotypic diversity and heterogeneity. The genus Bacillus currently comprises 268 species and 7 subspecies although a few of these have been assigned to other genera, commonly found in the environment and as laboratory contaminants but a few of the species have been known to cause infections in humans. 2. Importance Two Bacillus species are considered medically significant: B. anthracis, which causes anthrax, and B. cereus, which causes a foodborne illness similar to that of Staphylococcus. B. subtilis is a very common laboratory contaminant. B. thuringeinsis var. israelensis is the source of Bti toxin used against plant pests. The Bti gene has been integrated into the genomes of corn, eggplant, tomatoes and other vegetables t6o protect them from various pests (GMO plants). B. stearothermophilus spores are used to test the efficiency of an autoclave condition (121 C, 15 pounds pressure/square inch, 15 minutes autoclaving). 3. Characteristics Bacillus species are Gram positive rods often arranged in pairs or chains with rounded or square ends and usually have a single endospore. The endospores are generally oval or sometimes round or cylindrical and are very resistant to adverse conditions. Sporulation is not repressed by exposure to air. Traditionally, Bacillus species was broadly divided in to three groups based on the morphology of the spore. 3.1.Group 1 – Gram positive, produce central or terminal, ellipsoidal or cylindrical spores that do not distend the sporangium. It comprises of two subgroups: - Large cell subgroup include Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus thuringiensis and Bacillus megaterium - Small cell subgroup include Bacillus pumilus, Bacillus subtilis and Bacillus licheniformis 3.2. Group 2 – Gram variable with central or ellipsoidal spores and swollen sporangia: Bacillus circulans and Bacillus coagulans. Bacillus alvei, Bacillus brevis and Bacillus macerans belonged to this group but have since been reclassified to other genera. 3.3. Group 3 – Gram variable, sporangia swollen with terminal or subterminal spores: Bacillus sphaericus In recent years, there has been a taxonomic development in two selected groups of the genus Bacillus. They are called the B. subtilis group and the B. cereus group. Many Bacillus species are haemolytic, a useful characteristic in differentiating them from B. anthracis (which is non-haemolytic). They are aerobic or facultatively anaerobic and most species are motile (a notable exception is Bacillus anthracis) by peritrichous flagella. Most species are oxidase positive, which may lead to confusion with Pseudomonas species, especially if the Bacillus species are poorly stained. They are usually catalase positive and metabolise carbohydrates by fermentation. ANTHRAX B. anthracis is almost invariably sensitive to penicillin whereas other species are generally resistant. If B. anthracis is suspected, specimens should be referred directly to the appropriate Reference Laboratory without doing any further work/manipulations. All form oval spores located centrally in an unswollen sporangium. B. anthracis spores in particular are highly resilient, surviving extremes of temperature, low- nutrient environments, and harsh chemical treatment over decades or centuries. Unlike the other members of the B. cereus group, B. anthracis is non-motile and nonhaemolytic on horse (or sheep’s) blood agar, 45 | P a g e  grows at 37°C, and forms typical grey/white flat colonies with “bee’s eye appearance” (that is, oval, slightly granular but not dry, about 2- 5mm in diameter) with irregular edges, which are characteristically tacky on teasing with a loop. The edges of B. anthracis are often described as ‘medusa head’, but this is a character that can be found throughout the B. cereus group. Spores do not form in host tissues unless the infected body fluids are exposed to air. When nutrients are exhausted, resistant spores form that can survive in soil for Identification of Bacillus species These spores then germinate when exposed to a nutrient rich environment, such as the tissues or blood of an animal or human host10. Virulent strains of B. anthracis produce a characteristic polypeptide capsule, which can be demonstrated by culture on a medium containing 0.7% bicarbonate which is incubated overnight in an atmosphere with a raised CO2 concentration. Alternatively a small volume of sterile defibrinated horse blood may be inoculated and incubated for 6 – 18hr. Colonies of capsulate B. anthracis appear mucoid and the capsule can be seen by the use of McFadyean’s polychrome methylene blue. Anthrax is a zoonotic disease. It causes three types of anthrax in humans- cutaneous, pulmonary and gastro-intestinal. Avirulent