PH 152 Medical and Public Health Microbiology Post Lab Notes PDF

PH152 MEDICAL AND PUBLIC HEALTH MICROBIOLOGY POST LAB EXERCISES 7: FUNGAL CULTURE AND MICROSCOPIC OBSERVATION  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION INTRODUCTION Medical Mycology - study and ident...

PH152 MEDICAL AND PUBLIC HEALTH MICROBIOLOGY POST LAB EXERCISES 7: FUNGAL CULTURE AND MICROSCOPIC OBSERVATION  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION INTRODUCTION Medical Mycology - study and identification of the pathogenic yeasts and molds, collectively called fungi. Mycoses = fungal infections - Most pathogenic fungi are exogenous, their natural habitats being water, soil, and organic debris. - The mycoses with the highest incidence—candidiasis and dermatophytosis—are caused by fungi that are part of the normal human microbiota and highly adapted to survival on the human host. PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Molds - multicellular, threadlike fungus. - filamentous colonies - the branching cylindric tubules is called hyphae. - the mass of intertwined hyphae that accumulates during active growth is a mycelium. Yeast - unicellular, budding fungus. - spherical to ellipsoid in shape - some species produce buds that characteristically fail to detach and become elongated; continuation of the budding process then produces a chain of elongated yeast cells called pseudohyphae.  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Asexual spores in molds: 1. Sporangiospores - spores that form within a sac called a sporangium. The sporangia are attached to stalks called sporangiophores. 2. Conidia - spores that form on specialized hyphae called conidiophores  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Asexual spores in molds: Conidia - spores that form on specialized hyphae called conidiophores  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Sexual spores in molds: 1. Zygospores - formed by the union of nuclear material from the hyphae of two different strains 2. Ascospores - produced in enclosures, which may be oval sacs or elongated tubes 3. Basidiospores - produced on club-shaped bodies  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Spores in yeast: Hyphae - Unlike molds, yeasts do not have true hyphae. Instead they form multicellular structures called pseudohyphae. Asexual Spores - The only asexual spore produced by yeasts is called a blastospore, or bud. These spores form as an outpouching of a cell by a budding process. If successive buds remain attached in the budding process, the result is the formation of a pseudohypha. PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Microscopic appearance of some of the more common molds PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Mycotic Diseases and Causative Agents  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Mycotic Diseases and Causative Agents 1. Superficial Mycoses - pathogenic fungi that cause infections of skin, hair, or nails are often referred to collectively as dermatophytes - 3 major genera: a. Trichophyton = which may infect hair, skin, nails, body, or feet. = “ringworm” infection = this genus is characterized by a develop cylindric, smooth-walled macroconidia b. Microsporum = distinctive multicellular macroconidia with echinulate walls. Both types of conidia are borne singly in these genera = ringworm infections of the hair and scalp, and also of the body. c. Epidermophyton = produces only macroconidia, which are smooth-walled, clavate, two- to four-celled, and formed in small clusters = ringworm of the body, including “athlete’s foot.” It does not affect hair or nails  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Mycotic Diseases and Causative Agents 1. Superficial Mycoses - These superficial fungal infections are called ringworms because the lesions are often circular in form. - The medical term for ringworm is tinea, followed by a word indicating the involved area, for example, tinea capitis (scalp), tinea corporis (body), or tinea pedis (feet).  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Mycotic Diseases and Causative Agents 2. Subcutaneous Mycoses - usually acquired when minor cuts or scratches introduce soil or plant debris (eg, splinters, thorns) containing the pathogenic fungus. - these mycoses are usually confined to the subcutaneous tissues, but in rare cases they become systemic and produce life-threatening disease. - Causative agents are either yeasts or display both a yeast and a mold phase (they are said to be dimorphic because of this). a. Sporotrichosis = Sporothrix schenckii (dimorphic fungus that lives on vegetation. It is associated with a variety of plants—grasses, trees, sphagnum moss, rose bushes, and other horticultural plants) b. Chromoblastomycosis = Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Rhinocladiella aquaspersa, and Cladophialophora carrionii (dematiaceous fungi which reside in soil and vegetation) c. Phaeohyphomycosis = Exophiala jeanselmei, Phialophora richardsiae, Bipolaris spicifera, and Wangiella dermatitidis. These species and others (eg, Exserohilum rostratum, Alternaria species, and Curvularia species) (exogenous molds that normally exist in nature) d. Mycetoma = several saprophytic species of fungi or actinomycetous bacteria that are normally found in soil.  