ISOLATION OF FUNGI FROM CLINICAL SAMPLES Fungi are significant, sometimes overlooked, human pathogens. Infection caused by the fungus ranges from mild to life threatening. Diagnosis is based on a combination of clinical and laboratory investigations. Laboratory procedure includes, Demonstration of fungi by microscopy, Identification by culture, Detection of specific humoral response and Detection of fungal antigens and metabolites in body fluids. Successful of laboratory fungal diagnosis depends on specimen selection, Specimen collection, Specimen transport, Processing, Microscopic examination, Culturing. Specimen Collect ion Specimen collection for the detection of an etiologic agent of mycoses is very similar to specimen collection for bacteria. But fungal detection requires large quantity of specimen than bacterial identification. Fingernails and toenails suspected of Dermatophytosis should be cleaned extensively with 70% ethanol. Swabs are not optimal specimen collection for fungal identification. Depends on the site of infection, sample collection method and specimen type may vary. For dermatophytic infection : Skin scrapings, Pus, Nail clippings, Hair plug not cut. Subcutaneuous infection: Pus, biopsy tissues, Aspirated fluid, Skin scrapings Systemic mycoses : Sputum, Bronchial washings, Exudates from cutaneous lesion, CSF, Urine, Tissue biopsy, Vaginal swab, Blood. Specimen Processing Liquid specimens may have low concentration of fungus and will require centrifugation to increase fungal cell concentration. Hard specimens should be mined before inoculation. Concent rat ion Large volume of fluid should be concentrated by centrifugation (2000 rpm for 10 min). Use the resulting pellet for culture and KOH examination. Body fluids, Urine, CSF, Sputum are used after digestion with N-acetyl L-cysteine. Mincing or homogenizat ion Biopsy samples, tissues and nails must be processed to increase the recovery. With sterile scalpels, mince specimens into small pieces in a petriplate with a few drops of sterile distilled water. If Histoplasma is suspected, specimen is homogenize by using tissue grinder after mincing. Media select ion Media selection depends on body site, nature of infection and nature of contamination. To prevent bacterial pathogens antibiotics should be used or reduce the pH of the medium Brain heart infusion agar with antibiotics, Potato Dextrose Agar, Mold agar, Sabouraud Glucose Agar, Niger seed agar, Yeast extract phosphate medium. , Buffered charcoal yeast extract agar, Rose Bengal chloramphenical agar. From the above list of mycotic media, any one or two media selected for cultivation. Inoculat ion Concentrated aspirated body fluids are directly inoculated on to the medium by making use of inoculation loop. Swabs are vortexed in distilled water before inoculation. Vortexed specimens are inoculated on medium after concentration. After inoculation hair and skin scrapings are pressed firmly on the surface of the medium. Nail should be pulverized using scalpels and press fragments firmly onto medium surface. Incubat ion All fungal media were incubated at 28 to 30°C for 4 weeks, because most fungi will produce colonies by the end of the 3rd Week.. Vaginal cultures may be incubated for only 7 days. Observe the media once in two days. Histoplasma capsulatum and Blastomyces dermatidis may require 4-8 weeks. Aerobic non clinical fungus may produce fruiting bodies within 72 hours. Examinat ion of fungal growth on primary media Read primary plates daily for the first week, every other day for the second week and twice weekly for the remaining two weeks. When growth appears, differentiate yeast from mold by microscopic examination. Once colonies are formed on the media, the organism should be viewed microscopically. Zygomycetes are observed under dissecting microscope. Yeast is observed by Wet preparation, Lacto phenol cotton blue and Indian ink preparation. Mold is observed by Wet preparation, Scotch tape , Tease preparation , Slide cultures, Lactophenol cotton blue staining , Ascospore. KOH Mount KOH may be used to examine hair, nails, skin scrapings, fluids, exudates or biopsies. The fungal structures such as hyphae, large yeast (Blastomyces), spherules, and sporangia may be distinguished. Examine slides with reduced light (narrow the iris diaphragm) and examine negative smears on several consecutive days. The fungal structures may be enhanced by using a phase-contrast microscope. Specimens placed in a drop of 15% KOH will dissolve at a greater rate than fungi because fungi have chitinous cell walls. The clearing effect throughout the clinical specimen can be accelerated by gently heating the KOH preparation. Visualization of fungi can be further enhanced by the addition of Parker Superquink permanent black ink to the preparation. Mycelium and spore were observed within tissue. KOH clears hard tissue and facilitates easy observation. KOH mount provides first hand information about fungal infection in tissues. KOH-DMSO-INK mount / stain Specimens with tissue placed in a drop of 15% KOH will dissolve at a greater rate than fungi because fungi have chitinous cell walls. The clearing effect throughout the clinical specimen can be accelerated by gently heating the KOH preparation. Addition of Ink to KOH enhances contrast. This method is most useful for detecting Malassezia furfur in skin scrapings. DMSO present in staining reagent eliminates the need of heat during staining Mycelial elements clearly demonstrated, which indicates the availability of fungal infection in particular tissue. Spore along with conidium describes mould species. KOH-Calcoflour fluorescent -stain Calcoflour white stain may be used for direct examination of most specimens using fluorescent microscopy. The cell walls of the fungi bind the stain and fluoresce blue-white or apple-green depending on the filter combination used. The use of calcofluor white (CFW), a fluorescent brightener used in the textile industry, with the addition of potassium hydroxide (KOH) will enhance the visualization of fungal elements in specimens for microscopic examination. The CFW nonspecifically binds to the chitin and cellulose in the fungal cell wall and fluoresces a bright green to blue. A substantial amount of non-specific fluorescence from human cellular materials and natural and synthetic fibers should be expected. The CFW highlights suspicious structures but the interpretation of the structures relies on traditional fungal morphologic features. India ink Preparations An India ink preparation can be used for the rapid detection of the encapsulated yeasts based on negative staining. The capsule repels the carbon particles of the Indian ink, giving a clear well-demonstrated halo around each encapsulated stain. for Histoplasma capsulatum Giemsa stain is used for examining intracellular structures and is applied to primary specimens of bone marrow tissue and WBC’s in which H.capsulatum is suspected. Necrotic cells in the specimen will have pink cytoplasm. Normal cell- light blue - violet lavender cytoplasm. Phagocytised yeast cells will stain light to dark blue, and each will have a clear halo around it. Look for purple pseudoencapsulated yeast forms of H.capsulatum inside PMN cells and monocytes.