strains may occur which do not produce a capsule or toxin and these may be misidentified as Bacillus cereus. FOOD POISONING Bacillus cereus is 1 x 3-4µm in size. They present as straight or slightly curved slender bacilli with square ends singly or in short chains. They are facultative anaerobes, and like other members of the genus Bacillus can produce protective endospores. Capsules are not formed, but spore and sporangial morphology are similar to those of B. anthracis. They are motile by means of peritrichous flagella and exhibit two types of motility including swimming and swarming, depending on the environment and are resistant to lysis by gamma-phage. On blood agar plate, they appear as weakly or strongly β-haemolytic large flat or slightly convex, irregular, dull grey colonies with a slight green tinge and are about 2-5mm in diameter. In some instances, smooth colonies develop either alone or in the midst of rough colonies13. They grow optimally at temperatures between 5°C and 50°C, and are capable of adapting to a wide range of environmental conditions. They are positive for metabolising carbohydrates, proteins and amino acids and can reduce nitrates to nitrites. In anaerobic respiration, B. cereus utilizes fermentation to generate energy. Classical features to distinguish the group 1 (which includes, Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus mycoides and Bacillus thuringiensis) from the other groups are their inability to produce acid from mannitol and their production of lecithinase. B. cereus is resistant to penicillin and gamma phage and this distinguishes it from B. anthracis. B. thuringiensis is very similar to B. cereus but can be differentiated by the presence of crystal formation5. Strains of B. weihenstephanensis may carry genes coding for endotoxins generally associated with Bacillus cereus. Some strains of B. cereus are harmful to humans and cause foodborne illness, while other strains can be beneficial as probiotics for animals. Bacillus subtilis group (They include B. subtilis subsp. subtilis, B. subtilis subsp. spizizenii, B. mojavensis, B. vallismortis, B. clausii, B. atrophaeus, B. amyloliquefaciens, B. licheniformis, B. sonorensis, B. firmus, B. lentus and B. sporothermodurans) On selective agar such as Polymixin egg yolk mannitol bromothymol blue agar (PEMBA) B. cereus (which is mannitol negative and hydrolyses lecithin) produces characteristic blue colonies with a zone of precipitation. Bacillus thuringiensis produces a similar reaction. B. cereus, unlike B. thuringiensis, does not produce cuboid or diamond shaped parasporal crystals in cultures on sporulation agar or nutrient agar. The crystals are demonstrated with phase contrast microscopy or staining with malachite green. Care must be taken to distinguish B. cereus from other organisms such as Staphylococcus aureus, Serratia 46 | P a g e  marcescens and Proteus vulgaris which also grow on PEMBA. These colonies can be differentiated from B. cereus by colonial morphology and color. They also produce an egg yolk clearing reaction in contrast to the precipitate produced by B. cereus. Identification is verified by Gram stain, lecithinase activity, motility, penicillin susceptibility and biochemistry. Clinically significant isolates including isolates from sterile sites and from stool specimens in cases of gastroenteritis should be referred to the Reference Laboratory for further confirmation. IDENTIFICATION 1.1 Microscopic Appearance: Large Gram positive rods, often in pairs or chains with rounded or square ends (which may have a single endospore). 1.1.1. McFadyean stain is used to stain the capsule of B. anthracis. 1.1.2. Giemsa stain Use to stain the capsule of B. anthracis. Capsules are only normally seen if B. anthracis is growing in blood serum or is present in very fresh tissue samples. Spore stain Use to stain the spores of Bacillus species. Spores will be light green and vegetative cell walls will pick up the counterstain safranin. The position of the spore in the cell differs with different species. Note: Older cultures should be used when performing spore stain because they are lacking in nutrients and in competitive living environment. 1.2 Primary Isolation Media Blood agar incubated in air/CO2 at 35°C-37°C for 24 – 48hr. Polymyxin, egg yolk, mannitol, bromothymol blue agar (PEMBA) – optional. 