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Mycotic Diseases and Causative Agents 3. Endemic/Systemic Mycoses - four primary systemic mycoses—coccidioidomycosis, histoplasmosis, blastomycosis, and paracoccidioidomycosis— is geographically restricted to specific areas of endemicity.  PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION Mycotic Diseases and Causative Agents 4. Opportunistic Mycoses - when the host’s immune defense mechanisms are decreased by illness (leukemias, lymphomas, acquired immunodeficiency syndrome) or by drugs (steroids, cancer chemotherapeutics, transplantation drugs), fungi (as well as other microorganisms) find the opportunity to invade and establish disease. a. Candidiasis = genus Candida = C albicans, C parapsilosis, C glabrata, C tropicalis, C guilliermondii, and C dubliniensis; most prevalent systemic mycosis b. Cryptococcosis = Cryptococcus neoformans and Cryptococcus gattii are environmental, basidiomycetous yeasts; Cryptococcus neoformans occurs in immunocompetent persons but more often in patients with HIV/AIDS, hematogenous malignancies, and other immunosuppressive conditions. c. Aspergillosis = A. fumigatus is the most common human pathogen, but many others, including A. flavus, A. niger, A. terreus, and A. lentulus may cause disease. These fungi produces abundant small conidia that are easily aerosolized. d. Mucormycosis (zygomycosis) = genera Rhizopus, Rhizomucor, Lichtheimia, Cunninghamella. Most prevalent species is Rhizopus oryzae. These fungi are ubiquitous thermotolerant saprobes. e. Penicillosis = Penicillium marneffei; this species has emerged as an endemic, opportunistic pathogen PH 152 POST LABORATORY DISCUSSION EXERCISE 7: FUNGAL CULTURE and MICROSCOPIC OBSERVATION PH152 MEDICAL AND PUBLIC HEALTH MICROBIOLOGY POST LAB | EXERCISE 8: ENTEROBACTERIACEAE  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS INTRODUCTION Gut/Intestinal Microflora Mostly anaerobic, few are facultative anaerobes Occur both in the lumen and attached to the mucosa, but do not normally penetrate the bowel wall Crucial component of the enterohepatic circulation. Koboziev, I., Reinoso Webb, C., Furr, K. L., & Grisham, M. B. (2014). Role of the enteric microbiota in intestinal homeostasis and inflammation. Free Radical Biology and Medicine, 68, 122–133. PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Functions of Gut/Intestinal Microflora Immune system regulation Protection against exogenous pathogens Digestion and nutrient metabolism  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Enteric Pathogens Exogenous Pathogens Endogenous Pathogens Acquired pathogens that Opportunistic Pathogens developed strategies to are microbes that can only compete with the intestinal cause infection when the community leading to host’s normal defenses are infection and/chronic not fully intact. disease.  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Characteristics Salmonella typhi Common Enteric Pathogens Capsule Negative (-ve) Catalase Positive (+ve) Citrate Negative (-ve) Salmonella species Flagella Positive (+ve) Gas Negative (-ve) Gelatin Hydrolysis Negative (-ve) - Gram-negative, flagellated facultatively anaerobic bacilli Gram Staining Negative (-ve) Growth in KCN Negative (-ve) characterized by O, H, and Vi antigens. H2S Positive (+ve) - Salmonellosis ranges clinically from the common Indole Negative (-ve) Motility Motile Salmonella gastroenteritis (diarrhea, abdominal cramps, and MR (Methyl Red) Positive (+ve) fever) to enteric fevers (including typhoid fever) MUG Test Negative (-ve) Nitrate Reduction Positive (+ve) Oxidase Negative (-ve) Pigment Negative (-ve) Shape Rod Spore Negative (-ve) TSIA (Triple Sugar Alkali/Acid Iron Agar) Urease Negative (-ve) VP (Voges Negative (-ve) Proskauer)  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Shigella species -Gram-negative, nonmotile, facultatively anaerobic, non-spore-forming rods, non-lactose fermenters. -utilize glucose and other carbohydrates, producing acid but not gas. - comprise four species and many serotypes - Causes shigellosis (watery diarrhea and dysentery)  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Characteristics Klebsiella pneumoniae Capsule +ve Catalase +ve Klebsiella pneumoniae