IDENTIFICATION OF YEAST Yeasts are a heterogenous group of fungi that superficially appear to be homogeneous. Yeasts grow in a conspicuous unicellular form that reproduces by fission, budding, or a combination of both. True yeasts reproduce sexually, developing ascospores or basidiospores under favorable conditions. Yeast-like fungi (imperfect yeasts) reproduce only by asexual means. The identification of these fungi is based upon a combination of morphological and biochemical criteria. Morphology is primarily used to establish the genera, whereas biochemical assimilations are used to differentiate the various species. Principal Criteria and Test s for Identifying Yeast s 1. Culture characteristics - Colony colour, shape, texture 2. Asexual structures: a. Shape and size of cells; b. Bipolar, fission, multipolar or unipolar “budding”; c. Absence or presence of arthroconidia, ballistoconidia, blastoconidia, clamp connections, endoconidia, germ tubes, hyphae, pseudohyphae, or sporangia and sporgangiospores. 3. Sexual structures - Arrangement, cell wall ornamentation, number, shape and size of ascospores or basidiospores 4. Physiological studies: a. Assimilation; c. Fermentation d. Nitrogen utilization e. Urea hydrolysis Cultural characters Clinical sample is streaked on Sabourad dextrose agar or Rose Bengal chloramphenicol agar plates. Plates were incubated at 25-30°C for 48 hours and observed for colony morphology, colour, shape and texture. Record the result and interpretate the results. CHROM agar test CHROM agar contains enzymatic substrates that are linked to chromogenic compounds. When specific enzymes cleave the substrates, the chromogenic substrates produce colour. The action of different enzymes produced by yeast species results in colour variations useful for the presumptive identification of yeasts. The test provides only presumptive identification of yeast eg: C.kruses, C.tropicalis, C.albicans. Cultures are streaked on the medium and incubate at 35°C in humidified dark chamber for 48-72 hours. After 72 hours observe colour change and interpretate the results. C.albicans: A medium sized, green, smooth matte colony with a very slight green halo ion the surrounding medium. C.tropicalois : Smooth medium sized matte colony, which is blue to blue gray with a pale pink edge. The colony may have a dark brown to purple halo, which diffuses into the agar. C.krusei : A large, spreading, rough pink colony with a pale pink to white edge. Germ tube test Candida are the members of the normal flora of skin. More than 100 species are available in candida. Among these Candida albicans cause most of the human infections. The germ tube test provides a simple, reliable and economical procedure for the presumptive identification of Candida albicans. About 95% of the clinical isolates produce germ tubes when incubated in serum at 35°C for 2.5-3 hours. A germ tube represent the initiation of a hypha directly from the yeast cell. They have parallel walls at their point of origin. Germ tube formation is influenced by the medium, inoculum size and temperature of incubation. Fresh normal pooled human sera or a commercially available germ tube solutionare to be used as the medium for the test. The inoculum should result in a very faintly turbid serum suspension. Over-inoculation will inhibit the development of germ tubes. Incubate in at 35°C-37°C for 2.5-3 hours. India ink preparat ions India ink can be added to specimens such as spinal fluids or exudates to provide a dark background that will highlight hyaline yeast cells and capsular material (halo effect). Hence, it should be used to examine specimens suspected of containing Cryptococcus neoformans. White blood cells may be distinguished from Cryptococcus neoformans because of the irregular edge of the halo and the pale cell wash Giemsa staining Giemsa stain is used for examining intracellular structures and is applied to primary specimens of22 bone marrow tissue and WBC’s in which H.capsulatum is suspected. Urease test The rapid urea hydrolysis test is used to screen isolates for Cryptococcus neoformans. Under these conditions, C. neoformans will rapidly hydrolyze urea, which results in a pink to red colour. Nit rate test Yeasts have the ability to use ammonium sulfate, asparagine, peptone, and urea aerobically as sole sources of nitrogen if adequate vitamins are provided. In contrast, aliphatic amines, potassium nitrate, sodium nitrate, and some amino acids are utilized selectively by different yeasts. Ascospore Induct ion and Detect ion One step in indentifying a yeast involves determining whether or not the isolate has the ability to form ascospores. Some yeasts will readily form ascospores on primary isolation medium, whereas others require special media. The ability to form ascospores varies from isolate to isolate and may be lost in old laboratory strains. If only one mating type of a heterothallic yeast is present, no ascospores will be formed. Ascospore media contain small amounts of carbohydrates; this restricts vegetative growth while enhancing ascospore formation. Modified kinyons Acid fast staining Modified acid-fast stains are recommended for demonstrating ascosspores. Unlike the Ziehl- Neelsen modified acid-fast stain, the modified Kinyoun acid-fast stain does not require heating the reagents used for staining Carbohydrate fermentat ion It is performed as like bacterial carbohydrate fermentation test. Carbohydrate assimilat ion This is performed using the medium phenol red agar base. On the medium spread 0.1mL of yeast culture and place carbohydrate disc. Incubate plates at 37°C for 24 to 48 hours and observed for colour change around disc. Table 66.1: Identification and differentiation features of Different Yeasts Organism Pseudo True Urease Blasto Ascospore Nitrate Germ hyphae hyphae conidia s tube Candida albicans Present Present Negative Present Absent Positive Positive Trichosporan Present Present Positive Present Absent Negative Negative Saccharomyces Present Absent Negative Present Present Negative Negative Crytococcus neoformans Present Absent Positive Present Absent Negative Negative Torulopsis Absent Absent Negative Present Absent Negative Negative Rhodotorulla Absent Absent Positive Present Absent Negative Negative Prototheca Absent Absent Negative Present Absent Negative Negative Geotrichum Absent Present Negative Absent Present Negative Negative Other Candida Absent Absent Positive Absent Present Negative Negative Hanseula Absent Absent Negative Absent Present Negative Negative Table 66.2: Differentiation features of Different Yeasts based on carbohydrate assimilation and fermentation

Assimilation Yeast/ Sugar Glucose Maltose Sucrose Lactose Galactose Melibiose Inositol Xylose Trehalose Candida albicans Positive Positive Positive Negative Positive Negative Negative Positive Positive Candida lipolytica Positive Negative Negative Negative Negative Negative Negative Negative Negative Candida krusei Positive Negative Negative Negative Negative Negative Negative Negative Negative Cryptococcus neoformans Positive Negative Negative Negative Negative Negative Negative Negative Negative Fermentation Glucose Maltose Sucrose Lactose Galactose Melibiose Inositol Xylose Trehalose Candida albicans Fermentative Fermentative Negative Negative Fermentative Negative Negative Negative Fermentative Candida lipolytica Negative Negative Negative Negative Negative Negative Negative Negative Negative Candida krusei Fermentative Negative Negative Negative Negative Negative Negative Negative Negative Cryptococcus neoformans Fermentative Fermentative Fermentative Negative Fermentative Negative Negative Negative Negative Figure 64.a – Microscopic morphology of fungus Figure 64b – Microscopic morphology of fungus

Figure 64c – Microscopic morphology of fungus Figure 64d – Microscopic and culture morphology of fungus

Figure 64e – Microscopic morphology of fungus

Figure 64d – Microscopic morphology of yeasts

Fig 64d : Microscopic morphology of yeasts 1. Yeast 2. Exophiala wernecki 3. Geotrichum 4. Malassia furfur 5. Phaeacecomyces 6. Rhodotorulla 7. Sporobolomyces 8. Sporothrix 9. Torulopsis 10. Hansiella