1.3 Colonial Appearance Colonial appearance varies with species and a brief description is given here. 1.3.Organism Haemolysis Characteristics of growth on horse blood agar or PEMBA after incubation at 35°C – 37°C for 18 – 24hr B. anthracis Non-haemolytic (may occasionally be weakly haemolytic) Blood agar - Colonies are flat and irregular, 2 – 5mm in diameter, grey/white in colour with a ground glass appearance. Colonies show a tenacity that allows them to be pulled up and stay upright on teasing with a loop. PEMBA - These can be misidentified as B. cereus on PEMBA (B. anthracis is a B. cereus with a plasmid). B. cereus group ( B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis) β- haemolytic Blood agar - Colonial appearance is similar to that of B. anthracis although B. cereus colonies both cream to white or grey and have a slight green tinge and B. mycoides are rhizoid or hairy looking adherent colonies which spread over the entire agar and cover the entire surface of the medium in 48 hours. PEMBA - Colonies are crenated, 5mm diameter, turquoise to peacock blue with a zone of egg yolk precipitation after 18-24hr incubation. Bacillus subtilis group β- haemolytic Blood agar - Colonies are large (2 - 7mm) with a frosted-glass appearance, but may become opaque. Colour varies. Variable colonial morphology - some species may produce mucoid or smooth or raised wrinkly colonies. PEMBA – Colonies are cream to light yellow with no zone of egg Identification of Bacillus species 1.4. Biochemical tests 1.4.1. Lecithinase production (TP 22 - Nagler Test) Inoculate an egg yolk agar plate and incubate at 35°C – 37° C for 18 – 24hr, then examine for a zone of egg yolk precipitation. B. anthracis, B. cereus, B. thuringiensis and B. mycoides are positive. Motility (TP 21 – 1.4.2. Motility Test. All Bacillus species are motile with the exception of B. anthracis and B. mycoides. 47 | P a g e  1.4.3. Penicillin susceptibility. All Bacillus species, with the exception of B. anthracis, are generally resistant to penicillin as determined by E-Test. 1.4.4. Crystal formation. This is used to differentiate B. cereus from B. thuringiensis. After growth on sporulation agar or on nutrient agar for at least 48hr, B. thuringiensis produces cuboid or diamond shaped parasporal crystals. 1.4.5. Matrix-Assisted Laser Desorption Ionisation - Time of Flight (MALDI-TOF) Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDITOF MS), which can be used to analyse the protein composition of a bacterial cell, has emerged as a new technology for species identification. This has been shown to be a rapid and powerful tool because of its reproducibility, speed and sensitivity of analysis. The advantage of MALDI-TOF as compared with other identification methods is that the results of the analysis are available within a few hours rather than several days. The speed and the simplicity of sample preparation and result acquisition associated with minimal consumable costs make this method well suited for routine and high-throughput use. 1.4.6. PFGE. A variety of rapid identification and sensitivity methods have been developed for isolates from clinical samples; these include molecular techniques such as Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST), and 16S rRNA gene sequencing. 1.4.7. (MLST) Multilocus sequence typing (MLST) is a tool that is widely used for phylogenetic typing of bacteria. MLST is based on PCR amplification and sequencing of internal fragments of a number (usually 6 or 7) of essential or housekeeping genes spread around the bacterial chromosome. MLST has been extensively used as the main typing method for analysing the genetic relationships within the whole B. cereus group population 1.4.8. Primary isolation plate Blood agar incubated in air/ CO2 at 35-37C for 24-48hr B. cereus group - flat, grey/green/cream haemolytic colonies, ground glass appearance, 2-5mm diameter. B. anthracis - grey/white non-haemolytic flat, colonies with irregular edges and resembles “medusa head”. If appropriate growth characteristics, colonial appearance and Gram stain of the culture, are demonstrated. Confirmation of Identification Following lecithinase activity, motility, penicillin susceptibility and crystal formation results. Ulcerating skin lesions with a black eschar Fulminating pneumonia (especially with widening of the mediastinum on X-ray) and in outbreaks of the same) Circumstances predisposing to infection with B. anthracis eg farming, horticulture, veterinary, dockyard, tannery, woollen textile or medical laboratory work Deliberate release Injecting drug users The medical microbiologist should also be informed of other Bacillus species (other than B. anthracis), presumed or confirmed in accordance with local protocol, when the request form bears relevant additional information for example: Penetrating injury, compound fracture or retained foreign body Infection of an indwelling medical devices, such as prosthetic valves, pacemaker, CSF shunt or peritoneal or vascular catheter Food poisoning Investigation of a possible outbreak Follow local protocols for reporting to the patient’s clinicians. 