Citrate +ve - commonly associated with UTIs, wounds and respiratory Flagella -ve infections Gas +ve -gram-negative, encapsulate, and non-motile bacterium. Gelatin Hydrolysis -ve -colonizes human mucosal surfaces of the oropharynx and Gram Staining -ve Growth in KCN +ve gastrointestinal (GI) tract. H2S -ve Indole -ve Motility -ve MR (Methyl Red) -ve MUG Test +ve Nitrate Reduction +ve OF (Oxidative- Fermentative Fermentative) Oxidase -ve Pigment -ve Shape Rod Spore -ve TSIA (Triple Sugar Iron A/A Agar) https://www.ncbi.nlm.nih.gov/books/NBK519004/ Urease +ve VP (Voges Proskauer) +ve  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Proteus species - An opportunistic pathogen - one of the common causes of urinary tract infections (UTIs) and are associated with infection-induced renal stones. - have pili (fimbriae) which are associated with adhesive properties and correlated with virulence. * Infections causes: pyogenic lesions, infections of the ear, respiratory tract infections, and nosocomial infections Proteus mirabilis in blood agar and MacConkey agar  PH 152 POST LABORATORY DISCUSSION Characteristics E.coli EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Catalase test Positive Oxidase test Negative E. coli Nitrate reduction test Methyl-Red (MR) test Positive Positive - Disease-producing E. coli known to be a common Voges-Proskauer (VP) test Negative Citrate utilization test Negative cause of “traveler’s diarrhea” (“turista”) and a variety of other Acetate utilization test Positive gastrointestinal diseases Indole test Positive Pyrrolidonyl-β-naphthylamide - Enterotoxigenic Escherichia coli (ETEC), (PYR) test Negative enterohemorrhagic Escherichia coli (EHEC), which is also known as H2S production test No Urease test Negative Shiga toxin-producing Escherichia coli (STEC), enteroinvasive Oxidative-fermentative (OF) test Fermentative Escherichia coli (EIEC), enteropathogenic Escherichia coli (EPEC), MUG test Positive and enteroaggregative Escherichia coli (EAEC) TSI reactions Acid/Acid, Gas, SIM No H2S, Indole +ve, motile Motility Motile Phenyl Pyruvic acid (PPA) test Negative Lysine decarboxylation test + Arginine decarboxylation test -/+ (strain variability) Ornithine decarboxylation test +/- (strain variability) ONPG + Sugar fermentation test Glucose Yes Sucrose No Lactose Yes Mannitol Yes https://www.ncbi.nlm.nih.gov/books/NBK564298/  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Other bacterial pathogens associated with intestinal diseases: Campylobacter jejuni Vibrio cholerae and Vibrio parahaemolyticus Yersinia Pseudomonas aeruginosa PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Table 1. Summary of enteric pathogens and methods of estimating incidence of infection. Kirk MD, Pires SM, Black RE, Caipo M, Crump JA, Devleesschauwer B, et al. (2015) World Health Organization Estimates of the Global and Regional Disease Burden of 22 Foodborne Bacterial, Protozoal, and Viral Diseases, 2010: A Data Synthesis. PLoS Med 12(12): e1001921. doi:10.1371/ journal.pmed.1001921 PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS https://www.cdc.gov/ncezid/dfwed/edeb/index.html Food and water borne diseases/intoxication Animal contact disease outbreak Antibiotics-associated diarrhea Antimicrobial resistance pathogens Cases with immunocompromised individuals  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Isolation and Identification 1. Isolation on Selective and Differential Media EMB (Eosin Methylene Blue) Agar - selective to Gram negative bacteria - Inhibitory to Gram + - differentiate lactose fermenters * metallic sheen/ blue black colonies = lactose fermenters * Dark purple = slow fermenters * pink colonies = non lactose fermenters https://laboratoryinfo.com/emb-agar-eosin-methylene-blue-agar/  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS MacConkey Agar - Gram negative bacteria - selective = bile salts and crystal violets - differentiate lactose fermenters * pink colonies = lactose fermenters * colorless colonies = non lactose fermenters https://laboratoryinfo.com/macconkey-agar/  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Salmonella-Shigella Agar - Salmonella and Shigella - selective = bile salts, sodium citrate, and bright green. - differentiate lactose fermenters * Red colonies = lactose fermenters * colorless colonies = non lactose fermenters Colonies of Salmonella https://www.labtestsguide.com/salmonella-shigella-agar spp. may appear with or without black centers (depending on the species isolated).  