IDENTIFICATION OF INTESTINAL PARASITES Introduction Protozoan and helminthes are two major groups of organisms. They are responsible for varieties of human intestinal infection. Majority of intestinal parasites are detected using stool as specimen. All parasites have distinctive morphology and is studied by microscopic examination of faecal materials. Faecal specimens shows cyst and trophozoites of protozoans, adult worm, segments, larva and eggs of helminthes. The following parasites may be detected by making use of microscopic techniques. They are Entamoeba histolytica, Giardia lamblia, Trichomonas hominis, Balantidium coli, Isospora bellei, Taenia solium, Fasciola hepatica, Schistosoma sp, Trichostrongylus,Capilaria, Ascaris lumbricoides, Paragonimus, Enterobius vermicularis, Faciola buski, T. trichura Fresh faecal specimen is important for the detection of parasitic infections. Faecal specimen should be examined within a day (formed stool -1 day, semiformed stool-1 hours, liquid stool-30 min). Faecal specimen should not incubated for parasitic examinations. Colour, consistency, presence of blood, mucus and segments are to be noted during faecal specimen examinations. Following methods are adopted to diagnose intestinal parasites. They are microscopic methods for morphological identification, concentration methods and culture methods. Microscopic methods include saline and iodine wet mount. Concentration methods are sedimentation method (formal ether, formal detergents) and floatation techniques

(Sodium chloride, ZnSO4 and sugar floatation method)

Microscopic methods - Saline wet mount, Iodine wet mount Concentration technique – Sedimentation, Floatation

Microscopic methods Saline wet mount Aim To examine the faecal specimens for parasitic ova, cyst, trophozoites etc. To identify intestinal cyst and trophozites of protozoan, eggs and larva of helminthes based on the morphological characters.

Background Information Stool microscopy is a rapid method employed for the detection of intestinal parasites. Saline wet mount is more advantageous than other wet mount procedure adopted to examine parasites. Saline wet mount maintain viability / motility status of the parasite. It also facilitates demonstration of chromotoidal bodies in the cyst.

Materials Required Compound microscope, microscopic slide & cover glasses, normal saline (it is prepared by adding 85 gms of NaCl in 100mL of distilled water), applicator sticks, Pasteur pipettes, stool Specimen etc..

Procedure Take clean microscopic slide. Add a drop of Normal saline on the glass slide with the help of Pasteur pipette. Take small portion of stool sample in applicator stick and emulsify it in the drop of saline. Place the cover glass over the saline preparation. Examine the preparation under 10x and 40x objective of the compound microscope. Record the findings with the description of morphological characteristics. Observation and Results Various morphological features of parasites are observed and recorded and identified(Fig 67a).

Iodine wet mount Aim To examine intestinal parasites To identify cyst, trophozoites, eggs, larva of intestinal parasites on the basis of morphology and internal structures.

Background Information Iodine wet mount is performed by making use of Dobell’s, O’Connor’s, lugols and Antoine’s Iodine solutions. It is mainly used for the detection of protozoan parasites. This method clearly demonstrates the presence of nuclei as brown dots. It also demonstrates the yellowish cytoplasm and brown glycogen mass present in the cyst. Disadvantages of this technique includes trophozoites are killed by iodine; chromatoidal bars in protozoan cysts are not clearly demonstrable.

Materials required Compound microscope, microscopic slide, cover glasses, applicator stick, pasteur pipette, iodine Solution (it is prepared by adding 1g potassium iodide and 15g of iodine crystals in 100mL of distilled water), Stool sample.

Procedure Box 67.1- Nature of helminthic eggs Place a drop of Iodine solution on a clean glass slide. lumbricoides, Trichuris trichura, taenia sp., Echinococcus sp. Emulsify the stool specimen with the help of applicator Schistosoma., Fasciola, Paragonimus stick. are bile stained eggs. Hook worm, Enterobious Use the cover glass to cover the emulsified stool sample vermicularis, Hymenolepsis nana are and blot excess fluid. non bile stained eggs Examine the sample under low power and high power objectives and record the findings.

Result Cyst, trophozoits, eggs of different Parasites (Fig 67a and b) are observed and identified.

Concentration methods Concentration of Stool Parasites Aim To demonstrate parasitic burden of an individual Background information Faecal concentration has become a routine procedure as a part of the complete ova and parasite examination and allows the detection of small numbers of organism that may be missed in direct wet smear.

Sedimentation method Principle Ethylacetate is used as an extractor of debris and fat from the faeces and leaves the parasites at the bottom of the suspension. The formalin-ethyl acetate sedimentation method is recommended, because it is the easiest to perform, allows recovery of the broadest range of organisms and is least subject to technical error.

Procedure Transfer ½ teaspoan of fresh stool into 10mL of 10% formalin in a vial or round bottom tube. Mix the stool and formalin throughly. Let the mixture stand a minimum of 30 minutes for fixation. Depending on the amount and viscosity of the specimen, stain a sufficient quantity through wet gauze into a conical 15mL centrifuge tube to give the desirable amount of sediment. Add 0.85%Nacl almost to the top of the tube and centrifuge for 10minutes at 2000rpm. The amount of sediment obtained should be ½ to 1mL. Decant the supernatant fluid and suspend sediment in saline. Add saline almost top of the tube and centrifuge again for 10minutes at 2000rpm. Decant the supernatant fluid and suspend the sediment on the bottom of the tube in 10% formalin. Fill the tube half full only. If the amount of sediment left in the bottom of the tube is very small or the original specimen contain lot of mucous, do not add ethyl acetate. Add 4-5mL ethyl acetate. Stopper the tube shake vigorously for at least 30 seconds. Centrifuge for 10minutes at 2000rpm. Four layers should result; A small amount of sediment in the bottom of the tube. A layer of formalin. A plug of Faecal debris on the top of formalin. A layer of ehtylacetate at the top. Free the debris by ringing the plug with an applicator stick; decant all of the supernatant fluid. After proper decanting, a drop of or two of fluid remaining on the side of the tube may run down into the sediment. Mix this fluid with sediment. If the sediment is still solid, add a drop or two of saline to the sediment mix, add a small amount of material to slides, add a cover slip and examine. Systematically scan using the 10x objective. The entire cover slip area should be examined.

Result Protozoan trophozoite and/or cyst helminthes eggs and larvae maybe seen and identified. Protozoan trophozoites are less likely to be seen (Fig 67a, b and c).

Floatation method Aim To concentrate parasites presents in the Faecal matter on the basis of specific gravity.

Principle The floatation procedure permits the separation of protozoan cyst and helmintic eggs from excess debris through the use of liquid with the help of specific gravity. The parasitic elements are recovered in the surface film and the debris remains in the bottom of the tube. This technique yields a preparation than the sedimentation procedure; however, some helminthes eggs do not concentrate well with the flotation method. The specific gravity of the zinc sulphate can be increased, although this usually causes more distortation in the organism organisms present and is not recommended for routine clinical use. To ensure the detection of all possible organisms, both the surface film and sediment must be examined. For most laboratories, this is not a practical approach. Specific gravity of hookworm egg-1.055 Specific gravity of zinc sulphate is over 1.2 The zinc sulfate flotation technique is a clinical method to determine the presence or absence of parasitic organisms and also the approximate parasite burden. The zinc sulfate flotation technique breaks up faeces and using differential specific gravities, isolates the protozoan cysts and helminth ova and larvae. MgSo4 and sheathers sucrose floatation are few other floatation methods used to concentrate stool specimens.