48 | P a g e CLOSTRIDIUM 1. The Genus Clostridium is composed of strictly anaerobic to aerotolerant spore-forming bacilli found in soil and in normal intestinal flora of humans and animals. Isolates are gram-positive bacilli, spore formers. Exotoxin(s) play an important role in disease pathogenesis. 2. Clostridium perfringens, Gas Gangrene and Related Clostridial Wound Infections Patients may present with a wound infection. Severity varies from invasion of live tissue with systemic toxemia to relatively benign superficial contamination of already necrotic tissue. 2.1. Clostridium perfringens causes mainly gas gangrene. The wound is contaminated by spores from the environment or the host's normal flora. The anaerobic tissue environment facilitates replication of the bacteria and secretion of toxins. Host defenses are essentially absent. There is little, if any, innate immunity. Clostridial wound infections are found worldwide. Clostridia are ubiquitous in the soil and in the normal microbial flora of humans and animals. 2.2. Gas gangrene is diagnosed by the recognition of a characteristic lesion coupled with tissue Gram stain and anaerobic bacterial culture. Wound infections are controlled by administration of antimicrobial agents (e.g., penicillin, chloramphenicol) coupled with tissue debridement (for more severe forms of clostridial wound infections). 2.3. A commonly used laboratory test for presumptive identification of C perfringens is the Nagler reaction which detects the presence of alpha-toxin (phospholipase-C), one of the most prominent toxins produced by C. perfringens. However, several other species of clostridia also have a positive Nagler reaction, and thus this test is not entirely specific for C. perfringens. 3.Clostridium tetani and tetanus 3.1.Tetanus is characterized by twitching of muscles around a wound, pain in neck and jaw muscles (trismus), and around the wound. Patients have no fever, but sweat profusely and exhibit muscle rigidity and spasms. 3.2.The organisms are bacilli with terminal spores (drumstick bacilli, lollipop bacilli, tackhead bacilli, tennis racket bacillus). C. tetani is the only species. There are no serotypes. 3.3.The infection is initiated as a result of contamination of a wound with C. tetani spores. The anaerobic tissue environment facilitates C. tetani replication and secretion of exotoxins. C. tetani actually produces two toxins: tetanolysin, a hemolysin that is inactivated by cholesterol and has no role in pathogenesis, and tetanospasmin, a spasmogenic toxin responsible for the classical symptoms of the disease. Tetanospasmin, fixes to inhibitory neurons and blocks the release of neurotransmitters, glycine and gamma-aminobutyric acid. Host defenses are essentially absent. There is little, if any, innate immunity and the disease does not produce immunity in the patient. Active immunity follows vaccination with tetanus toxoid. C. tetani is found worldwide. Ubiquitous in soil, it is occasionally found in intestinal flora of humans and animals. 3.3.Diagnosis is primarily by the clinical symptoms. The wound may not be obvious. Furthermore, C. tetani is recovered from only one-third of all implicated wounds. Gram-stained smear of a swab from the wound can be of help. 3.4. The administration of tetanus toxoid is a preventive measure. It can confer artificial passive acquired immunity. C. tetani infection is treated with antimicrobial agents (metronidazole or penicillin) and by local 49 | P a g e wound debridement. Mothers before delivery of their babies are immunized with anti-tetanus vaccine to provide protection to the mothers and their infants from having tetanus. 4. Clostridium botulinum and botulism 4.1.These infections may have early gastrointestinal symptoms. The cranial nerves are initially affected, followed by descending, symmetric paralysis of motor nerves, with critical involvement of the respiratory tree. Muscle paralysis may occur. 4.2. These organisms are bacilli with oval, subterminal spores. C. botulinum consists of several biochemically distinct groups of organisms that produce botulinum toxin. Seven types of neurotoxins are designated A, B, C, D, E, F, and G, some of which have been shown to be encoded on bacteriophage DNA. 4.3. There are three forms: 4.3.1. adult botulism, caused by ingestion of preformed toxin in food; (2) 4.3.2. infant botulism, in which the organism replicates and secretes toxin in the intestinal tract; 4.3.3.wound botulism, in which the organism replicates in the wound and secretes toxin. Toxin binds to neuromuscular junctions of parasympathetic nerves and interferes with acetylcholine release, causing flaccid muscle paralysis. 