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Cetrimide Agar - Pseudomonas aeruginosa - selective = magnesium chloride and potassium sulphate - yellow-green to blue color colonies which indicates the production of pyocyanin of P. aeruginosa https://microbiologyinfo.com/cetrimide-agar-composition-principle-uses-preparation-and-colony-morphology/ Limitations: 1. Occasionally some enterics will exhibit a slight yellowing of the medium; however, this coloration is easily distinguished from fluorescein production because this yellowing does not fluoresce. 2. Some non-fermenters and some aerobic spores formers may exhibit a water-soluble tan to brown pigmentation on this medium. Serratia strains may exhibit a pink pigmentation.  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS 2. Biochemical Testing - IMVIC * Indole = enzyme tryptophanase, which can cleave tryptophan to produce indole, pyruvic acid, and ammonia. * MR Test = produce mixed acids from glucose fermentation. https://microbenotes.com/imvic-tests/ * VP Test = presence of acetoin, suggesting the bacterium follows the 2,3- * Citrate Test = use citrate as the only carbon source butanediol fermentation pathway.  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS - TSI = Fermentation of sugars; Gas Production; Hydrogen Sulfide (H2S) Production - Urea = enzyme urease and is capable of hydrolyzing urea into https://www.onlinebiologynotes.com/tsi-triple-sugar-iron-test-objective-principle-procedure-and-result/ ammonia and carbon dioxide 3. Serological Testing – Detection of antigen https://microbiologyinfo.com/urease-test-principle-media-procedure-and-result/  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Coliform -gram-negative, facultative anaerobic bacteria found in the intestines of warm-blooded animals, including humans. They can ferment lactose to produce acid and gas. Used as indicators of fecal contamination in water and food. Examples include Escherichia coli, Klebsiella spp., and Enterobacter spp. Non-Coliform - gram-negative, but they do not ferment lactose to produce gas. Examples include Salmonella, Shigella, and Yersinia. These organisms are often pathogenic and can cause gastrointestinal diseases.  PH 152 POST LABORATORY DISCUSSION EXERCISE 8: ENTEROBACTERIACEAE AND GRAM NEGATIVE INTESTINAL PATHOGENS Dichotomous Key of Identifying Enterobacteriaceae Source: Chapter 80 Gram-Negative Intestinal Pathogens of Benson Microbiological Applications Lab Manual PH152 MEDICAL AND PUBLIC HEALTH MICROBIOLOGY POST LAB EXERCISES 9: Urine Culture Techniques and Urinary Tract Pathogens PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens INTRODUCTION Urine is a sterile waste product composed of water-soluble nitrogen products that is formed in the kidneys, stored in the urinary bladder, and is excreted from the body through the urethra. Three main steps: 1. Filtration 2. Reabsorption 3. Secretion.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Urine Composition Approximately 91-96% water, with the remaining 4-9% consisting of inorganic salts, urea, organic compounds, and organic ammonium salts. The percentage composition vary depending on factors such as hydration status, diet, medications, and underlying medical conditions.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Urinalysis The routine urine examination. - Most useful tool for the clinicians as an indicator or health or disease. - Particularly, used in renal metabolic disorders. - Often done for patients admitted to the hospital. - The routine urine analysis is divided into four main groups: 1. Physico-chemical properties. 2. Chemical examination. 3. Microscopic examination. 4. Bacterial screening  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Urine Collection Urine must be collected in a sterile bottle. - The routine urine examination must be conducted within 30 min. - In case of delay, urine must be refrigerated. Delay in sample examination will result in: Decreased pH by the utilization of glucose by bacteria. Increased pH by the conversion of urea to ammonia by bacteria. In increased pH, the tendency of phosphates to precipitate will increase. Oxidation of urobilinogen to urobilin. It may give false negative result for jaundice.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Color of normal urine Normal urine is clear and the color ranges from pale yellow to deep amber. it is the result of a pigment called urochrome. the higher the concentration of urine, the deeper is the color. the concentration of urine is highest in the morning specimen (overnight urine) and is lowest in a specimen passed an hour after much fluid has been taken. colored urines occur in certain diseases or metabolic disorders, and after the administration of many drugs. - B vitamins turn urine an eyepopping neon yellow BUT may also indicate liver disease. - Porphyria, a disease that affects your skin and nervous system, turns urine the color of port wine.