Materials required Formalin (5 or 10%), 0.85% NaCl, Zinc Sulphate (33%). Slide, Coverslip, Test tube

Procedure Mix one to a few grams of faeces with 10 times its volume of distilled water. Strain 10 mL through gauze into a centrifuge tube. Centrifuge at 2500 RPM for one minute. Discard the supernatant and add 2-3 mL of water to the sediment, breaking up the sediment. Repeat this procedure until the supernatent is nearly clear (Usually 2-4 washings). Pour off the supernatant and mix the sediment with 2 mL of zinc sulfate (38.6 g/100 mL of distilled water). Fill the centrifuge tube to within 5 mm of the top. Centrifuge again for 5 minutes. Let the sample stand for 5 minutes. Using a bacteriological loop, remove several loopfuls and examine microscopically. Add a drop of Lugol's iodine to stain. Examine for protozoan cysts and helminth egg capsules and larvae.

Observation Protozoan trophozoites and cyst and helminthic eggs and larvae may be seen and identified

Result Based on the morphology and size parasitic burden is identified as ------Fig – intestinal Protozoans

Fig 67b – Intestinal, Tissue protozoans and helminthes Fig67c. Intestinal helminthes eggs

170 IDENTIFICATION OF BLOOD AND TISSUE INTESTINAL PARASITES Introduction Different types of protozoans and lesser number of helminthes played a vital role in blood and tissue infections. Plasmodium sp, Tryphanosoma sp,Leishmania sp, T. vaginalis, W. bancrofty, Loa loa, Schistosoma, Paragonimus, Enterobius vermicularis, Taenia sp, are some of the parasites usually detected using blood, urine, sputum, CSF, bone marrow, spleen, skin tissue, deodinal aspirate, corneal scrapes samples. Thick and thin blood smears from peripheral blood is used to screen blood parasites. Microscopic methods are the best methods to screen. Stained blood films are the most reliable and efficient means for definitive diagnosis. Blood parasite can be detected by means of giemsa and wrights staining. It is used to differentiate nuclear or cytoplasmic morphology of platelets, RBCs, WBCs and parasites. should be prepared to observe clear morphology of blood parasites. There are two types of blood films , they are thin blood film and thick blood film. Stained blood films are the most reliable and efficient means for definitive diagnosis of nearly all blood parasites. For some parasites it is recommended to prepare a thin film in one slide, a thick film on other slide and combination on third slide.

Thin Blood film preparation Aim To prepare thin blood film for parasite examinations. Background information This is identical to a differential blood cell count film. It provides a good area for examining the morphology of parasites and RBCs and is used to confirm identity of parasites. This is less sensitive than thick film. Materials Required Blood from finger puncture or EDTA blood, Slide 2 nos, Lancet or syringe, Methanol Procedure Place a drop of blood onto the center of the slide about ½ inch from the end. Holding a second clean slide at a 40° angle, touch the angled end to the midlength area of the specimen slide. Pull the angled slide back into the blood, and allow the blood to almost fill the end area of the angled slide. Continue contact with the blood under the lower edge, quickly and steadily moving the angled slide until the blood is used up. Label the slide appropriately and allow it to air dry. If the film is stained with giemsa stain after the film is completely dried, fix it with methonal and allow to air dry Observation Thin blood smear is stained with giemsa/wrights/ leishmans staining procedure

Thick blood film preparation Aim To prepare thick blood smear for examination of parasitic infection. Principle The thick film essentially condenses into an area suitable for examination above 20 times more blood than thin film. The RBCs are lysed during the staining process so that only parasites, platelets and WBCs remain visible. It is used to differentiate a low parasitemia. Procedure For finger puncture blood Touch a clean glass slide to a large drop of blood standing on the finger until the circle of blood is nearly 1.8 - 2cm. For vein puncture blood Place a drop of blood in the centre of the slide. Using either the corner of the another slide or an applicator stick, spread the blood into a circle about 1.8-2cm. Allow the film to air dry. Do not fix the thick film. If it is stained with methanol based stain dip smear in buffered water for 10 min. Observation Thin blood smear is stained with giemsa/wrights/ leishmans staining procedure

Giemsa staining Aim To stain and differentiate blood parasites. Principle In 1891, Giemsa stain was discovered by Ramonowsky the Russian protozoologist. Giemsa stain is also called Ramonowsky stain. Until 1960s, Giemsa stain is used to describe chromosomes of cells. Giemsa stain is used to differentiate nuclear & cytoplasmic morphology of platelets (pink) RBC(pink), nuclei and granules of PMN (purple), parasite (blue), flagella of parasites (red). Giemsa stain is most useful to stain thick blood film. Giemsa stain must be diluted with buffered water ph 6.5 or 7 or 7.2 before using. Materials required Specimen - Finger puncture blood or vein puncture blood containing EDTA (0.020 g/10 mL of blood). Reagents - Giemsa stain and Giemsa buffer (Phosphate Buffer) Glass slides, alcohol, Glass marker, Applicator stick. Equipment -Microscope, binocular with mechanical stage; calibrated ocular micrometer. Procedure Wear gloves when performing this procedure. Thin blood films (only) Fix air-dried film in absolute methanol by dipping the film briefly (two dips) in a Coplin jar containing absolute methanol. Remove and let air dry. Stain with diluted Giemsa stain (1:20, vol/vol) for 20 min (For a 1:20 dilution, add 2 mL of stock Giemsa to 40 mL of buffered water in a Coplin jar). Wash by briefly dipping the slide in and out of a Coplin jar of buffered water (one or two dips). Note: Excessive washing will decolourize the film. Let air dry in a vertical position. Thick blood films (only) Allow film to air dry thoroughly for several hours or overnight. Do not dry films in an incubator or by heat, because this will fix the blood and interfere with the lysing of the RBCs. Note: If a rapid diagnosis of malaria is needed, thick films can be made slightly thinner than usual, allowed to dry for 1 h, and then stained. Do not fix. Stain with diluted Giemsa stain (1:50, vol/vol) for 50 min (For a 1:50 dilution, add 1mL of stock Giemsa to 50 mL of buffered water in a Coplin jar). Wash by placing film in buffered water for 3 to 5 min. Let air dry in a vertical position. Observation If Plasmodium is present, the cytoplasm stains blue and the nuclear material stains red to purple. Schüffner’s stippling and other inclusions in the RBCs infected by Plasmodium spp. stain red. Nuclear and Cytoplasmic colours that are seen in the malarial parasites will also be seen in the trypanosomes and any intracellular leishmaniae that are present. The sheath of microfilariae may or may not stain with Giemsa, while the body will usually appear blue to purple. Results Report any parasite, including the stage(s) seen (do not use abbreviations). Examples: Plasmodium falciparum rings and gametocytes, rings only. Plasmodium vivax rings, trophozoites, schizonts, and gametocytes. Wuchereria bancrofti microfilariae. Trypanosoma brucei gambiense/rhodesiense trypomastigotes. Trypanosoma cruzi trypomastigotes. Leishmania donovani amastigotes