4.4. No host defenses are known. C. botulinum is distributed worldwide, and is ubiquitous in soil. Consumption of uncoocked can goods and meat produced such as sausages and Improper heating of canned foods is a major factor in botulism food poisoning. 4.5. Diagnosis is from the clinical symptoms, especially gastrointestinal and neurological symptoms, coupled with laboratory confirmation. A finding of normal spinal fluid helps to eliminate the possible diagnosis of numerous other central nervous system disorders. The best means of control is to eliminate the toxin source via proper food handling. Once the food poisoning is diagnosed, treatment measures should include an attempt to neutralize unbound toxin. Supportive care is of primary importance. 5. Clostridium difficile: Antibiotic-Associated Diarrhea, Pseudomembranous Colitis 5.1. Patients can present with a spectrum of disease that varies from uncomplicated antibiotic-associated diarrhea to antibiotic-associated pseudomembranous colitis that may be fatal. 5.1. This species consists of bacilli with large, oval, subterminal spores. C. difficile is the only species. There are no defined serotypes. Toxigenic and nontoxigenic strains exist. The former produce varying amounts of toxin A (enterotoxin) and toxin B (cytotoxin). Broad spectrum antibiotic therapy eliminates much competing normal flora, permitting intestinal overgrowth of toxigenic C. difficile. There are no defined host defenses. C difficile is a component of the normal intestinal flora of a small percentage of healthy adults and of a relatively large percentage of healthy neonates. It also may be found in the environment, especially in hospitals. The presence of antibiotic therapy, diarrhea, and pseudomembranes by colonoscopy help establish the severity of disease, coupled with the demonstration of organisms and/or toxin in feces. Metronidazole and vancomycin should be used therapeutically. However, relapses can occur. Supportive therapy may be needed. 50 | P a g e 6. Other Pathogenic Clostridia 6.1. Clostridium perfringens causes food poisoning and necrotizing enteritis. 6.2. C. sordellii causes bacteremia, endometritis and nonbacteremic infections. 6.3. C septicum is correlated with the presence of cancer. 6.4. C tertium is associated with bacteremia. CORYNEBACTERIUM There are currently 112 species and 11 subspecies in this genus. 1. Corynebacterium taxonomy 1.1. 55 species are occasional or extremely rare causes of infection in humans or are transmitted to humans by zoonotic contact, with the remaining species having been recovered solely from animals or birds, the environment, water, foodstuffs or synthetic materials. 1.2. The potentially toxigenic corynebacteria comprise C. diphtheriae, C. pseudotuberculosis and C. ulcerans. 1.3. C. diphtheriae consists of four biovars: gravis, mitis, intermedius and belfanti. 2.Characteristics 2.1.Corynebacterium species are Gram positive non-motile rods, often with clubbed ends, occurring singly or in pairs. Some cells may stain unevenly giving a beaded appearance. Their size is between 2-6µm in length and 0.5µm in diameter. They group together in a characteristic way, which has been described as the form of a "V", "palisades", or "Chinese letters". Metachromatic granules are usually present representing stored phosphate regions. 2.2.They are aerobic or facultatively anaerobic and exhibit a fermentative metabolism (carbohydrates to lactic acid) under certain conditions. 2.3.They are fastidious organisms, growing slowly even on enriched medium. Agar containing blood and potassium tellurite, such as Hoyle's tellurite medium, serves as a selective and differential medium. On blood agar, they form small greyish colonies with a granular appearance, mostly translucent, but with opaque centers, convex, with continuous borders. Their optimum growth temperature is 37°C. 2.4. C. diphtheriae grows as pinpoint grey/black colonies on Hoyle’s tellurite agar in 16-18hr and produces characteristic colonies after 48hr. Isolates of potentially toxigenic Corynebacterium species will also grow on blood agar. Colonial morphology varies among the species. C. ulcerans and C. pseudotuberculosis colonies may be slightly β-haemolytic on blood agar. C. diphtheriae, C. ulcerans and C. pseudotuberculosis are facultatively anaerobic, non-sporing, non-capsulated and non-acid-fast. These organisms are non-motile and catalase positive. C. ulcerans and C. pseudotuberculosis are both urease positive which may be used to distinguish them presumptively from C. diphtheriae. 