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Color of normal urine Most changes in urine color are harmless and temporary and may be due to: – Certain foods = beets may turn urine red – Dyes in foods/drinks – Supplements = vitamins – Prescription drugs Unusual urine color can indicate an infection or serious illness. PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Color of normal urine Different colors of urine pale yellow (straw) light yellow yellow green-yellow (olive) red-yellow red red-brown brown-black black milky PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Turbidity  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Turbidity Substances that cause cloudiness but that are not considered unhealthy include: – mucous, – sperm and prostatic fluid, – cells from the skin, – normal urine crystals, and – contaminants (like body lotions and powders). Other substances that can make urine cloudy (such as red blood cells, white blood cells, yeast or bacteria) indicate a condition that requires attention.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Odor Normally urine smells aromatic due to the presence of volatile organic acids. In some cases, an unusual or strong urine odor may be due to benign conditions that are not harmful, such as eating certain foods or taking certain medications and volatile acids. The urine of patients with diabetes mellitus may have a fruity (acetone) odor because of ketosis. Urine which is infected with Gram-negative organisms often has a distinctive unpleasant smell.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Specific Gravity SG is a measure of the density of the dissolved chemicals in the specimen. There are direct relationship between concentration of substance in urine (concentration of urine) and SG. The normal specific gravity (correctly called relative density) of a pooled 24 hour urine sample is between 1.025 and 1.010.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties pH On a normal mixed diet the urine is usually acid, generally varying in pH between 5.5 and 8.0, with a mean of 6 in 24 hours. Acidic Urine : Diabetic ketosis, fevers. Alkaline Urine: - A vegetarian diet which causes a tendency to alkalosis. -It may also be grossly increased by bacterial infection of the urinary tract.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Chemical Examination Reagent strips are used only once and discarded. Testing – Perform within 1 hour after collection – Allow refrigerated specimens to return to room temperature. – Dip strip in fresh urine and compare color of pads to the color chart after appropriate time period. – Instruments are available which detect color changes electronically PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Reagent Strips  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Glucose Presence of glucose (glycosuria) indicates that the blood glucose level has exceeded the renal threshold. Useful to screen for diabetes.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Bilirubin Bilirubin is a byproduct of the breakdown of hemoglobin. Normally contains no bilirubin. Presence may be an indication of liver disease, bile duct obstruction or hepatitis. Since the bilirubin in samples is sensitive to light, exposure of the urine samples to light for a long period of time may result in a false negative test result.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Ketones Ketones are excreted when the body metabolizes fats incompletely (ketonuria)  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Blood Presence of blood may indicate infection, trauma to the urinary tract or bleeding in the kidneys. False positive readings most often due to contamination with menstrual blood.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Protein Presence of protein (proteinuria) is an important indicator of renal disease. False negatives can occur in alkaline or dilute urine or when primary protein is not albumin.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Urobilinogen Urobilinogen is a degradation product of bilirubin formed by intestinal bacteria. It may be increased in hepatic disease or hemolytic disease  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Nitrite Nitrite formed by gram negative bacteria converting urinary nitrate to nitrite  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Leukocytes Leukocytes (white blood cells) usually indicate infection. Leucocyte esterase activity is due to presence of WBCs in urine while nitrites strongly suggest bacteriuria. PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Normal Values Negative results for glucose, ketones, bilirubin, nitrites, leukocyte esterase and blood. Protein negative or trace. pH 5.5-8.0 Urobilinogen 0.2-1.0 Ehrlich units  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Physico-chemical Properties Parameter Normal Abnormal Color