Wrights staining Aim To stain thin blood film for the differentiation of blood parasites. Principle Wright stain is the combination of acid dye (Eosin) and a basic dye () for use of staining the blood smear. It highlights the differences among different types blood leukocytes for easier recognition of eosinophils and basophils.Wrights stain cen be used to stain thin blood films. Wrights stain also called ramanovsky dye. Materials required Wright stain – grind 0.9g of wright stain powderwith a portion of methanol in clean mortor, as the dye is dissolved in the methanol and pour this to tightly stoppered glass bottle. Shake vigorously several times daily for atleast 5 days. Filter through whatman no.1 paper into a brown bottle , shelf life is 36 months. Stock Buffer

Alkaline buffer - Na2HPO4-9.5g; Distilled water –1000mL

Acid buffer - NaH2PO4-9.2; Distilled water –1000mL Buffered water - Acid Buffer-50mL; Alkaline buffer- 50mL; Distilled water –900mL; pH- 6.8±0.1 Coplin jar, Morter and Pestel, filter paper, volumetric flask, Microscope, Brown bottle, pH meter, staining rack etc., Procedure Prepare thin and thick blood film. Add stain drop by drop. Count the number of drops needed to cover the surface. Let stand 1 – 3 minutes. Add the same drop of buffered water and mix the stain. After 4 – 8 minutes, flood the stain from the slide with buffered water. Wipe the stain. Air dry. Observed under microscope. Observation If Plasmodium is present, the cytoplasm stains blue and the nuclear material stains red to purple. Schüffner’s stippling and other inclusions in the RBCs infected by Plasmodium sp. Stains red. Nuclear and Cytoplasmic colours that are seen in the malarial parasites will also be seen in the Trypanosomes and any intracellular leishmaniae that are present. The sheath of microfilariae may or may not stain with Giemsa, while the body will usually appear blue to purple. Results Report any parasite, including the stage(s) seen (do not use abbreviations). Examples: Plasmodium falciparum rings and gametocytes, rings only. Plasmodium vivax rings, trophozoites, schizonts, and gametocytes. Wuchereria bancrofti microfilariae. Trypanosoma brucei gambiense/rhodesiense trypomastigotes. Trypanosoma cruzi trypomastigotes. Leishmania donovani amastigotes

Leishmans staining Aim To differentiate blood parasites Principle It is also called Romanowskys stain. Leishman stain is a combination of eosin and methylene blue. It also contain oxidation product of methylene blue called azures. These azures provide further contrast in smears of peripheral blood. Materials required Leishman's stain – it is prepared by grinding 150μg of leishman stain powder in Morter and Pestle by adding 100mL methanol. This stain matures in 2 or 3 days. pH 6.8 phosphate buffer Working stain: Leishman's stain diluted 1:4 with buffer Xylene Procedure For thin smear Cover the smear with 5-10 drops of leishman working stain and stained for 2 minutes. Add Buffered water to dilute the stain and allowed for 10 – 15 minutes. Wash the stain with buffered distilled water. Dry the slide and examine under high power objectives. For thick smear Perform staining as like thin smear Finally, immerse the smear in water until red colour disappeares. Observation If Plasmodium organisms are present, the cytoplasm stains blue and the nuclear material stains red to purple. Schüffner’s stippling and other inclusions in the RBCs infected by Plasmodium spp. stain red. Nuclear and Cytoplasmic colours that are seen in the malarial parasites will also be seen in the trypanosomes and any intracellular leishmaniae that are present. The sheath of microfilariae may or may not stain with Giemsa, while the body will usually appear blue to purple. Results Report any parasite, including the stage(s) seen (do not use abbreviations). Examples: Plasmodium falciparum rings and gametocytes, rings only. Plasmodium vivax rings, trophozoites, schizonts, and gametocytes. Wuchereria bancrofti microfilariae. Trypanosoma brucei gambiense/rhodesiense trypomastigotes. Trypanosoma cruzi trypomastigotes. Leishmania donovani amastigotes Note : Leishmen and Wright stain has both fixatives & stain. Giemsa stain , has only staining reagent. Dehaemoglobination should be done on thick blood film. This is done by immersing slide in distilled water for few minutes. JSB (Jaswant singh bhattacharjee) stain also used for staining of blood film. Thick blood film should not be fixed because fixation with methanol prevents lysis of RBC & dehaemoglobilization.

Figure 68.1- Malarial parasite

IDENTIFICATION OF BACTERIA Identification of bacteria is the most vital topic in microbiology. The methods of bacterial identification fall into three categories. They are phenotypic methods, immunological methods and genotyping methods. Phenotypic methods Morphological features of colony (Macroscopy) and bacterial cells (Microscopy) i.e., cultural variation of bacterial colony & morphological variation of bacterial cells. It also includes physiological and biochemical character of the bacteria. Microscopic morphology include a combination of cell shape, size, Gram stain, acid fast reactions, special structures e.g. endospores, granule and capsule. Macroscopic morphology are traits that can be accessed with the naked eye e.g. appearance of colony including texture, shape, pigment, speed of growth and growth pattern in broth. Physiology/Biochemical characteristic are traditional mainstay of bacterial identification. These include enzymes (catalase, oxidase, decarboxylase), fermentation of sugars, capacity to digest or metabolize complex polymers and sensitivity to drugs can be used in identification. The successful identification of microbe depends on: Using the proper aseptic techniques. Correctly obtaining the specimen. Correctly handling the specimen Quickly transporting the specimen to the lab. Once the specimen reaches the lab it is cultured and identified.

Immunological methods Immunological methods involve the interaction of a microbial antigen with an antibody (produced by the host immune system). Testing for microbial antigen or the production of antibodies is often easier than test for the microbe itself. . Genotypic methods Genotypic methods involve examining the genetic material of the organisms and has revolutionized bacterial identification and classification. Genotypic methods include PCR (RT-PCR, RAPD-PCR),use of nucleic acid probes, RFLP and plasmid fingerprinting. Increasingly genotypic techniques are becoming the sole means of identifying many microorganisms because of its speed and accuracy

Phenotypic Methods of Identification Microbiologists use 5 basic techniques to grow, examine and characterize microorganisms in the lab. They are called the 5 ‘I’s: inoculation, incubation, isolation, inspection and identification. Inoculation - To culture microorganisms a tiny sample (inoculum) is introduced into medium (inoculation). Isolation – it involves the separating one species from another. Incubation: once the media is inoculated it is incubated which means putting the culture in a controlled environment (incubation) to allow for multiplication. After incubation the organisms are inspected and identified phenotypically, immunologically or genetically.