2.5.Strains of these species can all harbor the phage borne diphtheria tox gene, which is required for the production of toxin. Toxigenic strains may cause diphtheria or diphtheria-like illness. Possible toxigenic strains of Corynebacterium species should be referred to the Reference Laboratory for detection of toxin production as soon as possible. C. diphtheriae are generally non-toxigenic. But once the bacterial cell is infected by a corynephage virus, the tox gene is injected by the phage into the bacterial cell. The tox gene is integrated into the bacterial genome and the host bacterium become lysogenized. As it multiplies, the tox gene is also duplicated. When expressed, the bacterium becomes toxigenic and causes diphtheria, manifested as grayish lesions (pseudomembrane) in the 51 | P a g e posterior area of the mouth near the tonsil. Cotton swab of these pseudomembrane can reveal the bacterial cells in Gram’s stained smear or using Loeffler’s methylene blue stain. Non toxigenic strains of corynebacteria eg C. ulcerans, C. jeikeium, C. striatum and non-toxigenic C. diphtheriae are also known to cause infections in humans including pulmonary infection, leukaemia and endocarditis. Both C. jeikeium and C. striatum are non-haemolytic, urease negative and catalase positive. 2.6. A cotton swab specimen is collected from lesions described as grayish pseudomembrane that appear near the tonsil in the posterior part of the oral cavity especially of children. This pseudomembrane is painful and causes difficulty of breathing. Isolates from primary culture are identified by: 2.6.1. Gram’s staining – Gram-positive non-spore forming bacilli appearing with Chinese characters, V, L, etc. 2.6.2. Can be stained with Loeffler’s methylene blue showing the metachromatic granules made of polyphosphates 2.6.3. On BAP, can show hemolysis; on Tellurite plate medium colonies can show gray or black color 2.6.3.Four preliminary tests (this includes nitrate, urease, catalase and pyrazinamidase tests) which permit the presumptive identification of the potentially toxigenic Corynebacterium species within 4hr. 2.6.4. Additional identification may be made using a commercial identification kit in conjunction with toxin testing. It is advisable that suspected toxigenic culture be tested serologically using The Elek test. 2.7. C. pseudotuberculosis 2.7.1. Can give a variable nitrate test result. This is because it consists of two biovars: biovar equi (from horses or cattle) that reduces nitrate and the biovar ovis (from sheep or goats) that fails to do so.. 2.7.2. Agar Media The classic colonial morphology apparently develops better on media containing sheep blood rather than horse in some Corynebacterium species. For example, the degree of haemolysis in Arcanobacterium haemolyticum, formerly known as Corynebacterium haemolyticum is far greater on sheep blood agar plate than most other corynebacterial.. 2.8. Safety Considerations C. diphtheriae, C. ulcerans and C. pseudotubercolosis are Hazard Group 2 organisms, and in some cases the nature of the work may dictate full Containment Level 3 conditions. All laboratories should handle specimens as if potentially high risk. All suspected isolates of potentially toxigenic corynebacteria should always be handled in a microbiological safety cabinet. For the urease test, a urea slope is considered safer than a liquid medium. C. diphtheriae and C. ulcerans cause severe and sometimes fatal diseases. Laboratory acquired infections have been reported. Diphtheria antitoxin for the treatment of clinical cases is available. Laboratory procedures that give rise to infectious aerosols must be conducted in a microbiological safety cabinet. MYCOBACTERIUM SPECIES 1. Tuberculosis (TB) is caused by a bacterium called Mycobacterium tuberculosis. The bacteria usually attack the lungs, but can attack any part of the body such as the kidney, spine, and brain. Not everyone infected with TB bacteria becomes sick. As a result, two TB-related conditions exist: (a) latent TB infection (LTBI) and (b) TB disease. If not treated properly, TB disease can be fatal. 2. Spread. TB bacteria are spread through the air from one person to another. 52 | P a g e  2.1. The TB bacteria are put into the air when a person with TB disease of the lungs or throat coughs, speaks, or sings. People nearby may breathe in these bacteria and become infected. When a person breathes in TB bacteria, the bacteria can settle in the lungs and begin to grow. From there, they can move through the blood to other parts of the body, such as the kidney, spine, and brain. 2.2. TB is NOT spread by shaking someone’s hand, sharing food or drink, touching bed linens or toilet seats, sharing toothbrushes, or even kissing. 