Yellow straw, amber, Dark amber – concentrated urine insufficient of fluid intake transparent Cloudy - infectious process Dark orange – drug, eg: pyridium Red or dark brow - disease process causing blood in urine Consistency Clear Mucus plug, viscid, thick - infectious process Odor Faint aromatic Offensive – infectious process Sterility No microorganisms present Microorganisms present = UTI pH 4.5-8.0 Over than 8.0 - UTI Under 4.5 - uncontrolled diabetes, starvation, dehydration Specific gravity 1.010-1.025 Over 1.025 – diabetes mellitus, under hydration Under 1.01 – diabetes insipidus, kidney disease, over hydration Urine glucose Not present Present – diabetes mellitus Urine ketones bodies Not present Present – diabetic coma, starvation, prolonged vomiting Urine blood Not present Occult – kidney disease Bright red – hemorrhage  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Microscopic Examination White blood cells Pyuria refers to the presence of elevated number of leukocytes (granulocytes): – Upper or lower UT infection – Glomerulonephrotis – Vaginal & cervical infections – External urethral meatus (men & women) Normal range: 0-2/HPF (5+ indicates an infection and 10+ indicates more severe conditions).  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Microscopic Examination Epithelial cells Normal urine – not present/unquantified Renal tubular epithelial cells, usually larger than granulocytes, contain a large round or oval nucleus are normally slough into the urine in small amounts. Positive – nephrotic syndrome, condition leading to tubular degeneration.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Microscopic Examination Urinary Crystals Normal urine – present but unquantified. Type of crystals: – Calcium oxalate – Triple phosphate – Amorphous phosphates – Uric acids Uric acid crystals (hyperuricemia) – Cyctine – Tyrosine – Leusine Elevated numbers of crystal may indicate: – Hypercalcemia – Cystine crystal –cystinuria May due to renal failure or other renal disease.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Microscopic Examination Casts Formed at the distal convoluted tubule or the collecting duct (distal nephron). Protein based cylindrical molds of the renal tubule Result of damage to the renal tubule Can contain cells and other material Dehydration and acidic urine especially predisposes to cast formation. Types of casts - Protein - RBC - WBC - Other  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Urinary Bacteria Abundant normal microbial flora of the vagina or external urethral meatus. Ability to rapidly multiply in urine standing room temperature. However, it should be interpreted in view of clinical symptoms. Bacterial culture must be done in case of bacteriuria  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Urinary Yeast Yeast cells may be contaminants or represent a true yeast infection. Often difficult to distinguish from red cells and amorphous crystals but can be distinguished by their tendency to bud. Most often they are Candida, which may colonize bladder, urethra or vagina. PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Microscopic Examination PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Microscopic Examination PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Microscopic Examination PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Microscopic Examination  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Urinary Tract Infection Urinary tract infections (UTIs) are caused by a wide range of pathogens, including Gram-negative and Gram-positive bacteria, as well as fungi. Urinary tract infections (UTI’s) are one of the most common infections treated by primary care physicians (PCP) in the world to date.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Urinary Tract Infection The urinary tract is divided into upper and lower tracts. Cystitis is an infection within the bladder and is the most common type of lower UTI. Urethritis is an infection with the ureters. Pyelonephritis is an infection within the kidneys. Pyelonephritis is more serious than urethritis or cystitis, but less common.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Types of Urinary Tract Infection 1. Uncomplicated UTIs - affect individuals who are otherwise healthy and have no structural or neurological urinary tract abnormalities - typically affect women, children and elderly patients who are otherwise healthy. 2. Complicated UTIs - UTIs associated with factors that compromise the urinary tract or host defense. - usually associated with indwelling catheters, urinary tract abnormalities, immunosuppression or exposure to antibiotics.