Specimen Collection Successful identification depends on how the specimen is collected, handled and stored. It is important that general aseptic procedures be used including sterile sample containers and sampling methods to prevent contamination of the specimen. E.g. Throat and nasopharyngeal swabs should not touch the cheek, tongue or salvia. After collection the specimen must be transported to the laboratory and stored appropriately (e.g. refrigeration) and processed. Processing of the specimen Processing of specimen include microscopic examination of specimen and prepare the media for the inoculation of specimen. During throat specimen processing using direct microscopic Examination of Gram stained smear. We may note the presence of polymorphonuclear cells and organisms. Similary wet mount preparation (KOH, Saline, Iodine), giemsa staining, lactophenol cotton blue staing may provide preliminart idea about type of organism found (bacteri, fungi, protozoa, helminthes), nature of contamination, grams nature of bacteria, whether the pathogen is intracellular or extracellular. Direct fluorescent antibody test (DFA) used to highlight the presences of microorganisms in a specimen. DFA test are available for Staphylococcus aureus, Streptococcus pyogenes, Neisseria gonorhoeae and Haemophilus influenza. Based on the results, technician submit preliminary report to the physician for treatment, microbiologist select the media and incubation condition for bacterial cultivation.

Media inoculation Sample is to be inoculated on any one type of the following medium. It will depend on the type of organism tobe isolated. Table ----- provides details of media used for specific microorganisms. Inoculate the media using asepting technique.

Incubation After inoculation, media must be incubated appropriately. Aerobic environment or micro aerophilic environment or anaerobic envirment are selected for incubation.

Inspection After 24 to 48 hours of incubation, media is looked for the growth and note the colony morphology and microscopic nature. After preliminary observation, pure cuture of the isolate will be made by making use of storage media on tube as slant or deep. Identification Generic level confirmed identification will be performed by using various physiological and biochemical tests.

Biochemical Tests Biochemical tests like Methyl Red Test, Nitrate Reduction, Starch Hydrolysis, Catalase Test, H2S production, Indole test, Oxidase test, Oxidation fermentation, Phenylalanine deaminase test are performed to identify bacteria. Prominent biochemical tests also include carbohydrate fermentation, acid or gas production and the hydrolysis of gelatin or starch. Carbohydrate Fermentation -This medium show fermentation (acid production) and gas formation. The small Durham tube for collecting gas bubbles.

Rapid Tests Rapid test are used for the bacteria. It consist of plastic strips with 20 μl of dehydrated ONPG (β galactosidase); ADH (arginine dihydrolase); biochemical substrates LDC (lysine decarboxylase); ODC (ornithine used to detect decarboxylase); CIT (citrate utilization); H S biochemical 2 (hydrogen disulphide production); URE (urease); TDA characteristics. ( tryptophan deaminase); IND (indole production); VP The biochemical (Voges Proskauer test for acetoin); GEL ( gelatin substrates are inoculated liquefaction); the fermentation of glucose (GLU), with pure cultures and mannitol (MAN), inositol (INO), sorbitol (SOR), suspended in rhamnose (RHA), sucrose (SAC); Melibiose (MEL), physiological saline. amygdalin (AMY), and arabinose (ARA); and OXI After 5 hrs-overnight (oxidase). the 20 tests are converted to 7-9 digital profile.

Bacteriophage Typing Phage typing is a method used for detecting single strains of bacteria. this method can be used for identification and differentiating of bacterial pathogens. Phage typing is a method used for detecting single strains of bacteria. It is used to trace the source of outbreaks of infections. The viruses that infect bacteria are called bacteriophages. and some of these can only infect a single strain of bacteria. These phages are used to identify different strains of bacteria within a single species. A culture of the strain is grown in the agar and dried. A grid is drawn on the base of the petri dish to mark out different regions. Inoculation of each square of the grid is done by a different phage. The phage drops are allowed to dry and are incubated: The susceptible phage regions will show a circular clearing where the bacteria have been lysed, and this is used in differentiation.

Identification of Unculturable Organisms Environmental researchers estimate that < 1% of microorganisms are culturable and therefore it is not possible to use phenotypic methods of identification. These microorganisms are called viable nonculturable (VNC). Flow Cytometry Flow cytometry allows single or multiple microorganisms detection an easy, reliable and fast way. In flow cytometry, microorganisms are identified on the basis of the cytometry parameters or by means of certain dyes called fluorochromes that can be used independently or bound to specific antibodies. The cytometer forces a suspension of cells through a laser beam and measures the light they scatter or the fluorescence the cell emits as they pass through the beam.The cytometer also can measure the cell’s shape, size and the content of the DNA or RNA.

Immunological Methods The study of antibody(Ab) - antigen(Ag) reactions in in vitro is called serology. Serological reactions are the basic of immunological identification and diagnostic methods.

Precipitation Reactions Precipitation is the interaction of a soluble Ag with a soluble Ab to for an insoluble complex.The complex formed is an aggregate of Ag and Ab. Precipitation reactions occurs maximally only when the optimal proportions of Ag and Ag are present. Precipitation can also be done in agar referred to as immunodiffusion. precipitation test uses antibodies to detect for streptococcal group antigens.

Agglutination Reactions Agglutination is the visible clumping of an Ag when mixed with a specific Ab. Agglutination tests are widely used because they are simple to perform, highly specific, inexpensive and rapid. Standardized tests are available for the determination of blood groups and identification of pathogens and their products. E.g. Blood typing and detection of mycoplasma pneumonia.

Indirect (passive) agglutination. Ab/Ag is adsorbed or chemically coupled to the cell, latex beads or charcoal particles which serves as an inert carrier. Latex Agglutination Tests are available for the detection of Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenza and Camplyobacter spp.

Fluorescent Antibodies Antibodies can be chemically modified with fluorescent dyes such as rhodamine B, fluorescent red, fluorescien isothiocynate and fluoresces yellow or green. Cells with bound fluorescent antibody emit a bright red, orange, yellow or green light depending on the dye used. There are two distinct fluorescent Antibody procedure. They are direct and indirect. In the direct method the fluorescent Ab is directed to surface Ag of the organism. In the indirect method a non-fluorescent Ab reacts with the organism's Ag and a fluorescent Ab reacts with the non-fluorescent Ag. Fluorescent Ab can be used to detect microorganisms directly in tissue, long before a primary isolation technique yield the suspected pathogen. Fluorescent Ab has been used for the detection of Bacillus anthracis and HIV virus.

ELISA Engval and Perlmann developed it in 1970. Enzyme-linked immunosorbent assay (ELISA) is a popular test to detect the presence an antigen or antibody in a sample. The ELISA has been used as a diagnostic tool in medicine. It is a useful tool for determining serum antibody concentrations (such as with the HIV test). It has also found applications in the food industry in detecting potential food allergens. There are three important types of ELISA. They are Competitive ELISA, Sandwich ELISA and Indirect ELISA.