2.3. TB disease in the lungs or throat can be infectious. This means that the bacteria can be spread to other people. TB in other parts of the body, such as the kidney or spine, is usually not infectious. 2.4. People with TB disease are most likely to spread it to people they spend time with every day. This includes family members, friends, and co-workers or schoolmates. 3. Testing. There are different kinds of tests that are used to detect TB bacteria in the body: 3.1. The TB skin test (TST) Mantaux test= a delayed reaction that tells the person had a history of tuberculosis 3.2. An Acid Fast Staining of sputum = presence of acid fast bacilli indicate present infection and possibility, transmission. 3.2.1. Ziehl-Neelsen staining method 3.2.2. Kinyoun staining method Sputum smear microscopy allows a rapid and reliable identification of patients with pulmonary tuberculosis (PTB) where there are more than 5000 bacilli/ml of sputum. If the sputum has less than 5000 bacilli/ml, smear microscopy is highly unlikely to diagnose PTB, thus has an overall low sensitivity for PTB. Another shortcoming of smear microscopy is its non-specificity, such that M. tuberculosis appears the same as non tuberculous mycobacteria (NTM). However, in areas of high TB prevalence, positive smears have a very high probability of being M. tuberculosis. It is recommended that all patients suspected of PTB should submit at least two sputum specimens. Studies have shown that, when collection and examination techniques are correctly conducted, about 80% of sputum smear-positive patients are found during the first sputum examination and over 15% more during the second. Successive, repeated examinations yield fewer positives. Usually, a first sample is collected at the time of the consultation when the patient is identified as a suspected TB case. A second sample is collected in the early morning the day after the initial consultation (and the patient brings the sample to the health facility if it is collected at home). In order to limit the number of visits to the health facility, “frontloaded microscopy” (also referred to as 'same day' or 'spot-spot' microscopy) can be performed. Two sputum specimens are collected one hour apart. This strategy has shown similar results to the standard strategy over two days (spot-morning-spot) in terms of diagnostic yield. The staining methods uses a technique where the mycobacteria retain a primary stain after exposure to decolourising acid-alcohol, hence the term “acid-fast bacilli” (AFB). The two most common methods of staining, which determine the acid-fast nature of the mycobacteria, are (1) Ziehl-Neelsen staining and (2) Auramine staining. Auramine staining has the advantage of permitting a more rapid slide reading. It is recommended in laboratories with a high workload defined as ≥ 20 slides per reader per day. It requires trained, experienced technicians and a fluorescent microscope. LED (light emitting-diodes) modules that can be adapted to ordinary 53 | P a g e microscopes or new LED microscopes are simpler, cheaper and safer alternatives to traditional mercury vapor lamp microscopes and do not require dark room. Concentration techniques increase the sensitivity of sputum smear microscopy and fluorescence and have also been shown to increase the detection up to 20% in some settings with high HIV prevalence. 3.2. An x-ray may show a lesion but is not confirmative for M. tuberculosis. 4. Characteristics of M. tuberculosis Mycobacteria are small rod-shaped bacilli that can cause a variety of diseases in humans. They can be thought of in three main groups: 4.1. Mycobacterium tuberculosis complex: this group includes M. tuberculosis, M. bovis, M. africanum, M. microti, and M. canetti. They all can cause “tuberculosis” in humans. All mycobacteria are classical acid-fast organisms and are named so because of the stains used in evaluation of tissue or sputum specimens (i.e. Ziehl- Neelsen stain). M. tuberculosis multiplies more slowly than the majority of bacteria; this is why tuberculosis has a slower evolution (causes disease weeks or even months to years after infection) than most other bacterial infections. M. tuberculosis is a strictly aerobic bacterium. It therefore multiplies better in pulmonary tissue (in particular at the apex, where oxygen concentration is higher) than in the deeper organs. The vast majority of tuberculosis is caused by M. tuberculosis, with the other organisms being relatively rare. Their treatment is similar (with M. bovis being innately resistant to pyrazinamide and M. africanum being innately resistant to thioacetazone). 4.2. Mycobacterium leprae causes leprosy(Hansen’s bacillus) detected by AFB staining of Skin snip. A little tissue is taken from the lesion, and stained on smear to see the AFB present.