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Types of Urinary Tract Infection 3. Recurrent UTI’s are described as at least 2 - 3 uncomplicated or complicated infections in a 6 month time period or 3 UTIs within a year. 4. Relapse UTI’s are infections which reoccur within the first two weeks of completing the initial antibiotic drug therapy for the first UTI.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Types of Urinary Tract Infection 5. Catheter-associated UTI’s (CAUTI’s) occur when a person has an indwelling urinary or supra-pubic catheter and or has recently been catheterized in the past 48 hours.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Risk Factors of UTI UTI’s are more common in women and girls than in men because their urethra is shorter than males. A female urethra is approximately 4 inches in length, and closer to the rectum, where more bacteria exists and can easy travel into the urinary tract.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Risk Factors of UTI Previous UTI’s. Family-related risk (a mother, sister, aunt, or grandmother with history of UTI’s). Changes in a female’s bacterial growth caused by medication used for vaginal infections, or during menopause. Age 65 years or older, with urinary incontinence, urinary retention, numerous hospitalizations and or institutionalizations. Diabetes (impaired insulin processing within the body). Neuropathy (incomplete emptying of the bladder due to diabetic related nerve damage).  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Risk Factors of UTI Men with an enlarged prostate or incomplete emptying of the bladder related to narrowing of the urethra. Young children being toilet trained, if not taught proper hygiene of wiping from front to back after bowel movements. Prolonged use of an indwelling urinary or suprapubic catheter and or having recently experienced urinary catheterization in the hospital or medical environment. PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Causes of UTI  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Causes of UTI - The most common causative agent for both uncomplicated and complicated UTIs is Uropathogenic Escherichia coli (UPEC). - Uncomplicated UTIs, UPEC is followed in prevalence by Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, group B Streptococcus (GBS), Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida spp. - Complicated UTIs, the order of prevalence for causative agents, following UPEC as most common, is Enterococcus spp., K. pneumoniae, Candida spp., S. aureus, P. mirabilis, P. aeruginosa and GBS.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Causes of UTI AipA, adhesion and invasion mediated by the Proteus autotransporter; CNF1, cytotoxic necrotizing factor 1; Ebp, endocarditis- and biofilm-associated; Epa, enterococcal polysaccharide antigen; Esp, enterococcal surface protein; ExoS, exoenzyme S; F1C pili, type 1-like immunological group C pili; HlyA, α-haemolysin; HpmA, haemolysin; MR/P, mannose-resistant Proteus-like; Msr, methionine sulfoxide reductase; NAF, non-agglutinating fimbria; ND, not determined; PMF, P. mirabilis-like fimbria; P pili, pyelonephritis-associated pili; Pta, Proteus toxic agglutinin; TaaP, trimeric autoagglutinin autotransporter of Proteus; UPEC, uropathogenic Escherichia coli.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Causes of UTI Feces from the rectum find their way into the urethra, which can occur when a female wipes her perianal area from the back to the front of the body.  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Causes of UTI Urine that is left sitting in the bladder after urination is referred to as stagnant urine residual which provides an environment for bacterial growth. Stagnant urine residual happens when the bladder is not emptied completely during urination, leaving a small amount of left over urine. Treatment for stagnant urine residual is an in-out (straight) catheterization. − Catheterization is considered a sterile procedure requiring a nurse to perform under a physician’s order  PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens Diagnosis of UTI If UTI is suspected an individual should be seen by a physician right away, either by the primary care physician (PCP) or in the hospital emergency room, based on the increased risk of the infection developing into sepsis. − Urinary tract infections are the second leading cause of sepsis, which if not treated immediately can quickly lead to death. Verification of UTI is based on the presence of bacteria and white blood cells in the urinary tract with the presents of physical symptoms. A person can have increased bacteria in their urine without having any physical symptoms. − This is referred to as Asymptomatic Bacteriuria (ASB), which is at times confused with UTI. PH 152 POST LABORATORY DISCUSSION EXERCISE 9: Urine Culture Techniques and Urinary Tract Pathogens