Immunoblot/Western Blot It involves separation of viral proteins by SDS PAGE (Sodium Dodecylsulphate – Polyacrylamide Gel Electrophoresis), transferring the resolved antigen to nitrocellulose membrane and identifying specific antigen through antigen and antibody reactions (Immuno blotting). It is used for the confirmation of HIV detected in ELISA. Genotypic methods Nucleic acid probes, PCR (RT-PCR, RAPD-PCR), Nucleic acid sequence analysis, rRNA analysis, RFLP,Plasmid fingerprinting are the Genotypic methods used for the microbe identification. Nucleic acid probes Nucleic acid hybridization is one of the most powerful tools available for microbe identification. Hybridization detects for a specific DNA sequence associated with an organism. The process uses a nucleic acid probe which is specific for that particular organism. The target DNA (from the organism) is attached to a solid matrix such as a nylon or nitrocellulose membrane. A single stranded probe is added and if there is sequence complementality between the target and the probe a positive hybridization signal will be detected. Hybridization is detected by a reporter molecule (radioactive, fluorescent, chemiluminescent) which is attached to the probe. Nucleic acid probes have been marketed for the identification of many pathogens such as N. gonorrhoeae. Polymerase Chain Reaction (PCR) PCR is widely used for the identification of microorganisms.Sequence specific primers are used with PCR in the amplification of DNA or RNA of specific pathogens.PCR allows for the detection even if only a few cells are present and can also be used on viable nonculturables. The presence of the appropriate amplified PCR product confirms the presence of the organisms. Primers are available for the identification of Niesseria gonorrhoeae, and to monitor food for the presence of Salmonella and Staphylococcus.

Real Time PCR and RT-PCR Currently many PCR tests employ real time PCR. This involves the use of fluorescent primers. The PCR machine monitors the incorporation of the primers and display an amplification plot which can be viewed continuously thru the PCR cycle. Real time PCR yields immediate results. Another application of PCR is RT-PCR (reverse trancriptase PCR). During RT-PCR an RNA template is used to generate cDNA and from this dsDNA is generated. The enzyme used is reverse transciptase. RT-PCR is used to detect for HIV and to monitor the progress of the disease.

RAPD-PCR Random amplified polymorphic DNA PCR uses a random primer (10-mer) to generate a DNA profile. The primer anneals to several places on the DNA template and generate a DNA profile which is used for microbe identification. RAPD has many advantages, they are, Pure DNA is not needed, Less labour intensive than RFLP. There is not need for prior DNA sequence data.RAPD has been used to fingerprint the outbreak of Listeria monocytogenes from milk.

DNA sequencing The determination of a small amount of DNA sequence can be used for microbial identification. The most common sequence used for microbe identification is DNA sequence of the 16S rRNA gene. PCR is used to amplify the 16S rRNA gene and the sequence determined. rRNA is a major component for ribosome and ribosome have the same function in protein synthesis in all cells. Computer analysis of 16S rRNA sequence has revealed the presence of signature sequences, short oligonucleotides unique to certain groups of organisms and useful in their identification. rRNA sequence can be used to fine tune identity at the species level e.g differentiating between Mycobacterium and Legionella. 16s rRNA sequence can also be used to identify microorganisms from a microbial community.

Restriction Fragment Length Polymorphism RFLP involves digestion of the genomic DNA of the organism with restriction enzymes. The restricted fragments are separated by agarose gel electrophoresis. The DNA fragments are transferred to a membrane and probed with probes specific for the desired organisms. A DNA profile emerges which can be used for microbe identification. Plasmid fingerprinting Plasmid fingerprinting identifies microbial species or similar strains as related strains often contain the same number of plasmids with the same molecular weight. Plasmid of many strains and species of E. coli, Salmonella, Camylobacter and Psseudomonas has demonstrated that this methods is more accurate than phenotypic methods such as biotyping, antibiotic resistance patterns, phage typing and serotyping. The procedure involves: The bacterial strains are grown, the cells lysed and harvested. The plasmids are separated by agarose gel electrophoresis The gels are stained with EtBr and the plasmids located and compared.

Collection of Various Specimens For Diangnosis

Specimen means a potion of body fluid or tissue, which represents actual infection status of an individual. The specimen must be material from the actual infection site and must be collected with a minimum of contamination from adjacent tissues, organs or secretions. Optimal times and total number of specimens for collection must be established for the best chance of recovery of causative microorganisms.A sufficient quantity of specimen must be obtained to perform the culture techniques. Appropriate collection devices, specimen containers and culture media must be used to ensure proper isolation.

Transporting Speed in transporting specimen is in prime importance because some laboratories refuse to accept specimen if they have been in transit too long. Certain specimen should be transported in a medium that preserves the microorganisms and helps to maintain the ratio of one organism to another. Special treatment is required for specimen when the organism is anaerobic. Anaerobic specimens are transported to the laboratory with in 10 minutes. If any delay, the specimen must be injected immediately in to an anaerobic vial. Vials should contain a transport medium with an indicator Methylene blue. For anaerobic culturing aspirates are very useful than swab. Commonly available transport medium are Amies transport medium Bile peptone transport medium Buffered glycerol saline base. CVTR medium Specimen preservative medium Transport charcoal medium Transport medium Wangs semisolid medium Cary blair medium Stuarts medium Alkaline peptone water Skin specimen Tissue obtained during surgical procedure, Aspirated material from an abscess or deep wound, Pus or exudates obtained from an infected site during surgery can be aspirated in to the syringe. Skin swab, Skin scrapings, Nail clippings, Hair.

Upper Respiratory Tract specimen Nasopharyngeal swab, Sodium alginate throat swab for pertusis, Sinus washings, Surgical biopsy, Swab of posterior pharynx, Swab of tonsil, Pernasal swab, Nasopharyngeal aspirates. Nasopharyngeal aspirates Gently pass a sterile catheter through one tonsil as far as the nasopharynx. Attach a sterile syringe to the catheter, and aspirate the specimen of mucopus. Dispense the specimen into a sterile container. Nasopharyngeal swab or throat swab. Bright light from over the shoulder of the patient should be focused in to the oral cavity. The patient is instructed to open the mouth at ‘aah’ position and breath deeply. The tongue is gently depressed with tongue blade to visualize the tonsillar fossae and posterior pharynx. The swab is extended between tonsillor pillars and behind the vulva. The tonsillar areas and posterior pharynx should be firmly rubbed with the swab. After collection the cotton swabs are placed into sterile Cary Blair transport media to prevent desiccation during transit to the laboratory. Lower Respiratory Track specimen For the pathogen isolated from LRT, samples are obtained through Needle Aspiration Method. In this method, a needle is inserted in to the throat region and collects sputum specimen. Needle aspiration includes Transtracheal Aspiration and lung aspiration. Other methods are bronchial washing and blood collection. Early morning sputum should be collected because that contains pooled overnight secretions in which pathogenic bacteria are more likely to be concentrated. For collection sterile wide mouthed jar with a tightly firmed screw cap lid can be used. Intestinal Tract specimen Stool or rectal swab or gastric aspirate or gastric biopsy. Gastric aspirate is collected with the help of Intubation. Sample collection from the hollow tube is called Intubation . Long sterile tube is attached with syringe and the tube is either swallowed or passed through a nostril in to the patient stomach. Specimens are withdrawn periodically. The most common intubation tube is Levin Tube.

Urinary Tract specimen Mid stream urine, Cathedral and Suprapubic aspiration. Urine is collected in sterile, dry, wide necked, leak proof container. About 20ml of sample should be collected. If immediate delivery to the laboratory is not possible, the urine should be refrigerated at 40C. If the delivery of more than 1 hour is anticipated, boric acid should be added to the urine. Specimen containing boric acid need not be refrigerated. Suprapubic aspiration is performed only of neonates, small children’s and occasionally for adults with clinically suspected UTI those who fail to establish diagnosis. This technique is best performed when the bladder is full. The suprapubic skin overlaying the urinary bladder is disinfected and tap is made. In the immediate site where the tap is to be made, about 1ml of anesthetic solution was injected subcutaneously. With the point of a sharply tapered surgical blade, make a small lance wound incision through the epidermis. Through this wound gently extend an 18-guage needle in to the urinary bladder and aspirate 10ml of urine into the syringe. Blood specimen Blood collection was performed by Needle aspiration procedure. Blood for culture should be taken by vein puncture. 10-20 ml of blood collected from adult 1-2.4 ml collected from young infants 2.4- 5 ml collected from old infants. Two-blood culture should be performed from each patient to confirm the causative agent. Genital Tract specimen FEMALE - Catheter aspirate, Aminocentesis fluid, Bartholin gland aspirate, Trans cervical aspirate, Fallopian aspirate, Swab of genital ulcer, Urethral discharge, Swab of posterior vagina. MALE - Urethral swab, Swab of genital ulcer, Penial discharge, Prostetic secretions CSF specimen Lumbar puncture, Brain abscess Selective cum differential media used for the isolation of bacteria S.No Pathogen Media 1 Escherichia coli Eosin Methylene Blue Agar Xylose Lysine Deoxy Cholate Gram negative rod, Agar motile Salmonella shigella agar Rajhans medium Hektoen enteric agar Violet red bile agar Tergitol 7 agar with TTC CLED with bromothymol blue Deoxy cholate agar Endoagar Mac conkey agar 2 Shigella spp Fluid selenite cystine broth* Fluid tetra thionate medium* G - rod nonmotile. Xylose Lysine Deoxy Cholate Agar Salmonella shigella agar Hektoen enteric agar Deoxy cholate citrate agar GN broth* 3 Salmonella spp. Hektoen enteric agar G - rod Deoxy cholate citrate agar Motile Fluid selenite cystine broth* Xylose lysine deoxy cholate agar Salmonella shigella agar Violet red bile agar GN broth* Bismuth sulphite agar CLED with bromothymol blue Brillient green agar 4 Klebsiella Macconkey agar G -rod, Tergitol 7 agar with TTC Non motile CLED with bromothymol blue Eosin methylene blue agar 5 Yersinia enterocolitica PSB enrichment broth* G -rod Yersinia selective medium CIA agar PSTA enrichment broth* 6 Proteus spp MacConkey agar G - rod Tergitol 7 agar with TTC motile CLED with bromothymol blue Deoxy cholate agar 7 Vibrio Alkaline peptone water* G - comma bacilli Monsor medium Motile TCBS agar Vibrio agar 8 Aeromonas Aeromonas isolation medium G -rod Furunculosis agar Motile Inositol brillient green bile agar Rippey cabelli agar Blood agar with 10 l/ml ampicillin 9 Pseudomonas Macconkey agar aeruginosa Tergitol 7 agar with TTC G -rod Cetrimide agar motile Pseudomonas isolation agar Malachite green agar 10 Campylobacter spp. Doyle's enrichment broth* G -rod Preston agar Motile Campy agar

11 Neisseria spp. Blood agar G - cocci Chocholate agar Non motile GC medium Modified Thayer Martin Agar New York City Agar Medium Columbia blood agar 12 Haemophillus spp. Eugonic agar G -rod Levinthols medium Motile Chocholate agar GC medium 13 Brucella spp Schaedler broth G -rod Brucella selective medium 14 Mycobacterium Petragnani medium Acid Fast Bacilli Lowenstein Jensen medium Middle brook medium Pfizer TB medium Dubos oleic agar Kirschner medium 15 Staphylococcus Blood agar G + cocci Mannitol salt agar Non motile Baired parker agar Vogel -Johnson agar DNase test medium Phenylethyl alcohol agar 16 Streptococcus Blood agar G + cocci Streptococcus enrichment Non motile broth* Edwards medium Neomycin blood agar Kanamycin esculin azide agar Synders test medium Todd -Hewitt medium 17 Enterococcus Azide dextrose broth G + cocci KF sterptococcus agar with Non motile TTC SF broth Slanez and Bartly medium 18 Corynebacteria Loeffler agar G + rod Cysteine tellurite blood agar Non motile Hoyle medium Tinddale medium 19 Bordetella Bordet –Gengou medium G- rod Rogen-Lowey medium 20 Listeria Listeria enrichment broth G- rod Modified Mc Bride Listeria agar Listeria selective medium 21 Legionella Buffered charcoal yeast extract G-rod agar Feeley Gorman broth Legionella agar 22 Bacillus anthrasis Columbia blood agar G+ rod Eugonic broth Eugonic agar 23 Clostridium Cooked meat medium G+rod Forget Fredett AGAR Wikins Chalgren Anaerobic Agar Reinforced clostridial agar Clostridium difficile agar Mc Clung agar 24 Bacteroides Wikins Chalgren Anaerobic G+rod Agar Bacteroides bile esculin agar L.D esculin agar Veal infusion agar 25 Mycoplasma Mycoplasma broth & agar Pleomorphic Cell free culture medium BYE agar

* Enrichment Broth Superantigens: Protein toxins that activate the immune system by binding to major histocompatibility complex (MHC) molecules and T-cell receptors (TCR) and stimulate large numbers of T cells to produce massive quantities of cytokines. Toxigenicity: The ability of a microorganism to produce a toxin that contributes to the development of disease. Virulence: The quantitative ability of an agent to cause disease. Virulent agents cause disease when introduced into the host in small numbers. Virulence involves adherence, persistence, invasion, and toxigenicity (see above). Adherence (adhesion, attachment): The process by which bacteria stick to the surfaces of host cells. After bacteria have entered the body, adherence is a major initial step in the infection process. The terms adherence , adhesion , and attachment are often used interchangeably. Carrier: A person or animal with asymptomatic infection that can be transmitted to another susceptible person or animal. Infection: Multiplication of an infectious agent within the body. Multiplication of the bacteria that are part of the normal fl ora of the gastrointestinal tract, skin, and so on is generally not considered an infection; on the other hand, multiplication of pathogenic bacteria (eg, Salmonella species)—even if the person is asymptomatic—is deemed an infection. Invasion: The process whereby bacteria, animal parasites, fungi, and viruses enter host cells or tissues and spread in the body. Microbiota: Microbial fl ora harbored by normal, healthy individuals. Nonpathogen: A microorganism that does not cause disease; may be part of the normal microbiota. Opportunistic pathogen: An agent capable of causing disease only when the host’s resistance is impaired (ie, when the patient is “immunocompromised”). Pathogen: A microorganism capable of causing disease. Pathogenicity: The ability of an infectious agent to cause disease. (See also virulence.)